Comparison of proton-specific ATPase activities in plume and root tissues of two co-occurring hydrocarbon seep tubeworm species Lamellibrachia luymesi and Seepiophila jonesi.

PubMed

Dattagupta, Sharmishtha; Redding, Meredith; Luley, Kathryn; Fisher, Charles

2009-01-01

Lamellibrachia luymesi and Seepiophila jonesi are co-occurring species of vestimentiferan tubeworms found at hydrocarbon seepage sites on the upper Louisiana slope of the Gulf of Mexico. Like all vestimentiferans, they rely on internal sulfide-oxidizing symbiotic bacteria for nutrition. These symbionts produce hydrogen ions as a byproduct of sulfide oxidation, which the host tubeworm needs to eliminate to prevent acidosis. The hydrothermal vent tubeworm Riftia pachyptila uses a high activity of P- and V-type H + -ATPases located in its plume epithelium to excrete protons. Unlike R. pachyptila , the seep species grow a posterior root, which they can use in addition to their plumes as a nutrient exchange surface. In this study we measured the ATPase activities of plume and root tissues collected from L. luymesi and S. jonesi , and used a combination of inhibitors to determine the relative activities of P- and V-type H + -ATPases. We found that the total H + -ATPase activity of their plumes was approximately 14 μmol h -1  g -1 wet weight, and that of their roots was between 5 and 7 μmol h -1  g -1 wet weight. These activities were more than ten times lower than those measured in R. pachyptila . We suggest that seep tubeworms might use passive channels to eliminate protons across their roots, in addition to ATP-dependant proton pumps located in their plumes and roots. In addition, we found strong differences between the types of ATPase activities in the plumes of L. luymesi and S. jonesi . While the H + -ATPase activity of L. luymesi plumes is dominated by P-type ATPases, S. jonesi has an unusually high activity of V-type H + -ATPases. We suggest that S. jonesi relies on its high V-type H + -ATPase activity to drive carbon dioxide uptake across its plume surface. L. luymesi , on the other hand, might rely partially on bicarbonate uptake across its root.

The mechanism of the calorigenic action of thyroid hormone. Stimulation of Na plus + K plus-activated adenosinetriphosphatase activity.

PubMed

Ismail-Beigi, F; Edelman, I S

1971-06-01

In an earlier study, we proposed that thyroid hormone stimulation of energy utilization by the Na(+) pump mediates the calorigenic response. In this study, the effects of triiodothyronine (T(3)) on total oxygen consumption (Q(OO2)), the ouabain-sensitive oxygen consumption [Q(OO2)(t)], and NaK-ATPase in liver, kidney, and cerebrum were measured. In liver, approximately 90% of the increase in Q(OO2) produced by T(3) in either thyroidectomized or euthyroid rats was attributable to the increase in Q(OO2)(t). In kidney, the increase in Q(OO2)(t) accounted for 29% of the increase in Q(OO2) in thyroidectomized and 46% of the increase in Q(OO2) in euthyroid rats. There was no demonstrable effect of T(3) in euthyroid rats on Q(OO2) or Q(OO2)(t) of cerebral slices. The effects of T(3) on NaK-ATPase activity in homogenates were as follows: In liver +81% from euthyroid rats and +54% from hypothyroid rats. In kidney, +21% from euthyroid rats and +69% from hypothyroid rats. T(3) in euthyroid rats produced no significant changes in NaK-ATPase or Mg-ATPase activity of cerebral homogenates. Liver plasma membrane fractions showed a 69% increase in NaK-ATPase and no significant changes in either Mg-ATPase or 5'-nucleotidase activities after T(3) injection. These results indicate that thyroid hormones stimulate NaK-ATPase activity differentially. This effect may account, at least in part, for the calorigenic effects of these hormones.

Kalpaamruthaa ameliorates mitochondrial and metabolic alterations in diabetes mellitus induced cardiovascular damage.

PubMed

Latha, Raja; Shanthi, Palanivelu; Sachdanandam, Panchanadham

2014-12-01

Efficacy of Kalpaamruthaa on the activities of lipid and carbohydrate metabolic enzymes, electron transport chain complexes and mitochondrial ATPases were studied in heart and liver of experimental rats. Cardiovascular damage (CVD) was developed in 8 weeks after type 2 diabetes mellitus induction with high fat diet (2 weeks) and low dose of streptozotocin (2 × 35 mg/kg b.w. i.p. in 24 hr interval). In CVD-induced rats, the activities of total lipase, cholesterol ester hydrolase and cholesterol ester synthetase were increased, while lipoprotein lipase and lecithin-cholesterol acyltransferase activities were decreased. The activities of lipid-metabolizing enzymes were altered by Kalpaamruthaa in CVD-induced rats towards normal. Kalpaamruthaa modulated the activities of glycolytic enzymes (hexokinase, phosphogluco-isomerase, aldolase and glucose-6-phosphate dehydrogenase), gluconeogenic enzymes (glucose-6-phosphatase and fructose-1, 6-bisphosphatase) and glycogenolytic enzyme (glycogen phosphorylase) along with increased glycogen content in the liver of CVD-induced rats. The activities of isocitrate dehydrogenase, succinate dehydrogenase, malate dehydrogenase, α-ketoglutarate dehydrogenase, Complexes and ATPases (Na(+)/K(+)-ATPase, Ca(2+)-ATPase and Mg(2+)-ATPase) were decreased in CVD-induced rats, which were ameliorated by the treatment with Kalpaamruthaa. This study ascertained the efficacy of Kalpaamruthaa for the treatment of CVD in diabetes through the modulation of metabolizing enzymes and mitochondrial dysfunction.

Lipid requirement of the membrane sodium-plus-potassium ion-dependent adenosine triphosphatase system.

PubMed Central

Wheeler, K P; Walker, J A; Barker, D M

1975-01-01

The dependence of the (Na-++K-+)-dependent ATPase (adenosine triphosphatase) (EC 3.6.1.3) on lipid has been examined in a number of different ways, with the use of various preparations from kidney tissue. The main findings were as follows. (1) The ATPase activities of the preparations examined were closely correlated with their total phospholipid content. (2) Extraction of the ATPase with deoxycholate or Lubrol W, combined with suitable salt-fractionation and washing procedures, removed phospholipid, cholesterol and enzymic activity in parallel; but activity was completely lost before all lipid had been removed. (3) The loss of activity could not be attributed to inhibition by residual detergent. (4) No selective removal of any particular phospholipid class by detergent could be detected. (5) Consistent reactivation of the Lubrol-extracted enzymes was obtained by adding dispersions of exogenous phospholipid, but only some, bearing a net negative charge, such as phosphatidylserine and phosphatidylglycerol, were effective. (6) The degree of reactivation was correlated with the amount of residual activity remaining after lipid depletion. (7) Partial purification of the ATPase, giving a 50-fold increase in specific activity, was not accompanied by selective enhancement of any particular class of phospholipid. We conclude that although the ATPase is dependent on phospholipid, only the reactivation results provide evidence for specificity. PMID:125082

[Effects of desulfurization waste on calcium distribution, Ca(2+)-ATPase activity, and antioxidant characteristics of rice leaf under alkali stress].

PubMed

Mao, Gui-Lian; Xu, Xing; Zeng, Jin; Yue, Zi-Hui; Yang, Shu-Juan

2012-02-01

To approach the action mechanisms of desulfurization waste on alleviating alkali stress-induced injury of rice, a pot experiment was conducted to study the variations of leaf total calcium content, calcium distribution, plasma membrane Ca(2+)-ATPase activity, and reactive oxygen content of rice seedlings under alkali stress after the application of desulfurization waste. In the control, a few calcium particulates scattered in the cell wall and chloroplasts, while applying desulfurization waste or CaSO4 increased the calcium particulates in the plasma membrane, intercellular space, cell wall, and vacuole significantly. With the increasing application rate of desulfurization waste or CaSO4, the leaf total calcium content increased, Ca(2+)-ATPase activity in plasma membrane and tonoplast presented an increasing trend, plasma membrane relative permeability, MDA content, and O2 production rate decreased, and SOD and POD activities increased. The desulfurization waste could relieve the alkali stress to rice in some extent, and the main reactive compound in the waste could be CaSO4.

The Mechanism of the Calorigenic Action of Thyroid Hormone

PubMed Central

Ismail-Beigi, Faramarz; Edelman, Isidore S.

1971-01-01

In an earlier study, we proposed that thyroid hormone stimulation of energy utilization by the Na+ pump mediates the calorigenic response. In this study, the effects of triiodothyronine (T3) on total oxygen consumption (Q OO2), the ouabain-sensitive oxygen consumption [Q OO2(t)], and NaK-ATPase in liver, kidney, and cerebrum were measured. In liver, ∼90% of the increase in Q OO2 produced by T3 in either thyroidectomized or euthyroid rats was attributable to the increase in Q OO2(t). In kidney, the increase in Q OO2(t) accounted for 29% of the increase in Q OO2 in thyroidectomized and 46% of the increase in Q OO2 in euthyroid rats. There was no demonstrable effect of T3 in euthyroid rats on Q OO2 or Q OO2(t) of cerebral slices. The effects of T3 on NaK-ATPase activity in homogenates were as follows: In liver +81% from euthyroid rats and +54% from hypothyroid rats. In kidney, +21% from euthyroid rats and +69% from hypothyroid rats. T3 in euthyroid rats produced no significant changes in NaK-ATPase or Mg-ATPase activity of cerebral homogenates. Liver plasma membrane fractions showed a 69% increase in NaK-ATPase and no significant changes in either Mg-ATPase or 5'-nucleotidase activities after T3 injection. These results indicate that thyroid hormones stimulate NaK-ATPase activity differentially. This effect may account, at least in part, for the calorigenic effects of these hormones. PMID:4252666

Reduced cholesterol levels in renal membranes of undernourished rats may account for urinary Na⁺ loss.

PubMed

Oliveira, Fabiana S T; Vieira-Filho, Leucio D; Cabral, Edjair V; Sampaio, Luzia S; Silva, Paulo A; Carvalho, Vera C O; Vieyra, Adalberto; Einicker-Lamas, Marcelo; Lima, Vera L M; Paixão, Ana D O

2013-04-01

It has been demonstrated that reabsorption of Na⁺ in the thick ascending limb is reduced and the ability to concentrate urine can be compromised in undernourished individuals. Alterations in phospholipid and cholesterol content in renal membranes, leading to Na⁺ loss and the inability to concentrate urine, were investigated in undernourished rats. Sixty-day-old male Wistar rats were utilized to evaluate (1) phospholipid and cholesterol content in the membrane fraction of whole kidneys, (2) cholesterol content and the levels of active Na⁺ transporters, (Na⁺ + K⁺)ATPase and Na⁺-ATPase, in basolateral membranes of kidney proximal tubules, and (3) functional indicators of medullary urine concentration. Body weight in the undernourished group was 73 % lower than in control. Undernourishment did not affect the levels of cholesterol in serum or in renal homogenates. However, membranes of whole kidneys revealed 56 and 66 % reduction in the levels of total phospholipids and cholesterol, respectively. Furthermore, cholesterol and (Na⁺ + K⁺)ATPase activity in proximal tubule membranes were reduced by 55 and 68 %, respectively. Oxidative stress remained unaltered in the kidneys of undernourished rats. In contrast, Na⁺-ATPase activity, an enzyme with all regulatory components in membrane, was increased in the proximal tubules of undernourished rats. Free water clearance and fractional Na⁺ excretion were increased by 86 and 24 %, respectively, and urinary osmolal concentration was 21 % lower in undernourished rats than controls. Life-long undernutrition reduces the levels of total phospholipids and cholesterol in membranes of renal tubular cells. This alteration in membrane integrity could diminish (Na⁺ + K⁺)ATPase activity resulting in reduced Na⁺ reabsorption and urinary concentrating ability.

Single point mutations distributed in 10 soluble and membrane regions of the Nicotiana plumbaginifolia plasma membrane PMA2 H+-ATPase activate the enzyme and modify the structure of the C-terminal region.

PubMed

Morsomme, P; Dambly, S; Maudoux, O; Boutry, M

1998-12-25

The Nicotiana plumbaginifolia pma2 (plasma membrane H+-ATPase) gene is capable of functionally replacing the H+-ATPase genes of the yeast Saccharomyces cerevisiae, provided that the external pH is kept above 5.0. Single point mutations within the pma2 gene were previously identified that improved H+-ATPase activity and allowed yeast growth at pH 4.0. The aim of the present study was to identify most of the PMA2 positions, the mutation of which would lead to improved growth and to determine whether all these mutations result in similar enzymatic and structural modifications. We selected additional mutants in total 42 distinct point mutations localized in 30 codons. They were distributed in 10 soluble and membrane regions of the enzyme. Most mutant PMA2 H+-ATPases were characterized by a higher specific activity, lower inhibition by ADP, and lower stimulation by lysophosphatidylcholine than wild-type PMA2. The mutants thus seem to be constitutively activated. Partial tryptic digestion and immunodetection showed that the PMA2 mutants had a conformational change making the C-terminal region more accessible. These data therefore support the hypothesis that point mutations in various H+-ATPase parts displace the inhibitory C-terminal region, resulting in enzyme activation. The high density of mutations within the first half of the C-terminal region suggests that this part is involved in the interaction between the inhibitory C-terminal region and the rest of the enzyme.

Smoking-induced alterations in platelet membrane fluidity and Na(+)/K(+)-ATPase activity in chronic cigarette smokers.

PubMed

Padmavathi, Pannuru; Reddy, Vaddi Damodara; Maturu, Paramahamsa; Varadacharyulu, Nallanchakravarthula

2010-06-30

Cigarette smoking is a recognized risk factor for cardiovascular diseases and has been implicated in the pathogenesis of atherosclerosis. Platelet adhesiveness and aggregation increases as a result of smoking. Cigarette smoking modifies haemostatic parameters via thrombosis with a consequently higher rate of cardiovascular events, but smoking-induced alterations of platelet membrane fluidity and other changes have not been studied. Thirty experimental and control subjects (mean age 35+/-8) were selected for the study. Experimental subjects had smoked 10+/-2 cigarettes per day for 7-10 years. The plasma lipid profile, platelet carbonyls, sulfhydryl groups, Na(+)/k(+)-ATPase activity, fluidity using a fluorescent probe 1,6-diphenyl-1,3,5-hexatriene (DPH), total cholesterol and phospholipids as well individual phospholipids were determined. Increases in the platelet membrane cholesterol phospholipid (C/P) ratio, phosphotidylethanolamine, phosphotidylserine with decreased phosphotidylcholine, Na(+)/k(+)-ATPase activity, fluidity and no significant change in phosphotidylinositol and sphingomylein, as well as increases in plasma total cholesterol, LDL-cholesterol, protein carbonyls with decreased HDL-cholesterol and sulfhydryl groups were observed in cigarette smokers. Platelet membrane total phospholipids were positively correlated with plasma LDL-cholesterol (r=0.568) and VLDL-cholesterol (r=0.614) in cigarette smokers. Increased plasma LDL-cholesterol, VLDL-cholesterol and total cholesterol might have resulted in the increased C/P ratio and decreased platelet membrane fluidity of cigarette smokers.

Glucose starvation increases V-ATPase assembly and activity in mammalian cells through AMP kinase and phosphatidylinositide 3-kinase/Akt signaling.

PubMed

McGuire, Christina M; Forgac, Michael

2018-06-08

The vacuolar H + -ATPase (V-ATPase) is an ATP-driven proton pump involved in many cellular processes. An important mechanism by which V-ATPase activity is controlled is the reversible assembly of its two domains, namely the peripheral V 1 domain and the integral V 0 domain. Although reversible assembly is conserved across all eukaryotic organisms, the signaling pathways controlling it have not been fully characterized. Here, we identify glucose starvation as a novel regulator of V-ATPase assembly in mammalian cells. During acute glucose starvation, the V-ATPase undergoes a rapid and reversible increase in assembly and activity as measured by lysosomal acidification. Because the V-ATPase has recently been implicated in the activation of AMP kinase (AMPK), a critical cellular energy sensor that is also activated upon glucose starvation, we compared the time course of AMPK activation and V-ATPase assembly upon glucose starvation. We observe that AMPK activation precedes increased V-ATPase activity. Moreover, the starvation-induced increase in V-ATPase activity and assembly are prevented by the AMPK inhibitor dorsomorphin. These results suggest that increased assembly and activity of the V-ATPase upon glucose starvation are dependent upon AMPK. We also find that the PI3K/Akt pathway, which has previously been implicated in controlling V-ATPase assembly in mammalian cells, also plays a role in the starvation-induced increase in V-ATPase assembly and activity. These studies thus identify a novel stimulus of V-ATPase assembly and a novel signaling pathway involved in regulating this process. The possible function of starvation-induced increase in lysosomal V-ATPase activity is discussed. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

Iron overload impact on P-ATPases.

PubMed

Sousa, Leilismara; Pessoa, Marco Tulio C; Costa, Tamara G F; Cortes, Vanessa F; Santos, Herica L; Barbosa, Leandro Augusto

2018-03-01

Iron is a chemical element that is active in the fundamental physiological processes for human life, but its burden can be toxic to the body, mainly because of the stimulation of membrane lipid peroxidation. For this reason, the action of iron on many ATPases has been studied, especially on P-ATPases, such as the Na + ,K + -ATPase and the Ca 2+ -ATPase. On the Fe 2+ -ATPase activity, the free iron acts as an activator, decreasing the intracellular Fe 2+ and playing a protection role for the cell. On the Ca 2+ -ATPase activity, the iron overload decreases the enzyme activity, raising the cytoplasmic Ca 2+ and decreasing the sarco/endoplasmic reticulum and the Golgi apparatus Ca 2+ concentrations, which could promote an enzyme oxidation, nitration, and fragmentation. However, the iron overload effect on the Na + ,K + -ATPase may change according to the tissue expressions. On the renal cells, as well as on the brain and the heart, iron promotes an enzyme inactivation, whereas its effect on the erythrocytes seems to be the opposite, directly stimulating the ATPase activity, or stimulating it by signaling pathways involving ROS and PKC. Modulations in the ATPase activity may impair the ionic transportation, which is essential for cell viability maintenance, inducing irreversible damage to the cell homeostasis. Here, we will discuss about the iron overload effect on the P-ATPases, such as the Na + ,K + -ATPase, the Ca 2+ -ATPase, and the Fe 2+ -ATPase.

ACYL group composition of lipids and the activities of (Na+ + K+)-ATPase, 5'-nucleotidase and gamma-glutamyltranspeptidase in salivary glands and kidneys of rats fed diets containing different dietary fats.

PubMed

Alam, S Q; Alam, B S

1983-07-05

Two nutritional models, an essential fatty acid deficiency model and the feeding of saturated versus unsaturated fats, were used in a feeding study in order to assess the relationship between tissue fatty acid composition and the activities of some membrane-associated enzymes. Purified diets containing 7% hydrogenated coconut oil, 7% corn oil, 10% safflower oil or butter were fed to rats for a total of 49 weeks (1 week of pregnancy, 3 weeks of lactation and 45 weeks post-weaning). Tissue homogenates from submandibular salivary glands and kidneys were analyzed for fatty acid composition of total lipids and phospholipids. Changes in fatty acid patterns typical of essential fatty acid deficiency such as an increase in the levels of 16:1 and 18:1, a decrease in 18:2 and 20:4 and an accumulation of 20:3 omega 9 were observed in salivary glands and kidneys of rats fed the deficient diet. Tissues of rats fed 10% butter also showed fatty acid compositional changes which were somewhat similar to those in essential fatty acid deficiency, but to a lesser degree. The activities of ouabain-sensitive (Na+ + K+)-ATPase were higher in homogenates of salivary glands and kidneys of the deficient rats and those fed butter as compared with their controls. The results suggest a relationship between the double bond index of fatty acids as an indication of membrane lipid fluidity and allosteric modification of (Na+ + K+)-ATPase activity. However, other explanations for the observed changes in (Na+ + K+)-ATPase activity cannot be ruled out. There were no diet-related differences in the activities of gamma-glutamyltranspeptidase or 5'-nucleotidase.

Embryogenic competence acquisition in sugarcane callus is associated with differential H+ pump abundance and activity.

PubMed

Passamani, Lucas Z; Bertolazi, Amanda A; Ramos, Alessandro C; Santa-Catarina, Claudete; Thelen, Jay J; Silveira, Vanildo

2018-06-22

Somatic embryogenesis is an important biological process in several plant species, including sugarcane. Proteomics approaches have shown that H + pumps are differentially regulated during somatic embryogenesis; however, the relationship between H + flux and embryogenic competence is still unclear. This work aimed to elucidate the association between extracellular H + flux and somatic embryo maturation in sugarcane. We performed a microsomal proteomics analysis and analyzed changes in extracellular H + flux and H + pump (P-H + -ATPase, V-H + -ATPase and H + -PPase) activity in embryogenic and non-embryogenic callus. A total of 657 proteins were identified, 16 of which were H + pumps. We observed that P-H + -ATPase and H + -PPase were more abundant in embryogenic callus. Compared with non-embryogenic callus, embryogenic callus showed higher H + influx, especially on maturation day 14, as well as higher H+ pump activity (mainly P-H+-ATPase and H+-PPase activity). H+-PPase appears to be the major H + pump in embryogenic callus during somatic embryo formation, functioning in both vacuole acidification and PPi homeostasis. These results provide evidence for an association between higher H + pump protein abundance and, consequently, higher H + flux and embryogenic competence acquisition in the callus of sugarcane, allowing for optimization of the somatic embryo conversion process by modulating the activities of these H + pumps.

Lobatamide C: total synthesis, stereochemical assignment, preparation of simplified analogues, and V-ATPase inhibition studies.

PubMed

Shen, Ruichao; Lin, Cheng Ting; Bowman, Emma Jean; Bowman, Barry J; Porco, John A

2003-07-02

The total synthesis and stereochemical assignment of the potent antitumor macrolide lobatamide C, as well as synthesis of simplified lobatamide analogues, is reported. Cu(I)-mediated enamide formation methodology has been developed to prepare the highly unsaturated enamide side chain of the natural product and analogues. A key fragment coupling employs base-mediated esterification of a beta-hydroxy acid and a salicylate cyanomethyl ester. Three additional stereoisomers of lobatamide C have been prepared using related synthetic routes. The stereochemistry at C8, C11, and C15 of lobatamide C was assigned by comparison of stereoisomers and X-ray analysis of a crystalline derivative. Synthetic lobatamide C, stereoisomers, and simplified analogues have been evaluated for inhibition of bovine chromaffin granule membrane V-ATPase. The salicylate phenol, enamide NH, and ortho-substitution of the salicylate ester have been shown to be important for V-ATPase inhibitory activity.

Comparison of the lipid composition of oat root and coleoptile plasma membranes: lack of short-term change in response to auxin

NASA Technical Reports Server (NTRS)

Sandstrom, R. P.; Cleland, R. E.

1989-01-01

The total lipid composition of plasma membranes (PM), isolated by the phase partitioning method from two different oat (Avena sativa L.) tissues, the root and coleoptile, was compared. In general, the PM lipid composition was not conserved between these two organs of the oat seedling. Oat roots contained 50 mole percent phospholipid, 25 mole percent glycolipid, and 25 mole percent free sterol, whereas comparable amounts in the coleoptile were 42, 39, and 19 mole percent, respectively. Individual lipid components within each lipid class also showed large variations between the two tissues. Maximum specific ATPase activity in the root PM was more than double the activity in the coleoptile. Treatment of coleoptile with auxin for 1 hour resulted in no detectable changes in PM lipids or extractable ATPase activity. Differences in the PM lipid composition between the two tissues that may define the limits of ATPase activity are discussed.

Rapamycin mitigates erythrocyte membrane transport functions and oxidative stress during aging in rats.

PubMed

Singh, Abhishek Kumar; Singh, Sandeep; Garg, Geetika; Rizvi, Syed Ibrahim

2018-02-01

Erythrocyte membrane is a suitable model to study various metabolic and physiological functions as it undergoes variety of biochemical changes during aging. An age-dependent modulatory effect of rapamycin on erythrocyte membrane functions is completely unknown. Therefore, the present study was undertaken to investigate the effect of rapamycin on age-dependent impaired activities of transporters/exchangers, altered levels of redox biomarkers, viz. protein carbonyl (PC), lipid hydroperoxides (LHs), total thiol (-SH), sialic acid (SA) and intracellular calcium ion [Ca 2+ ]i, and osmotic fragility of erythrocyte membrane. A significant reduction in membrane-bound activities of Na + /K + -ATPase (NKA) and Ca 2+ -ATPase (PMCA), and levels of -SH and SA was observed along with a simultaneous induction in Na + /H + exchanger (NHE) activity and levels of [Ca 2+ ]i, PC, LH and osmotic fragility in old-aged rats. Rapamycin was found to be a promising age-delaying drug that significantly reversed the aging-induced impaired activities of membrane-bound ATPases and altered levels of redox biomarkers.

Impact of long-term moderate hypercapnia and elevated temperature on the energy budget of isolated gills of Atlantic cod (Gadus morhua).

PubMed

Kreiss, Cornelia M; Michael, Katharina; Bock, Christian; Lucassen, Magnus; Pörtner, Hans-O

2015-04-01

Effects of severe hypercapnia have been extensively studied in marine fishes, while knowledge on the impacts of moderately elevated CO2 levels and their combination with warming is scarce. Here we investigate ion regulation mechanisms and energy budget in gills from Atlantic cod acclimated long-term to elevated PCO2 levels (2500 μatm) and temperature (18°C). Isolated perfused gill preparations were established to determine gill thermal plasticity during acute exposures (10-22°C) and in vivo costs of Na(+)/K(+)-ATPase activity, protein and RNA synthesis. Maximum enzyme capacities of F1Fo-ATPase, H(+)-ATPase and Na(+)/K(+)-ATPase were measured in vitro in crude gill homogenates. After whole animal acclimation to elevated PCO2 and/or warming, branchial oxygen consumption responded more strongly to acute temperature change. The fractions of gill respiration allocated to protein and RNA synthesis remained unchanged. In gills of fish CO2-exposed at both temperatures, energy turnover associated with Na(+)/K(+)-ATPase activity was reduced by 30% below rates of control fish. This contrasted in vitro capacities of Na(+)/K(+)-ATPase, which remained unchanged under elevated CO2 at 10°C, and earlier studies which had found a strong upregulation under severe hypercapnia. F1Fo-ATPase capacities increased in hypercapnic gills at both temperatures, whereas Na(+)/K(+)ATPase and H(+)-ATPase capacities only increased in response to elevated CO2 and warming indicating the absence of thermal compensation under CO2. We conclude that in vivo ion regulatory energy demand is lowered under moderately elevated CO2 levels despite the stronger thermal response of total gill respiration and the upregulation of F1Fo-ATPase. This effect is maintained at elevated temperature. Copyright © 2014 Elsevier Inc. All rights reserved.

Attenuation of erythrocyte membrane oxidative stress by Sesbania grandiflora in streptozotocin-induced diabetic rats.

PubMed

Sureka, Chandrabose; Ramesh, Thiyagarajan; Begum, Vavamohaideen Hazeena

2015-08-01

The aim of the present study was to investigate the protective effects of Sesbania grandiflora flower (SGF) extract on erythrocyte membrane in Streptozotocin (STZ)-induced diabetic rats. Adult male albino rats of Wistar strain, weighing 190-220 g, were made diabetic by an intraperitonial administration of STZ (45 mg/kg). Normal and diabetic rats were treated with SGF, and diabetic rats were also treated with glibenclamide as drug control, for 45 days. In this study plasma insulin and haemoglobin levels were decreased and blood glucose, glycosylated haemoglobin, protein oxidation, lipid peroxidation markers, and osmotic fragility levels were increased in diabetic rats. Moreover, erythrocytes antioxidant enzymes such as superoxide dismutase, catalase, glutathione peroxide, glutathione reductase, glutathione-S-transferase, and glucose-6-phosphate dehydrogenase activities and non-enzymatic antioxidants such as vitamin C, vitamin E, reduced glutathione (GSH), and oxidized glutathione (GSSG) levels were altered. Similarly, the activities of total ATPases, Na(+)/K(+)-ATPase, Ca(2+)-ATPase, and Mg(2+)-ATPase were also decreased in the erythrocytes of diabetic rats. Administration of SGF to STZ-induced diabetic rats reduced blood glucose and glycosylated haemoglobin levels with increased levels of insulin and haemoglobin. Moreover, SGF reversed the protein and lipid peroxidation markers, osmotic fragility, membrane-bound ATPases activities, and antioxidant status in STZ-induced diabetic rats. These results suggest that SGF could provide a protective effect on diabetes by decreasing oxidative stress-associated diabetic complications.

Loss of Vacuolar H+-ATPase (V-ATPase) Activity in Yeast Generates an Iron Deprivation Signal That Is Moderated by Induction of the Peroxiredoxin TSA2 *

PubMed Central

Diab, Heba I.; Kane, Patricia M.

2013-01-01

Vacuolar H+-ATPases (V-ATPases) acidify intracellular organelles and help to regulate overall cellular pH. Yeast vma mutants lack V-ATPase activity and allow exploration of connections between cellular pH, iron, and redox homeostasis common to all eukaryotes. A previous microarray study in a vma mutant demonstrated up-regulation of multiple iron uptake genes under control of Aft1p (the iron regulon) and only one antioxidant gene, the peroxiredoxin TSA2 (Milgrom, E., Diab, H., Middleton, F., and Kane, P. M. (2007) Loss of vacuolar proton-translocating ATPase activity in yeast results in chronic oxidative stress. J. Biol. Chem. 282, 7125–7136). Fluorescent biosensors placing GFP under transcriptional control of either an Aft1-dependent promoter (PFIT2-GFP) or the TSA2 promoter (PTSA2-GFP) were constructed to monitor transcriptional signaling. Both biosensors were up-regulated in the vma2Δ mutant, and acute V-ATPase inhibition with concanamycin A induced coordinate up-regulation from both promoters. PTSA2-GFP induction was Yap1p-dependent, indicating an oxidative stress signal. Total cell iron measurements indicate that the vma2Δ mutant is iron-replete, despite up-regulation of the iron regulon. Acetic acid up-regulated PFIT2-GFP expression in wild-type cells, suggesting that loss of pH control contributes to an iron deficiency signal in the mutant. Iron supplementation significantly decreased PFIT2-GFP expression and, surprisingly, restored PTSA2-GFP to wild-type levels. A tsa2Δ mutation induced both nuclear localization of Aft1p and PFIT2-GFP expression. The data suggest a novel function for Tsa2p as a negative regulator of Aft1p-driven transcription, which is induced in V-ATPase mutants to limit transcription of the iron regulon. This represents a new mechanism bridging the antioxidant and iron-regulatory pathways that is intimately linked to pH homeostasis. PMID:23457300

[Effect of Cu2+ and Zn2+ ions in Ca-ATPase activity isolated from Pachymerus nucleorum (Fabricius) (Coleoptera: Chrysomelidae, Bruchinae) larvae].

PubMed

Dias, Decivaldo S; Coelho, Milton V

2007-01-01

ATPases, an important target of insecticides, are enzymes that hydrolyze ATP and use the energy released in that process to accomplish some type of cellular work. Pachymerus nucleorum (Fabricius) larvae possess an ATPase, that presents high Ca-ATPase activity, but no Mg-ATPase activity. In the present study, the effect of zinc and copper ions in the activity Ca-ATPase of that enzyme was tested. More than 90% of the Ca-ATPase activity was inhibited in 0.5 mM of copper ions or 0.25 mM of zinc ions. In the presence of EDTA, but not in the absence, the inhibition by zinc was reverted with the increase of calcium concentration. The inhibition by copper ions was not reverted in the presence or absence of EDTA. The Ca-ATPase was not inhibited by treatment of the ATPase fraction with copper, suggesting that the copper ion does not bind directly to the enzyme. The results suggest that zinc and copper ions form a complex with ATP and bind to the enzyme inhibiting its Ca-ATPase activity.

Apyrase activity and adenosine diphosphate induced platelet aggregation inhibition by the salivary gland proteins of Culicoides variipennis, the North American vector of bluetongue viruses.

PubMed

Pérez de León, A A; Tabachnick, W J

1996-02-01

Salivary gland homogenates of Culicoides variipennis, the primary vector of bluetongue (BLU) viruses in North America, were analyzed for apyrase activity. Apyrase (ATP diphosphohydrolase, EC 3.6.1.5) is an anti-hemostatic and anti-inflammatory salivary enzyme of most hematophagous arthropods. The enzyme activity was measured by the release of orthophosphate using ATP, ADP, and AMP as substrates with Ca2+ as the divalent cation. ATPase (11.5 +/- 1 mU/pair of glands), ADPase (7.3 +/- 0.7 mU/pair of glands), and insignificant (P < 0.05) AMPase (0.07 mU/pair of glands) activities were detected in female salivary glands. Male salivary glands contained lower amounts of ATPase and ADPase activity (P < 0.05). The ATPase and ADPase activities were greatest at pH 8.5, and were similarly activated by Mg2+. Molecular sieving HPLC of salivary gland homogenates generated a single peak which coincided with ATPase and ADPase, but no AMPase, activity; the protein has an estimated molecular mass of 35,000 Da. ATPase and ADPase activity, and total protein concentration, were reduced (P < 0.05) in the salivary glands of females after taking a blood meal from a sheep. Salivary gland homogenates also inhibited ADP-induced platelet aggregation in vitro. It is concluded that the salivary ATPase and ADPase activities of C. variipennis reside in one enzyme, and that this enzyme is likely an apyrase. The apyrase activity is thought to be responsible for the inhibition of ADP-induced platelet aggregation, as indicated by the apparent discharge of apyrase from salivary glands into the host during blood feeding. This suggests that apyrase is one of the salivary proteins present in C. variipennis acting as antigens in the development of Culicoides hypersensitivity in ruminants and horses. Apyrase may inhibit an inflammatory response at the feeding site through the subsequent degradation of its end-product, AMP, to adenosine, a potent anti-inflammatory substance, by the ecto-5' nucleotidase activity of neutrophils.

Structural relatedness of three ion-transport adenosine triphosphatases around their active sites of phosphorylation.

PubMed

Walderhaug, M O; Post, R L; Saccomani, G; Leonard, R T; Briskin, D P

1985-03-25

Three membrane-bound adenosine triphosphatases were investigated for homology in the sequence of four amino acids about the active site of phosphorylation. The ATPases were as follows: sodium-potassium-dependent ATPase from dog kidney, Na,K-ATPase; hydrogen-potassium-dependent ATPase from hog gastric mucosa, H,K-ATPase, an ATPase similar to Na,K-ATPase; and an ATPase activity in the plasma membrane of corn, Zea mays, roots (CR-ATPase), a higher plant ATPase. A membrane preparation containing an ATPase of Acholeplasma laidlawii, a prokaryote, (AL) was also investigated. For most of the experiments, the preparations were phosphorylated from [gamma-32P]ATP, denatured in acid, and subjected to proteolytic digestion. Radioactive phosphopeptides were separated by high voltage paper electrophoresis and characterized by sensitivity to chemical reagents. In gastric H,K-ATPase, the aspartate residue at the active site was determined directly by labeling with [3H]borohydride. A common sequence around the active site was found for Na,K-ATPase, H,K-ATPase, and CR-ATPase. This sequence, -Cys-(Ser/Thr)-Asp(P)-Lys-, is similar to that in the calcium ion-transport ATPase of sarcoplasmic reticulum. The AL membrane preparation showed an acylphosphate that turned over rapidly after a chase of labeled membranes with unlabeled ATP. The corresponding sequence was different from that of the three ATPases. An acylphosphate was on two polypeptides with molecular weights of about 80,000 and 60,000; these appear not to correspond to subunits of a Na+-stimulated ATPase in this organism (Lewis, R. N. A. H., and McElhaney, R. N. (1983) Biochim. Biophys. Acta 735, 113-122).

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jung, D.W.; Laties, G.G.

Potato mitochondria (Solanum tuberosum var. Russet Burbank), which readily phosphorylate ADP in oxidative phosphorylation, show low levels of ATPase activity which is stimulated neither by Mg/sup 2 +/, 2,4-dinitrophenol, incubation with respiratory substrates, nor disruption by sonication or treatment with Triton X-100, individually or in concert. Treatment of disrupted potato mitochondria with trypsin stimulates Mg/sup 2 +/-dependent, oligomycin-sensitive ATPase activity 10- to 15-fold, suggesting the presence of an ATPase inhibitor protein. Trypsin-induced ATPase activity was unaffected by uncoupler. Oligomycin-sensitive ATPase activity decreases as exposure to trypsin is increased. Incubation at alkaline pH or heating at 60/sup 0/C for 2 minutesmore » also activates ATPase of sonicated potato mitochondria. Disruption of cauliflower (Brassica oleracea), red sweet potato (Ipomoea batatas), and carrot (Daucus carota) mitochondria increases ATPase activity, which is further enhanced by treatment with trypsin. The significance of the tight association of the inhibitor protein and ATPase in potato mitochondria is not clear.« less

Overproduction, purification, and ATPase activity of the Escherichia coli RuvB protein involved in DNA repair.

PubMed Central

Iwasaki, H; Shiba, T; Makino, K; Nakata, A; Shinagawa, H

1989-01-01

The ruvA and ruvB genes of Escherichia coli constitute an operon which belongs to the SOS regulon. Genetic evidence suggests that the products of the ruv operon are involved in DNA repair and recombination. To begin biochemical characterization of these proteins, we developed a plasmid system that overproduced RuvB protein to 20% of total cell protein. Starting from the overproducing system, we purified RuvB protein. The purified RuvB protein behaved like a monomer in gel filtration chromatography and had an apparent relative molecular mass of 38 kilodaltons in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, which agrees with the value predicted from the DNA sequence. The amino acid sequence of the amino-terminal region of the purified protein was analyzed, and the sequence agreed with the one deduced from the DNA sequence. Since the deduced sequence of RuvB protein contained the consensus sequence for ATP-binding proteins, we examined the ATP-binding and ATPase activities of the purified RuvB protein. RuvB protein had a stronger affinity to ADP than to ATP and weak ATPase activity. The results suggest that the weak ATPase activity of RuvB protein is at least partly due to end product inhibition by ADP. Images PMID:2529252

BIOCHEMICAL AND ULTRASTRUCTURAL PROPERTIES OF A MITOCHONDRIAL INNER MEMBRANE FRACTION DEFICIENT IN OUTER MEMBRANE AND MATRIX ACTIVITIES

PubMed Central

Chan, T. L.; Greenawalt, John W.; Pedersen, Peter L.

1970-01-01

Treatment of the inner membrane matrix fraction of rat liver mitochondria with the nonionic detergent Lubrol WX solubilized about 70% of the total protein and 90% or more of the following matrix activities: malate dehydrogenase, glutamate dehydrogenase, and isocitrate dehydrogenase (NADP). The Lubrol-insoluble fraction was enriched in cytochromes, phospholipids, and a Mg++-stimulated ATPase activity. Less than 2% of the total mitochondrial activity of monoamine oxidase, an outer membrane marker, or adenylate kinase, an intracristal space marker could be detected in this inner membrane fraction. Electron micrographs of negatively stained preparations showed vesicles (≤0.4 µ diameter) literally saturated on the periphery with the 90 A ATPase particles. These inner membrane vesicles, which appeared for the most part to be inverted with respect to the normal inner membrane configuration in intact mitochondria, retained the succinicoxidase portion of the electron-transport chain, an intact phosphorylation site II with a high affinity for ADP, and the capacity to accumulate Ca++. A number of biochemical properties characteristic of intact mitochondria and the inner membrane matrix fraction, however, were either absent or markedly deficient in the inner membrane vesicles. These included stimulation of respiration by either ADP or 2,4-dinitrophenol, oligomycin-sensitive ADP-ATP exchange activity, atractyloside sensitivity of adenine nucleotide requiring reactions, and a stimulation of the Mg++-ATPase by 2,4-dinitrophenol. PMID:4254678

[Effect of baicalin on ATPase and LDH and its regulatory effect on the AC/cAMP/PKA signaling pathway in rats with attention deficit hyperactivity disorder].

PubMed

Zhou, Rong-Yi; Wang, Jiao-Jiao; You, Yue; Sun, Ji-Chao; Song, Yu-Chen; Yuan, Hai-Xia; Han, Xin-Min

2017-05-01

To study the effect of baicalin on synaptosomal adenosine triphosphatase (ATPase) and lactate dehydrogenase (LDH) and its regulatory effect on the adenylate cyclase (AC)/cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) signaling pathway in rats with attention deficit hyperactivity disorder (ADHD). A total of 40 SHR rats were randomly divided into five groups: ADHD model, methylphenidate hydrochloride treatment (0.07 mg/mL), and low-dose (3.33 mg/mL), medium-dose (6.67 mg/mL), and high-dose (10 mg/mL) baicalin treatment (n=8 each). Eight WKY rats were selected as normal control group. Percoll density gradient centrifugation was used to prepare brain synaptosomes and an electron microscope was used to observe their structure. Colorimetry was used to measure the activities of ATPase and LDH in synaptosomes. ELISA was used to measure the content of AC, cAMP, and PKA. Compared with the normal control group, the ADHD model group had a significant reduction in the ATPase activity, a significant increase in the LDH activity, and significant reductions in the content of AC, cAMP, and PKA (P<0.05). Compared with the ADHD model group, the methylphenidate hydrochloride group and the medium- and high-dose baicalin groups had a significant increase in the ATPase activity (P<0.05), a significant reduction in the LDH activity (P<0.05), and significant increases in the content of AC, cAMP, and PKA (P<0.05). Compared with the methylphenidate hydrochloride group, the high-dose baicalin group had significantly greater changes in these indices (P<0.05). Compared with the low-dose baicalin group, the high-dose baicalin group had a significant increase in the ATPase activity (P<0.05); the medium- and high-dose baicalin groups had a significant reduction in the LDH activity (P<0.05) and significant increases in the content of AC, cAMP, and PKA (P<0.05). Compared with the medium-dose baicalin group, the high-dose baicalin group had a significant increase in the ATPase activity (P<0.05). Both methylphenidate hydrochloride and baicalin can improve synaptosomal ATPase and LDH activities in rats with ADHD. The effect of baicalin is dose-dependent, and high-dose baicalin has a significantly greater effect than methylphenidate hydrochloride. Baicalin exerts its therapeutic effect possibly by upregulating the AC/cAMP/PKA signaling pathway.

Tributyltin sensitivity of vacuolar-type Na(+)-transporting ATPase from Enterococcus hirae.

PubMed

Chardwiriyapreecha, Soracom; Inoue, Tomohiro; Sugimoto, Naoko; Sekito, Takayuki; Yamato, Ichiro; Murata, Takeshi; Homma, Michio; Kakinuma, Yoshimi

2009-10-01

Tributyltin chloride (TBT), an environmental pollutant, is toxic to a variety of eukaryotic and prokaryotic organisms. Some members of F-ATP synthase (F-ATPase)/vacuolar type ATPase (V-ATPase) superfamily have been identified as the molecular target of this compound. TBT inhibited the activities of H(+)-transporting or Na(+)-transporting F-ATPase as well as H(+)-transporting V-ATPase originated from various organisms. However, the sensitivity to TBT of Na(+)-transporting V-ATPase has not been investigated. We examined the effect of TBT on Na(+)-transporting V-ATPase from an eubacterium Enterococus hirae. The ATP hydrolytic activity of E. hirae V-ATPase in purified form as well as in membrane-bound form was little inhibited by less than 10 microM TBT; IC50 for TBT inhibition of purified enzyme was estimated to be about 35 microM. Active sodium transport by E. hirae cells, indicating the in vivo activity of this V-ATPase, was not inhibited by 20 microM TBT. By contrast, IC50 of H(+)-transporting V-ATPase of the vacuolar membrane vesicles from Saccharomyces cerevisiae was about 0.2 microM. E. hirae V-ATPase is thus extremely less sensitive to TBT.

Biochemical Changes in Erythrocytes as a Molecular Marker of Cell Damage during Long-Term Simvastatin Treatment.

PubMed

Mikashinovich, Z I; Belousova, E S

2016-08-01

Long-term administration of simvastatin to rats, irrespective of the baseline cholesterol levels, induced biochemical changes in erythrocytes attesting to hypoxic damage (accumulation of lactate and 2,3-diphosphoglycerate), disturbances in ATP-dependent mechanisms of ion homeostasis regulation (decrease in total ATPase and Ca(2+)-ATPase activities), and antioxidant enzymes system imbalance. These changes can be considered as a sensitive indicator and molecular basis of cell damage during long-term administration of statins.

A more accurate profile of Achyrocline satureioides hypocholesterolemic activity.

PubMed

Espiña, Débora Corrêa; Carvalho, Fabiano Barbosa; Zanini, Daniela; Schlemmer, Josiane Bizzi; Coracini, Juliane Dors; Rubin, Maribel Antonello; Morsch, Vera Maria; Schetinger, Maria Rosa Chitolina; Leal, Daniela Bitencourt Rosa; Baiotto, Cléia Rosani; Jaques, Jeandre Augusto dos Santos

2012-06-01

The aim of this study was to investigate the effect of the aqueous extract (AE) of Achyrocline satureioides on serum lipid profile, liver oxidative profile and Na(+),K(+)-ATPase activity of rats submitted to a hyperlipidic diet. The animals were divided into four groups: control (C), AE 10% (A(10)), hyperlipidic (H) and hyperlipidic/AE 10% (HA(10)). In serum, we measured the levels of total cholesterol (TC), high-density lipoprotein, very-low-density lipoprotein, low-density lipoprotein (LDL) and triglyceride (TG). In liver homogenates, we measured the thiobarbituric acid reactive substances, the carbonyl proteins, the non-protein thiols (NPSHs) and the activity of superoxide dismutase, catalase (CAT) and Na(+),K(+)-ATPase. We observed a significant increase in the TC and LDL levels in the H group. A. satureioides prevented these effects, decreased the TG levels in the HA(10) group and increased the NPSH levels in the A(10) and HA(10) groups. The H group showed an increase in the carbonyl protein level and a decrease in CAT and Na(+),K(+)-ATPase activities. With the use of this model, results show that increased levels of lipids are related to a redox imbalance in the liver, which is also related to the inhibition of Na(+),K(+)-ATPase activity, and that chronic administration of the AE of A. satureioides is capable of changing this profile. Copyright © 2012 John Wiley & Sons, Ltd.

On archaebacterial ATPase from Halobacterium saccharovorum

NASA Technical Reports Server (NTRS)

Kristjansson, H.; Ponnamperuma, C.; Hochstein, L.; Altekar, W.

1984-01-01

The energy transducing ATPase from Halobacterium saccharovorum was studied in order to define the origin of energy transducing systems. The ATPase required high salt concentration (4M NaCl) for activity; activity was rapidly lost when NaCl was below 1 Molar. At low salt concentration, the membrane bound ATPase activity could be stabilized in presence of spermine. However, following solubilization spermine was ineffective. Furthermore, F1 ATPase activity was stabilized by ammonium sulfate even when the NaCl concentration was less than 1 Molar. These studies suggest that stabilization by hydrophobic interactions preceded ionic ones in the evolution of the energy transducing ATPases.

Hyperthyroidism increases the uncoupled ATPase activity and heat production by the sarcoplasmic reticulum Ca2+-ATPase.

PubMed

Arruda, Ana Paula; Da-Silva, Wagner S; Carvalho, Denise P; De Meis, Leopoldo

2003-11-01

The sarcoplasmic reticulum Ca2+-ATPase is able to modulate the distribution of energy released during ATP hydrolysis, so that a portion of energy is used for Ca2+ transport (coupled ATPase activity) and a portion is converted into heat (uncoupled ATPase activity). In this report it is shown that T4 administration to rabbits promotes an increase in the rates of both the uncoupled ATPase activity and heat production in sarcoplasmic reticulum vesicles, and that the degree of activation varies depending on the muscle type used. In white muscles hyperthyroidism promotes a 0.8-fold increase of the uncoupled ATPase activity and in red muscle a 4-fold increase. The yield of vesicles from hyperthyroid muscles is 3-4-fold larger than that obtained from normal muscles; thus the rate of heat production by the Ca2+-ATPase expressed in terms of g of muscle in hyperthyroidism is increased by a factor of 3.6 in white muscles and 12.0 in red muscles. The data presented suggest that the Ca2+-ATPase uncoupled activity may represent one of the heat sources that contributes to the enhanced thermogenesis noted in hyperthyroidism.

Abscisic acid induction of vacuolar H+-ATPase activity in mesembryanthemum crystallinum is developmentally regulated

PubMed

Barkla; Vera-Estrella; Maldonado-Gama; Pantoja

1999-07-01

Abscisic acid (ABA) has been implicated as a key component in water-deficit-induced responses, including those triggered by drought, NaCl, and low- temperature stress. In this study a role for ABA in mediating the NaCl-stress-induced increases in tonoplast H+-translocating ATPase (V-ATPase) and Na+/H+ antiport activity in Mesembryanthemum crystallinum, leading to vacuolar Na+ sequestration, were investigated. NaCl or ABA treatment of adult M. crystallinum plants induced V-ATPase H+ transport activity, and when applied in combination, an additive effect on V-ATPase stimulation was observed. In contrast, treatment of juvenile plants with ABA did not induce V-ATPase activity, whereas NaCl treatment resulted in a similar response to that observed in adult plants. Na+/H+ antiport activity was induced in both juvenile and adult plants by NaCl, but ABA had no effect at either developmental stage. Results indicate that ABA-induced changes in V-ATPase activity are dependent on the plant reaching its adult phase, whereas NaCl-induced increases in V-ATPase and Na+/H+ antiport activity are independent of plant age. This suggests that ABA-induced V-ATPase activity may be linked to the stress-induced, developmentally programmed switch from C3 metabolism to Crassulacean acid metabolism in adult plants, whereas, vacuolar Na+ sequestration, mediated by the V-ATPase and Na+/H+ antiport, is regulated through ABA-independent pathways.

Na/K-ATPase regulates bovine sperm capacitation through raft- and non-raft-mediated signaling mechanisms.

PubMed

Rajamanickam, Gayathri D; Kastelic, John P; Thundathil, Jacob C

2017-11-01

Highly dynamic lipid microdomains (rafts) in the sperm plasma membrane contain several signaling proteins that regulate sperm capacitation. Na/K-ATPase isoforms (testis-specific isoform ATP1A4 and ubiquitous isoform ATP1A1) are abundant in bovine sperm plasma membrane. We previously reported that incubation of bovine sperm with ouabain, a specific Na/K-ATPase ligand, induced tyrosine phosphorylation of several sperm proteins during capacitation. The objective of this study was to investigate the roles of lipid rafts and non-rafts in Na/K-ATPase enzyme activity and signaling during bovine sperm capacitation. Content of ATP1A4 and, to a lesser extent, ATP1A1 was increased in raft and non-raft fractions of capacitated sperm, although non-raft enzyme activities of both isoforms were higher than the corresponding activities in rafts from capacitated sperm. Yet, ATP1A4 was the predominant isoform responsible for total Na/K-ATPase activity in both rafts and non-rafts. A comparative increase in phosphorylation of signaling molecules was observed in both raft (CAV1) and non-raft (EGFR and ERK1/2) membrane fractions during capacitation. Although SRC was phosphorylated in both membrane fractions, the non-raft fraction possessed more of this activated form. We also inferred, by immunoprecipitation, that ATP1A4 interacted with CAV1 and EGFR in the raft fraction, whereas interactions of ATP1A4 with SRC, EGFR, and ERK1/2 occurred in the non-raft fraction of ouabain-capacitated sperm; conversely, ATP1A1 interacted only with CAV1 in both fractions of uncapacitated and capacitated sperm. In conclusion, both raft and non-raft cohorts of Na/K-ATPase isoforms contributed to phosphorylation of signaling molecules during bovine sperm capacitation. © 2017 Wiley Periodicals, Inc.

Oxidative stress and Na,K-ATPase activity differential regulation in brainstem and forebrain of Wistar Audiogenic rats may lead to increased seizure susceptibility.

PubMed

Parreira, Gabriela Machado; Resende, Maria Daniela Aparecida; Garcia, Israel José Pereira; Sartori, Daniela Bueno; Umeoka, Eduardo Henrique de Lima; Godoy, Lívea Dornela; Garcia-Cairasco, Norberto; Barbosa, Leandro Augusto; Santos, Hérica de Lima; Tilelli, Cristiane Queixa

2018-01-15

The Wistar Audiogenic Rat (WAR) is a well-characterized seizure-prone, inbred rodent strain that, when acutely stimulated with high-intensity sounds, develops brainstem-dependent tonic-clonic seizures that can evolve to limbic-like, myoclonic (forebrain) seizures when the acoustic stimuli are presented chronically (audiogenic kindling). In order to investigate possible mechanisms underlying WAR susceptibility to seizures, we evaluated Na,K-ATPase activity, Ca-ATPase activity, Mg-ATPase activity, lipid membrane composition and oxidative stress markers in whole forebrain and whole brainstem samples of naïve WAR, as compared to samples from control Wistar rats. We also evaluated the expression levels of α1 and α3 isoforms of Na,K-ATPase in forebrain samples. We observed increased Na,K-ATPase activity in forebrain samples and increased oxidative stress markers (lipid peroxidation, glutathione peroxidase and superoxide dismutase) in brainstem samples of WAR. The Ca-ATPase activity, Mg-ATPase activity, lipid membrane composition and expression levels of α1 and α3 isoforms of Na,K-ATPase were unaltered. In view of previous data showing that the membrane potentials from naïve WAR's neurons are less negative than that from neurons from Wistar rats, we suggest that Na,K-ATPase increased activity might be involved in a compensatory mechanism necessary to maintain WAR's brains normal activity. Additionally, ongoing oxidative stress in the brainstem could bring Na,K-ATPase activity back to normal levels, which may explain why WAR's present increased susceptibility to seizures triggered by high-intensity sound stimulation. Copyright © 2017 Elsevier B.V. All rights reserved.

The Arabidopsis Chaperone J3 Regulates the Plasma Membrane H+-ATPase through Interaction with the PKS5 Kinase[C][W

PubMed Central

Yang, Yongqing; Qin, Yunxia; Xie, Changgen; Zhao, Feiyi; Zhao, Jinfeng; Liu, Dafa; Chen, Shouyi; Fuglsang, Anja T.; Palmgren, Michael G.; Schumaker, Karen S.; Deng, Xing Wang; Guo, Yan

2010-01-01

The plasma membrane H+-ATPase (PM H+-ATPase) plays an important role in the regulation of ion and metabolite transport and is involved in physiological processes that include cell growth, intracellular pH, and stomatal regulation. PM H+-ATPase activity is controlled by many factors, including hormones, calcium, light, and environmental stresses like increased soil salinity. We have previously shown that the Arabidopsis thaliana Salt Overly Sensitive2-Like Protein Kinase5 (PKS5) negatively regulates the PM H+-ATPase. Here, we report that a chaperone, J3 (DnaJ homolog 3; heat shock protein 40-like), activates PM H+-ATPase activity by physically interacting with and repressing PKS5 kinase activity. Plants lacking J3 are hypersensitive to salt at high external pH and exhibit decreased PM H+-ATPase activity. J3 functions upstream of PKS5 as double mutants generated using j3-1 and several pks5 mutant alleles with altered kinase activity have levels of PM H+-ATPase activity and responses to salt at alkaline pH similar to their corresponding pks5 mutant. Taken together, our results demonstrate that regulation of PM H+-ATPase activity by J3 takes place via inactivation of the PKS5 kinase. PMID:20418496

Mitochondria-rich cells adjustments and ionic balance in the Neotropical fish Prochilodus lineatus exposed to titanium dioxide nanoparticles.

PubMed

Carmo, Talita L L; Azevedo, Vinícius C; Siqueira, Priscila R; Galvão, Tiago D; Santos, Fabrício A; Martinez, Cláudia B R; Appoloni, Carlos R; Fernandes, Marisa N

2018-07-01

Manufactured titanium dioxide nanoparticles (TiO 2 -NP) have been intensely applied in numerous industrial products and may be a risk for aquatic systems as they are not completely removed from domestic and industrial wastes after water treatment. This study evaluated the osmo- and ionic balance, Na + /K + -ATPase, H + -ATPase and carbonic anhydrase activities and the mitochondria-rich cells (MRC) in the gills and kidney of the Neotropical fish Prochilodus lineatus after 2 (acute) and 14 (subchronic) days of exposure to nominal 0, 1, 5, 10 and 50 mg L -1 TiO 2 -NP. The nominal concentrations corresponded to 0.0, 0.6, 1.6, 2.7 and 18.1 mg L -1 suspended TiO 2 -NP, respectively, in the water column one hour after NP introduction and were maintained for at least 24 h. Acute exposure to TiO 2 -NP decreased plasma osmolality and Ca 2+ levels. Na + /K + -ATPase, H + -ATPase and carbonic anhydrase activities were inhibited in the gills, but not in the kidney. Total MRC density did not change in gills and kidneys. At gill surface, total MRC density decreased in fish exposed to 50 mg L -1 TiO 2 -NP and the total MRC fractional surface area unchanged although, there were some changes in the fractional area of MRC with apical microvilli (MRCm) and MRC with apical sponge-like structure (MRCs). MRCm was more abundant than MRCs. After subchronic exposure, there was no change in plasma osmolality, ionic balance and enzyme activities. Total gill MRC density increased in the filament epithelium and renal tubules. In the gills, MRC contacting water exhibited some adjustments. Total MRC and fractional surface area unchanged, but there was an increase of MRCs contacting water at gill surface after exposure to10 and 50 mg L -1 TiO 2 -NP. MRC proliferation in filament epithelium and in renal tubules as well as the increasing MRCs at gill surface may have contributed to avoid change in plasma osmolality, ionic balance and enzyme activities and suggested a cellular physiological and morphological response to restore and maintain osmotic and ionic homeostasis after subchronic exposure. Copyright © 2018 Elsevier B.V. All rights reserved.

The AMPK-v-ATPase-pH axis: A key regulator of the pro-fibrogenic phenotype of human hepatic stellate cells.

PubMed

Marrone, Giusi; De Chiara, Francesco; Böttcher, Katrin; Levi, Ana; Dhar, Dipok; Longato, Lisa; Mazza, Giuseppe; Zhang, Zhenzhen; Marrali, Martina; Iglesias, Anabel Fernández-; Hall, Andrew; Luong, Tu Vinh; Viollet, Benoit; Pinzani, Massimo; Rombouts, Krista

2018-04-17

Liver fibrosis and cirrhosis are characterized by activation of hepatic stellate cells (HSC) which is associated with higher intracellular pH (pHi). The vacuolar H + adenosine-tri-phosphatase (v-ATPase) multi-subunit complex is a key regulator of intracellular pH homeostasis. The present work was aimed at investigating the functional role of v-ATPase in primary human HSC (hHSC) activation and its modulation by specific AMPK subunits. Here, we demonstrated that the expression of different v-ATPase subunits was increased in in vivo and in vitro activated hHSC, compared to non-activated hHSC. Specific inhibition of v-ATPase with Bafilomycin and KM91104 induced a down-regulation of the HSC fibrogenic gene profile, which coincided with increased lysosomal pH, decreased pHi, activation of AMPK, reduced proliferation, and a lower metabolic activity. Similarly, pharmacological activation of AMPK by treatment with Diflunisal, A769662 and ZLN024, reduced the expression of v-ATPase subunits and pro-fibrogenic markers. V-ATPase expression was differently regulated by AMPKα1 and AMPKα2, as demonstrated in mouse embryo fibroblasts (MEF) specific deficient for AMPKα subunits. In addition, the activation of v-ATPase in hHSC was shown to be AMPKα1 dependent. Accordingly, pharmacological activation of AMPK in AMPKα1-depleted hHSC prevented v-ATPase downregulation. Finally, we showed that v-ATPase expression was increased in fibrotic livers from Bile Duct Ligated mice and in human cirrhotic livers. The down-regulation of v-ATPase might represent a new promising target for the development of anti-fibrotic strategies. This article is protected by copyright. All rights reserved. © 2018 by the American Association for the Study of Liver Diseases.

Conditions of activation of yeast plasma membrane ATPase.

PubMed

Sychrová, H; Kotyk, A

1985-04-08

The in vivo activation of the H+-ATPase of baker's yeast plasma membrane found by Serrano in 1983 was demonstrated with D-glucose aerobically and anaerobically (as well as in a respiration-deficient mutant) and, after suitable induction, with maltose, trehalose, and galactose. The activated but not the control ATPase was sensitive to oligomycin. No activation was possible in a cell-free extract with added glucose. The ATPase was not activated in yeast protoplasts which may account for the absence of glucose-stimulated secondary active transports in these wall-less cells and provide support for a microscopic coupling between ATPase activity and these transports in yeast cells.

Abscisic Acid Induction of Vacuolar H+-ATPase Activity in Mesembryanthemum crystallinum Is Developmentally Regulated1

PubMed Central

Barkla, Bronwyn J.; Vera-Estrella, Rosario; Maldonado-Gama, Minerva; Pantoja, Omar

1999-01-01

Abscisic acid (ABA) has been implicated as a key component in water-deficit-induced responses, including those triggered by drought, NaCl, and low- temperature stress. In this study a role for ABA in mediating the NaCl-stress-induced increases in tonoplast H+-translocating ATPase (V-ATPase) and Na+/H+ antiport activity in Mesembryanthemum crystallinum, leading to vacuolar Na+ sequestration, were investigated. NaCl or ABA treatment of adult M. crystallinum plants induced V-ATPase H+ transport activity, and when applied in combination, an additive effect on V-ATPase stimulation was observed. In contrast, treatment of juvenile plants with ABA did not induce V-ATPase activity, whereas NaCl treatment resulted in a similar response to that observed in adult plants. Na+/H+ antiport activity was induced in both juvenile and adult plants by NaCl, but ABA had no effect at either developmental stage. Results indicate that ABA-induced changes in V-ATPase activity are dependent on the plant reaching its adult phase, whereas NaCl-induced increases in V-ATPase and Na+/H+ antiport activity are independent of plant age. This suggests that ABA-induced V-ATPase activity may be linked to the stress-induced, developmentally programmed switch from C3 metabolism to Crassulacean acid metabolism in adult plants, whereas, vacuolar Na+ sequestration, mediated by the V-ATPase and Na+/H+ antiport, is regulated through ABA-independent pathways. PMID:10398716

The influence of thapsigargin on Na,K-ATPase activity in cultured nonpigmented ciliary epithelial cells.

PubMed

Mito, T; Kuwahara, S; Delamere, N A

1995-08-01

Experiments were conducted to test the influence of thapsigargin on the NaK-ATPase activity of cultured cells (ODM2) derived from human nonpigmented ciliary epithelium. The rate of ouabain-sensitive ATP hydrolysis (Na,K-ATPase activity) was diminished in cells that had been pretreated with thapsigargin then permeabilized. Following 20 min exposure of intact cells to thapsigargin, the cells were permeabilized with digitonin and the rate of ouabain-sensitive ATP hydrolysis (Na,K-ATPase activity) was measured immediately in a calcium-free buffer. In permeabilized cells that had been pretreated with 1 microM thapsigargin for 20 min, the rate of ouabain-sensitive ATP hydrolysis (Na,K-ATPase activity) was reduced by 38%. Pretreatment with lesser concentrations of thapsigargin caused smaller changes of Na,K-ATPase activity. The decrease of Na,K-ATPase activity was the same whether or not calmodulin antagonists W7 or trifluoperazine were present during the thapsigargin pretreatment period. This inhibitory effect upon the Na,K-ATPase may serve to limit the extent of sodium pump activation that takes place in intact cells when thapsigargin causes sodium pump stimulation by a mechanism that appears to involve changes in cytoplasmic ion levels when potassium channels open.

Amino Acid Availability Modulates Vacuolar H+-ATPase Assembly*

PubMed Central

Stransky, Laura A.; Forgac, Michael

2015-01-01

The vacuolar H+-ATPase (V-ATPase) is an ATP-dependent proton pump composed of a peripheral ATPase domain (V1) and a membrane-integral proton-translocating domain (V0) and is involved in many normal and disease processes. An important mechanism of regulating V-ATPase activity is reversible assembly of the V1 and V0 domains. Increased assembly in mammalian cells occurs under various conditions and has been shown to involve PI3K. The V-ATPase is necessary for amino acid-induced activation of mechanistic target of rapamycin complex 1 (mTORC1), which is important in controlling cell growth in response to nutrient availability and growth signals. The V-ATPase undergoes amino acid-dependent interactions with the Ragulator complex, which is involved in recruitment of mTORC1 to the lysosomal membrane during amino acid sensing. We hypothesized that changes in the V-ATPase/Ragulator interaction might involve amino acid-dependent changes in V-ATPase assembly. To test this, we measured V-ATPase assembly by cell fractionation in HEK293T cells treated with and without amino acids. V-ATPase assembly increases upon amino acid starvation, and this effect is reversed upon readdition of amino acids. Lysosomes from amino acid-starved cells possess greater V-ATPase-dependent proton transport, indicating that assembled pumps are catalytically active. Amino acid-dependent changes in both V-ATPase assembly and activity are independent of PI3K and mTORC1 activity, indicating the involvement of signaling pathways distinct from those implicated previously in controlling assembly. By contrast, lysosomal neutralization blocks the amino acid-dependent change in assembly and reactivation of mTORC1 after amino acid starvation. These results identify an important new stimulus for controlling V-ATPase assembly. PMID:26378229

Beneficial influence of ellagic acid on biochemical indexes associated with experimentally induced colon carcinogenesis.

PubMed

Syed, Umesalma; Ganapasam, Sudhandiran

2017-01-01

To elucidate the key biochemical indexes associated with 1, 2-dimethylhydrazine (DMH)-induced colon carcinogenesis and the modulatory efficacy of a dietary polyphenol, ellagic acid (EA). Wistar rats were chosen to study objective, and were divided into 4 groups; Group 1-control rats; Group 2-rats received EA (60 mg/kg body weight/day, orally); rats in Group 3-induced with DMH (20 mg/kg body weight) subcutaneously for 15 weeks; DMH-induced Group 4 rats were initiated with EA treatment. We examined key citric acid cycle enzymes such as isocitrate dehydrogenase, alpha-ketoglutarate dehydrogenase, succinate dehydrogenase, malate dehydrogenase and the activities of respiratory chain enzymes NADH dehydrogenase and Cytochrome-C-oxidase and membrane-bound enzyme profiles (Na +/K + ATPase, Ca 2+ ATPase and Mg 2+ ATPase), activities of lysosomal proteases such as β-D-glucuronidase, β-galactosidase and N-acety-β-D-glucosaminidase and cellular thiols (oxidized glutathione, protein thiols, and total thiols). It was found that administration of DMH to rats decreased both mitochondrial and membrane-bound enzymes activities, increased activities of lysosomal enzymes and further modulates cellular thiols levels. Treatment with EA significantly restored the mitochondrial and ATPases levels and further reduced lysosomal enzymes to near normalcy thereby restoring harmful effects induced by DMH. EA treatment was able to effectively restore the detrimental effects induced by DMH, which proves the chemoprotective function of EA against DMH-induced experimental colon carcinogenesis.

Rapid activation of gill Na+,K+-ATPase in the euryhaline teleost Fundulus heteroclitus

USGS Publications Warehouse

Mancera, J.M.; McCormick, S.D.

2000-01-01

The rapid activation of gill Na+,K+-ATPase was analyzed in the mummichog (Fundulus heteroclitus) and Atlantic salmon (Salmo salar) transferred from low salinity (0.1 ppt) to high salinity (25-35 ppt). In parr and presmolt, Salmo salar gill Na+,K+-ATPase activity started to increase 3 days after transfer. Exposure of Fundulus heteroclitus to 35 ppt seawater (SW) induced a rise in gill Na+,K+-ATPase activity 3 hr after transfer. After 12 hr, the values dropped to initial levels but showed a second significant increase 3 days after transfer. The absence of detergent in the enzyme assay resulted in lower values of gill Na+,K+-ATPase, and the rapid increase after transfer to SW was not observed. Na+,K+-ATPase activity of gill filaments in vitro for 3 hr increased proportionally to the osmolality of the culture medium (600 mosm/kg > 500 mosm/kg > 300 mosm/kg). Osmolality of 800 mosm/kg resulted in lower gill Na+,K+-ATPase activity relative to 600 mosm/kg. Increasing medium osmolality to 600 mosm/kg with mannitol also increased gill Na+,K+-ATPase. Cycloheximide inhibited the increase in gill Na+,K+-ATPase activity observed in hyperosmotic medium in a dose-dependent manner (10-4 M > 10-5 M > 10-6 M). Actinomycin D or bumetanide in the culture (doses of 10-4 M, 10-5 M, and 10-6 M) did not affect gill Na+,K+-ATPase. Injection of fish with actinomycin D prior to gill organ culture, however, prevented the increase in gill Na+,K+-ATPase activity in hyperosmotic media. The results show a very rapid and transitory increase in gill Na+,K+-ATPase activity in the first hours after the transfer of Fundulus heteroclitus to SW that is dependent on translational and transcriptional processes. (C) 2000 Wiley-Liss, Inc.

Loss of vacuolar H+-ATPase (V-ATPase) activity in yeast generates an iron deprivation signal that is moderated by induction of the peroxiredoxin TSA2.

PubMed

Diab, Heba I; Kane, Patricia M

2013-04-19

Vacuolar H(+)-ATPases (V-ATPases) acidify intracellular organelles and help to regulate overall cellular pH. Yeast vma mutants lack V-ATPase activity and allow exploration of connections between cellular pH, iron, and redox homeostasis common to all eukaryotes. A previous microarray study in a vma mutant demonstrated up-regulation of multiple iron uptake genes under control of Aft1p (the iron regulon) and only one antioxidant gene, the peroxiredoxin TSA2 (Milgrom, E., Diab, H., Middleton, F., and Kane, P. M. (2007) Loss of vacuolar proton-translocating ATPase activity in yeast results in chronic oxidative stress. J. Biol. Chem. 282, 7125-7136). Fluorescent biosensors placing GFP under transcriptional control of either an Aft1-dependent promoter (P(FIT2)-GFP) or the TSA2 promoter (P(TSA2)-GFP) were constructed to monitor transcriptional signaling. Both biosensors were up-regulated in the vma2Δ mutant, and acute V-ATPase inhibition with concanamycin A induced coordinate up-regulation from both promoters. PTSA2-GFP induction was Yap1p-dependent, indicating an oxidative stress signal. Total cell iron measurements indicate that the vma2Δ mutant is iron-replete, despite up-regulation of the iron regulon. Acetic acid up-regulated P(FIT2)-GFP expression in wild-type cells, suggesting that loss of pH control contributes to an iron deficiency signal in the mutant. Iron supplementation significantly decreased P(FIT2)-GFP expression and, surprisingly, restored P(TSA2)-GFP to wild-type levels. A tsa2Δ mutation induced both nuclear localization of Aft1p and P(FIT2)-GFP expression. The data suggest a novel function for Tsa2p as a negative regulator of Aft1p-driven transcription, which is induced in V-ATPase mutants to limit transcription of the iron regulon. This represents a new mechanism bridging the antioxidant and iron-regulatory pathways that is intimately linked to pH homeostasis.

Neuroprotective effects of idebenone against pilocarpine-induced seizures: modulation of antioxidant status, DNA damage and Na(+), K (+)-ATPase activity in rat hippocampus.

PubMed

Ahmed, Maha Ali Eissa

2014-02-01

The current study investigated the neuroprotective activity of idebenone against pilocarpine-induced seizures and hippocampal injury in rats. Idebenone is a ubiquinone analog with antioxidant, and ATP replenishment effects. It is well tolerated and has low toxicity. Previous studies reported the protective effects of idebenone against neurodegenerative diseases such as Friedreich's ataxia and Alzheimer's disease. So far, the efficacy of idebenone in experimental models of seizures has not been tested. To achieve this aim, rats were randomly distributed into six groups. Two groups were treated with either normal saline (0.9 %, i.p., control group) or idebenone (200 mg/kg, i.p., Ideb200 group) for three successive days. Rats of the other four groups (P400, Ideb50 + P400, Ideb100 + P400, and Ideb200 + P400) received either saline or idebenone (50, 100, 200 mg/kg, i.p.) for 3 days, respectively followed by a single dose of pilocarpine (400 mg/kg, i.p.). All rats were observed for 6 h post pilocarpine injection. Latency to the first seizure, and percentages of seizures and survival were recorded. Surviving animals were sacrificed, and the hippocampal tissues were separated and used for the measurement of lipid peroxides, total nitrate/nitrite, glutathione and DNA fragmentation levels, in addition to catalase and Na(+), K(+)-ATPase activities. Results revealed that in a dose-dependent manner, idebenone (100, 200 mg/kg) prolonged the latency to the first seizure, elevated the percentage of survival and diminished the percentage of pilocapine-induced seizures in rats. Significant increases in lipid peroxides, total nitrate/nitrite, DNA fragmentation levels and catalase activity, in addition to a significant reduction in glutathione level and Na(+), K(+)-ATPase activity were observed in pilocarpine group. Pre-administration of idebenone (100, 200 mg/kg, i.p.) to pilocarpine-treated rats, significantly reduced lipid peroxides, total nitrate/nitrite, DNA fragmentation levels, and normalized catalase activity. Moreover, idebenone prevented pilocarpine-induced detrimental effects on brain hippocampal glutathione level, and Na(+), K(+)-ATPase enzyme activity in rats. Data obtained from the current investigation emphasized the critical role of oxidative stress in induction of seizures by pilocarpine and elucidated the prominent neuroprotective and antioxidant activities of idebenone in this model.

Inhibition of ATPase activity in rat synaptic plasma membranes by simultaneous exposure to metals.

PubMed

Carfagna, M A; Ponsler, G D; Muhoberac, B B

1996-03-08

Inhibition of Na+/K+-ATPase and Mg2+-ATPase activities by in vitro exposure to Cd2+, Pb2+ and Mn2+ was investigated in rat brain synaptic plasma membranes (SPMs). Cd2+ and Pb2+ produced a larger maximal inhibition of Na+/K+-ATPase than of Mg2+-ATPase activity. Metal concentrations causing 50% inhibition of Na+/K+-ATPase activity (IC50 values) were Cd2+ (0.6 microM) < Pb2+ (2.1 microM) < Mn2+ (approximately 3 mM), and the former two metals were substantially more potent in inhibiting SPM versus synaptosomal Na+/K+-ATPase. Dixon plots of SPM data indicated that equilibrium binding of metals occurs at sites causing enzyme inhibition. In addition, IC50 values for SPM K+-dependent p-nitrophenylphosphatase inhibition followed the same order and were Cd2+ (0.4 microM) < Pb2+ (1.2 microM) < Mn2+ (300 microM). Simultaneous exposure to the combinations Cd2+/Mn2+ or Pb2+/Mn2+ inhibited SPM Na+/K+-ATPase activity synergistically (i.e., greater than the sum of the metal-induced inhibitions assayed separately), while Cd2+/Pb2+ caused additive inhibition. Simultaneous exposure to Cd2+/Pb2+ antagonistically inhibited Mg2+-ATPase activity while Cd2+/Mn2+ or Pb2+/Mn2+ additively inhibited Mg2+-ATPase activity at low Mn2+ concentrations, but inhibited antagonistically at higher concentrations. The similar IC50 values for Cd2+ and Pb2+ versus Mn2+ inhibition of Na+/K+-ATPase and the pattern of inhibition/activation upon exposure to two metals simultaneously support similar modes of interaction of Cd2+ and Pb2+ with this enzyme, in agreement with their chemical reactivities.

Characteristics of the Mg2+-ATPase Activity Associated with the Membrane-Bound Maize Coupling Factor 1

PubMed Central

Cohen, William S.

1989-01-01

The membrane-bound coupling factor of maize mesophyll thylakoids is a latent ATPase. Mg2+-ATPase activity can be induced in the light with either dithiothreitol or low concentrations of trypsin. Maize thylakoids that are activated with light plus trypsin exhibit considerably higher levels of activity in Na2SO3-dependent Mg2+-ATPase assays compared to thylakoids that are light and dithiothreitol activated (1400 micromoles per milligram of chlorophyll per hour versus 200 micromoles per milligram of chlorophyll per hour). Treatment with light and dithiothreitol or light plus trypsin were also required to demonstrate high levels of octyl glucoside-dependent Mg2+-ATPase activity in maize mesophyll thylakoids. Only small differences in octyl glucoside-dependent Mg2+-ATPase activity were observed in preparations that were activated in the light with either trypsin or dithiothreitol. Mg2+-ATPase activity can also be induced in maize mesophyll chloroplasts by illuminating intact preparations under appropriate conditions. Little or no ATPase activity was observed in the absence of illumination or in the presence of light plus methyl viologen. The active state decayed in the dark with a t½ of 6 to 7 minutes at room temperature. Based on the effect of the thiol oxidant, o-iodosobenzoate, and the uncoupler, nigericin, on the kinetics of deactivation of ATPase activity in intact maize chloroplasts, it appears that the activation process requires a transmembrane proton gradient and reduction of a key disulfide bridge in the gamma of chloroplast coupling factor one. PMID:16667119

ATPase activity and light scattering of acto-heavy meromyosin: dependence on ATP concentration and on ionic strength.

PubMed

Dancker, P

1975-01-01

1. The dependence on ATP concentration of ATPase activity and light scattering decrease of acto-HMM could be described at very low ionic strength by one hyperbolic adsorption isotherm with a dissociation constant of 3 X 10(-6)M. Hence the increase of ATP ase activity was paralleled by a decrease in light scattering. At higher values of ionic strength ATPase activity stopped rising before HMM was completely saturated with ATP. Higher ionic strength prevented ATPase activity from further increasing when the rigor links (links between actin and nucleotide-free myosin), which have formerly protected the ATPase against the suppressing action of higher ionic strength have fallen below a certain amount. This protecting influence of rigor links did not require tropomyosin-troponin. 2. For complete activation of ATPase activity by actin less actin was needed when HMM was incompletely saturated with ATP than when it was completely saturated with ATP. 3. The apparent affinity of ATP to regulated acto-HMM (which contained tropomyosin-troponin) was lower than to unregulated acto-HMM (which was devoid of tropomyosin-troponin). In the presence of rigor complexes (indicated by an incomplete decrease of light scattering) the ATPase activity of regulated acto-HMM was higher than that of unregulated acto-HMM. At increasing ATP concentrations the ATPase activity of regulated acto-HMM stopped rising at a similar degree of saturation with ATP as the ATPase activity of unregulated acto-HMM at the same ionic strength.

Comparison of developmental gradients for growth, ATPase, and fusicoccin-binding activity in mung bean hypocotyls

NASA Technical Reports Server (NTRS)

Basel, L. E.; Cleland, R. E.

1992-01-01

A comparison has been made of the developmental gradients along a mung bean (Vigna radiata L.) hypocotyl of the growth rate, plasma membrane ATPase, and fusicoccin-binding protein (FCBP) activity to determine whether they are interrelated. The hook and four sequential 7.5 millimeter segments of the hypocotyl below the hook were cut. A plasma membrane-enriched fraction was isolated from each section by aqueous two-phase partitioning and assayed for vanadate-sensitive ATPase and FCBP activity. Each gradient had a distinctive and different pattern. Endogenous growth rate was maximal in the second section and much lower in the others. Vanadate-sensitive ATPase activity was maximal in the third section, but remained high in the older sections. Amounts of ATPase protein, shown by specific antibody binding, did not correlate with the amount of vanadate-sensitive ATPase activity in the three youngest sections. FCBP activity was almost absent in the first section, then increased to a maximum in the oldest sections. These data show that the growth rate is not determined by the ATPase activity, and that there are no fixed ratios between the ATPase and FCBP.

Transport stress induces weight loss and heart injury in chicks: disruption of ionic homeostasis via modulating ion transporting ATPases

PubMed Central

Xia, Jun; Li, Xue-Nan; Ge, Jing; Zhang, Cong; Li, Jin-Long

2017-01-01

Transportation is inevitable in the poultry industry, and it can induce stress to chicks in varying degrees, such as mild discomfort, sometimes even death. However, the research about the effects of transport stress on the weight loss and heart injury of chicks is lacking. To elucidate the underlying mechanism of transport stress-induced effects, chicks were transported for 2h, 4h and 8h. The creatinine kinase (CK) activities, the ionic contents, the ATPases activities and the transcription of the ATPase associated subunits in chick heart were detected. The results showed that transport stress increased the weight loss and the CK activity, disturbed the ionic (K+, Ca2+, Mg2+) homeostasis and inhibited the ATPase (Na+-K+-ATPase, Ca2+-ATPase, Mg2+-ATPase and Ca2+-Mg2+-ATPase) activities, increased the ATP content and downregulated the gene expression levels of the ATPase associated subunits in heart. In conclusion, transport stress disturbed the ionic homeostasis via modulating ion transporting ATPases and the transcriptions of the associated subunits, and ultimately induced weight loss and heart injury in chicks. PMID:28445983

Sperm Na+, K+-ATPase and Ca2+-ATPase activity: A preliminary study of comparison of swim up and density gradient centrifugation methods for sperm preparation

NASA Astrophysics Data System (ADS)

Lestari, Silvia W.; Larasati, Manggiasih D.; Asmarinah, Mansur, Indra G.

2018-02-01

As one of the treatment for infertility, the success rate of Intrauterine Insemination (IUI) is still relatively low. Several sperm preparation methods, swim-up (SU) and the density-gradient centrifugation (DGC) are frequently used to select for better sperm quality which also contribute to IUI failure. Sperm selection methods mainly separate the motile from the immotile sperm, eliminating the seminal plasma. The sperm motility involves the structure and function of sperm membrane in maintaining the balance of ion transport system which is regulated by the Na+, K+-ATPase, and Ca2+-ATPase enzymes. This study aims to re-evaluate the efficiency of these methods in selecting for sperm before being used for IUI and based the evaluation on sperm Na+,K+-ATPase and Ca2+-ATPase activities. Fourteen infertile men from couples who underwent IUI were involved in this study. The SU and DGC methods were used for the sperm preparation. Semen analysis was performed based on the reference value of World Health Organization (WHO) 2010. After isolating the membrane fraction of sperms, the Na+, K+-ATPase activity was defined as the difference in the released inorganic phosphate (Pi) with and without the existence of 10 mM ouabain in the reaction, while the Ca2+-ATPase was determined as the difference in Pi contents with and without the existence of 55 µm CaCl2. The prepared sperm demonstrated a higher percentage of motile sperm compared to sperm from the whole semen. Additionally, the percentage of motile sperm of post-DGC showed higher result than the sperm from post-SU. The velocity of sperm showed similar pattern with the percentage of motile sperm, in which the velocity of prepared sperm was higher than the sperm from whole semen. Furthermore, the sperm velocity of post-DGC was higher compared to the sperm from post-SU. The Na+, K+-ATPase activity of prepared sperm was higher compared to whole semen, whereas Na+, K+-ATPase activity in the post DGC was higher than post SU. The Ca2+-ATPase activity of prepared sperm was higher compared to whole semen, whereas Ca2+-ATPase activity in the post DGC was higher than post SU. The SU and the DGC methods were able to perform sperm selection by showing a high result of Na+, K+-ATPase and Ca2+-ATPase activities, moreover DGC method selected the sperm with high activities of both the Na+, K+-ATPase and Ca2+-ATPase better compared to SU method.

Cytophotometric study of ATPase activity in brain neurons and gliocytes of paradoxical sleep-deprived rats.

PubMed

Klenikova, V A; Taranova, N P

1989-01-01

Conditions were chosen for optimal demonstration of ATPases in brain slices by a modified method of Wachstein and Meisel, and the reaction was shown to obey the Bouguer-Beer laws, confirming that ATPase activity can be determined quantitatively in single cells by cytophotometry. In rats in a state of relative physiological rest specific activity of both Na+, K+-ATPase and Mg++-ATPase in gliocytes of the hippocampus and dorsal raphe was found to be considerably higher than in neurons. Deprivation of the paradoxical phase of sleep of the rats for 24 h led to a significant increase in Na+, K+-ATPase activity in hippocampal neurons and to a decrease in its activity in gliocytes of the hippocampus and dorsal nucleus raphe. It is suggested that these changes in Na+, K+-ATPase activity may be due to some extent to a change in excitability of neurons and depolarization of the glia when sleep is disturbed.

A novel deficiency of mitochondrial ATPase of nuclear origin.

PubMed

Houstek, J; Klement, P; Floryk, D; Antonická, H; Hermanská, J; Kalous, M; Hansíková, H; Hout'ková, H; Chowdhury, S K; Rosipal, T; Kmoch, S; Stratilová, L; Zeman, J

1999-10-01

We report a new type of fatal mitochondrial disorder caused by selective deficiency of mitochondrial ATP synthase (ATPase). A hypotrophic newborn from a consanguineous marriage presented severe lactic acidosis, cardiomegaly and hepatomegaly and died from heart failure after 2 days. The activity of oligomycin-sensitive ATPase was only 31-34% of the control, both in muscle and heart, but the activities of cytochrome c oxidase, citrate synthase and pyruvate dehydrogenase were normal. Electrophoretic and western blot analysis revealed selective reduction of ATPase complex but normal levels of the respiratory chain complexes I, III and IV. The same selective deficiency of ATPase was found in cultured skin fibroblasts which showed similar decreases in ATPase content, ATPase hydrolytic activity and level of substrate-dependent ATP synthesis (20-25, 18 and 29-33% of the control, respectively). Pulse-chase labelling of patient fibroblasts revealed low incorporation of [(35)S]methionine into assembled ATPase complexes, but increased incorporation into immunoprecipitated ATPase subunit beta, which had a very short half-life. In contrast, no difference was found in the size and subunit composition of the assembled and newly produced ATPase complex. Transmitochondrial cybrids prepared from enucleated fibroblasts of the patient and rho degrees cells derived from 143B. TK(-)human osteosarcoma cells fully restored the ATPase activity, ATP synthesis and ATPase content, when compared with control cybrids. Likewise, the pattern of [(35)S]methionine labelling of ATPase was found to be normal in patient cybrids. We conclude that the generalized deficiency of mitochondrial ATPase described is of nuclear origin and is caused by altered biosynthesis of the enzyme.

A Kinetic Characterization of the Gill (Na+, K+)-ATPase from the Semi-terrestrial Mangrove Crab Cardisoma guanhumi Latreille, 1825 (Decapoda, Brachyura).

PubMed

Farias, Daniel L; Lucena, Malson N; Garçon, Daniela P; Mantelatto, Fernando L; McNamara, John C; Leone, Francisco A

2017-10-01

We provide a kinetic characterization of (Na + , K + )-ATPase activity in a posterior gill microsomal fraction from the semi-terrestrial mangrove crab Cardisoma guanhumi. Sucrose density gradient centrifugation reveals two distinct membrane fractions showing considerable (Na + , K + )-ATPase activity, but also containing other microsomal ATPases. The (Na + , K + )-ATPase, notably immuno-localized to the apical region of the epithelial pillar cells, and throughout the pillar cell bodies, has an M r of around 110 kDa and hydrolyzes ATP with V M  = 146.8 ± 6.3 nmol Pi min -1  mg protein -1 and K M  = 0.05 ± 0.003 mmol L -1 obeying Michaelis-Menten kinetics. While stimulation by Na + (V M  = 139.4 ± 6.9 nmol Pi min -1  mg protein -1 , K M  = 4.50 ± 0.22 mmol L -1 ) also follows Michaelis-Menten kinetics, modulation of (Na + , K + )-ATPase activity by MgATP (V M  = 136.8 ± 6.5 nmol Pi min -1 mg protein -1 , K 0.5  = 0.27 ± 0.04 mmol L -1 ), K + (V M  = 140.2 ± 7.0 nmol Pi min -1 mg protein -1 , K 0.5  = 0.17 ± 0.008 mmol L -1 ), and NH 4 + (V M  = 149.1 ± 7.4 nmol Pi min -1 mg protein -1 , K 0.5  = 0.60 ± 0.03 mmol L -1 ) shows cooperative kinetics. Ouabain (K I  = 52.0 ± 2.6 µmol L -1 ) and orthovanadate (K I  = 1.0 ± 0.05 µmol L -1 ) inhibit total ATPase activity by around 75%. At low Mg 2+ concentrations, ATP is an allosteric modulator of the enzyme. This is the first study to provide a kinetic characterization of the gill (Na + , K + )-ATPase in C. guanhumi, and will be useful in better comprehending the biochemical underpinnings of osmoregulatory ability in a semi-terrestrial mangrove crab.

Dietary calcium induced cytological and biochemical changes in thyroid.

PubMed

Chandra, Amar K; Goswami, Haimanti; Sengupta, Pallav

2012-09-01

Certain epidemiological studies revealed correlation between hard water consumption (with high calcium) and thyroid size of the population, though the possible alterations in thyroid physiology upon calcium exposure are still inconclusive. Adult male Wistar strain rats were subjected to calcium treatment at the doses of 0.5g%, 1.0g% and 1.5g% calcium chloride (CaCl(2)) for 60 days. The parameters studied were - thyroid gland weight, histopathology, histomorphometry; thyroid peroxidase (TPO), 5'-deiodinase I (DI), sodium-potassium adenosine triphosphatase (Na(+)-K(+)-ATPase) activities; serum total and free thyroxine (tT4, fT4), total and free triiodothyronine (tT3, fT3), thyroid stimulating hormone (TSH) levels. Enlargement of thyroid with hypertrophic and hyperplastic changes, retarded TPO and 5'-DI but enhanced Na(+)-K(+)-ATPase activities, augmented serum total and free T4 and TSH but decreased total and free T3 levels and low T3/T4 ratio (T3:T4) were observed in the treated groups. All these findings indicate development of goitrogenesis upon exposure to excessive dietary calcium. Copyright © 2012 Elsevier B.V. All rights reserved.

Inhibition of partially purified K+/H+-ATPase from guinea-pig isolated and enriched parietal cells by substituted benzimidazoles.

PubMed Central

Beil, W.; Sewing, K. F.

1984-01-01

The cellular and subcellular distributions of adenosinetriphosphatases (ATPases) were examined in guinea-pig gastric mucosal cells. All cell types displayed Mg2+-ATPase and bicarbonate (HCO3-)-stimulated ATPase activity. K+-ATPase was located only in fractions derived from parietal cells. Differential and density-gradient centrifugation of material prepared from parietal cells revealed that K+-ATPase activity was located in a tubulo-vesicular membrane fraction. Enzyme activity was ten fold greater in this fraction than in a crude parietal cell homogenate. The substituted benzimidazoles, omeprazole and picoprazole, inhibited K+-ATPase (IC50 1.8 +/- 0.5 mumol l-1 and 3.1 +/- 0.4 mumol l-1, respectively). Detailed kinetic analysis indicated that these compounds were non-competitive and reversible inhibitors of the enzyme. In contrast cimetidine and verapamil were without effect on the enzyme. The relevance of the inhibition of K+-ATPase to the antisecretory activity of the benzimidazoles, in experimental animals and man, is discussed. PMID:6146367

Two widely expressed plasma membrane H(+)-ATPase isoforms of Nicotiana tabacum are differentially regulated by phosphorylation of their penultimate threonine.

PubMed

Bobik, Krzysztof; Duby, Geoffrey; Nizet, Yannick; Vandermeeren, Caroline; Stiernet, Patrick; Kanczewska, Justyna; Boutry, Marc

2010-04-01

The plasma membrane H(+)-ATPases PMA2 and PMA4 are the most widely expressed in Nicotiana plumbaginifolia, and belong to two different subfamilies. Both are activated by phosphorylation of a Thr at the penultimate position and the subsequent binding of 14-3-3 proteins. Their expression in Saccharomyces cerevisiae revealed functional and regulatory differences. To determine whether different regulatory properties between PMA2 and PMA4 exist in plants, we generated two monoclonal antibodies able to detect phosphorylation of the penultimate Thr of either PMA2 or PMA4 in a total protein extract. We also raised Nicotiana tabacum transgenic plants expressing 6-His-tagged PMA2 or PMA4, enabling their individual purification. Using these tools we showed that phosphorylation of the penultimate Thr of both PMAs was high during the early exponential growth phase of an N. tabacum cell culture, and then progressively declined. This decline correlated with decreased 14-3-3 binding and decreased plasma membrane ATPase activity. However, the rate and extent of the decrease differed between the two isoforms. Cold stress of culture cells or leaf tissues reduced the Thr phosphorylation of PMA2, whereas no significant changes in Thr phosphorylation of PMA4 were seen. These results strongly suggest that PMA2 and PMA4 are differentially regulated by phosphorylation. Analysis of the H(+)-ATPase phosphorylation status in leaf tissues indicated that no more than 44% (PMA2) or 32% (PMA4) was in the activated state under normal growth conditions. Purification of either isoform showed that, when activated, the two isoforms did not form hetero-oligomers, which is further support for these two H(+)-ATPase subfamilies having different properties.

Aldosterone stimulates vacuolar H+-ATPase activity in renal acid-secretory intercalated cells mainly via a protein kinase C-dependent pathway

PubMed Central

Winter, Christian; Kampik, Nicole B.; Vedovelli, Luca; Rothenberger, Florina; Păunescu, Teodor G.; Stehberger, Paul A.; Brown, Dennis; John, Hubert

2011-01-01

Urinary acidification in the collecting duct is mediated by the activity of H+-ATPases and is stimulated by various factors including angiotensin II and aldosterone. Classically, aldosterone effects are mediated via the mineralocorticoid receptor. Recently, we demonstrated a nongenomic stimulatory effect of aldosterone on H+-ATPase activity in acid-secretory intercalated cells of isolated mouse outer medullary collecting ducts (OMCD). Here we investigated the intracellular signaling cascade mediating this stimulatory effect. Aldosterone stimulated H+-ATPase activity in isolated mouse and human OMCDs. This effect was blocked by suramin, a general G protein inhibitor, and GP-2A, a specific Gαq inhibitor, whereas pertussis toxin was without effect. Inhibition of phospholipase C with U-73122, chelation of intracellular Ca2+ with BAPTA, and blockade of protein kinase C prevented the stimulation of H+-ATPases. Stimulation of PKC by DOG mimicked the effect of aldosterone on H+-ATPase activity. Similarly, aldosterone and DOG induced a rapid translocation of H+-ATPases to the luminal side of OMCD cells in vivo. In addition, PD098059, an inhibitor of ERK1/2 activation, blocked the aldosterone and DOG effects. Inhibition of PKA with H89 or KT2750 prevented and incubation with 8-bromoadenosine-cAMP mildly increased H+-ATPase activity. Thus, the nongenomic modulation of H+-ATPase activity in OMCD-intercalated cells by aldosterone involves several intracellular pathways and may be mediated by a Gαq protein-coupled receptor and PKC. PKA and cAMP appear to have a modulatory effect. The rapid nongenomic action of aldosterone may participate in the regulation of H+-ATPase activity and contribute to final urinary acidification. PMID:21832245

Alteration of aluminium inhibition of synaptosomal (Na(+)/K(+))ATPase by colestipol administration.

PubMed

Silva, V S; Oliveira, L; Gonçalves, P P

2013-11-01

The ability of aluminium to inhibit the (Na(+)/K(+))ATPase activity has been observed by several authors. During chronic dietary exposure to AlCl3, brain (Na(+)/K(+))ATPase activity drops, even if no alterations of catalytic subunit protein expression and of energy charge potential are observed. The aluminium effect on (Na(+)/K(+))ATPase activity seems to implicate the reduction of interacting protomers within the oligomeric ensemble of the membrane-bound (Na(+)/K(+))ATPase. The activity of (Na(+)/K(+))ATPase is altered by the microviscosity of lipid environment. We studied if aluminium inhibitory effect on (Na(+)/K(+))ATPase is modified by alterations in synaptosomal membrane cholesterol content. Adult male Wistar rats were submitted to chronic dietary AlCl3 exposure (0.03 g/day of AlCl3) and/or to colestipol, a hypolidaemic drug (0.31 g/day) during 4 months. The activity of (Na(+)/K(+))ATPase was studied in brain cortex synaptosomes with different cholesterol contents. Additionally, we incubate synaptosomes with methyl-β-cyclodextrin for both enrichment and depletion of membrane cholesterol content, with or without 300 μM AlCl3. This enzyme activity was significantly reduced by micromolar AlCl3 added in vitro and when aluminium was orally administered to rats. The oral administration of colestipol reduced the cholesterol content and concomitantly inhibited the (Na(+)/K(+))ATPase. The aluminium inhibitory effect on synaptosomal (Na(+)/K(+))ATPase was reduced by cholesterol depletion both in vitro and in vivo. Copyright © 2013 Elsevier Inc. All rights reserved.

Changes in the level and activation state of the plasma membrane H(+)-ATPase during aging of red beet slices.

PubMed

Papini, R; De Michelis, M I

1997-07-01

The effect of aging on the plasma membrane (PM) H(+)-ATPase of red beet (Beta vulgaris L.) parenchyma discs was analyzed in PM purified by aqueous two-phase partitioning. Aging increased both the activity in the amount of immunodetectable H(+)-ATPase in the PM. The activity assayed at slightly alkaline pH values increased earlier and more strongly than that assayed at acidic pH values, so that the pH curve of the enzyme from aged beet discs was shifted toward more alkaline values. Aging decreased the stimulation of the PM H(+)-ATPase activity by controlled trypsin treatments or by lysophosphatidylcholine. After trypsin treatment the pH dependence of H(+)-ATPase from dormant or aged beet discs became equal. These results indicate that aging not only increases the level of H(+)-ATPase in the PM, but also determines its activation, most likely by modifying the interaction between the autoinhibitory carboxyl-terminal domain and the catalytic site. When the PM H(+)-ATPase activity was assayed at a slightly alkaline pH, the tyrosine modifier N-acetylimidazole inhibited the H(+)-ATPase in the PM from dormant beet discs much less than in the PM from aged discs, suggesting that modification of a tyrosine residue may be involved in the activation of the PM H(+)-ATPase induced by aging. The results are discussed with regard to aging-induced development of transmembrane transport activities.

Plant Defense Response to Fungal Pathogens (Activation of Host-Plasma Membrane H+-ATPase by Elicitor-Induced Enzyme Dephosphorylation).

PubMed Central

Vera-Estrella, R.; Barkla, B. J.; Higgins, V. J.; Blumwald, E.

1994-01-01

Elicitor preparations containing the avr5 gene products from race 4 of Cladosporium fulvum and tomato (Lycopersicon esculentum L.) cells near isogenic for the resistance gene Cf5 were used to investigate events following the treatment of host plasma membranes with elicitor. A 4-fold increase in H+-ATPase activity, coincident with the acidification of the extracellular medium, was detected immediately after elicitor treatment. The elicitor-induced stimulation of the plasma membrane H+-ATPase was inhibited by okadaic acid but not by staurosporine, suggesting that protein dephosphorylation was required for increased H+-ATPase activity. This observation was confirmed by [gamma]-32P labeling and immunodetection of the plasma membrane H+-ATPase. Effects of guanidine nucleotide analogs and mastoparan on the ATPase activity suggested the role of GTP-binding proteins in mediating the putative elicitor-receptor binding, resulting in activation of a phosphatase(s), which in turn stimulates the plasma membrane H+-ATPase by dephosphorylation. PMID:12232073

Characterization and effect of light on the plasma membrane H(+) -ATPase of bean leaves

NASA Technical Reports Server (NTRS)

Linnemeyer, P. A.; Van Volkenburgh, E.; Cleland, R. E.

1990-01-01

Proton excretion from bean (Phaseolus vulgaris L.) leaf cells is increased by bright white light. To test whether this could be due, at least in part, to an increase in plasma membrane (PM) ATPase activity, PM vesicles were isolated from primary leaves by phase partitioning and used to characterize PM ATPase activity and changes in response to light. ATPase activity was characterized as magnesium ion dependent, vanadate sensitive, and slightly stimulated by potassium chloride. The pH optimum was 6.5, the Km was approximately 0.30 millimolar ATP, and the activity was about 60% latent. PM vesicles were prepared from leaves of plants grown for 11 days in dim red light (growing slowly) or grown for 10 days in dim red light and then transferred to bright white-light for 1 day (growing rapidly). For both light treatments, ATPase specific activity was approximately 600 to 700 nanomoles per milligram protein per minute, and the latency, Km, and sensitivity to potassium chloride were also similar. PM vesicles from plants grown in complete darkness, however, exhibited a twofold greater specific activity. We conclude that the promotion of leaf growth and proton excretion by bright white light is not due to an increase in ATPase specific activity. Light does influence ATPase activity, however; both dim red light and bright white light decreased the ATPase specific activity by nearly 50% as compared with dark-grown leaves.

Mechanisms of L-Triiodothyronine-Induced Inhibition of Synaptosomal Na+-K+-ATPase Activity in Young Adult Rat Brain Cerebral Cortex

PubMed Central

Sarkar, Pradip K.; Biswas, Avijit; Ray, Arun K.; Martin, Joseph V.

2013-01-01

The role of thyroid hormones (TH) in the normal functioning of adult mammalian brain is unclear. Our studies have identified synaptosomal Na+-K+-ATPase as a TH-responsive physiological parameter in adult rat cerebral cortex. L-triiodothyronine (T3) and L-thyroxine (T4) both inhibited Na+-K+-ATPase activity (but not Mg2+-ATPase activity) in similar dose-dependent fashions, while other metabolites of TH were less effective. Although both T3 and the β-adrenergic agonist isoproterenol inhibited Na+-K+-ATPase activity in cerebrocortical synaptosomes in similar ways, the β-adrenergic receptor blocker propranolol did not counteract the effect of T3. Instead, propranolol further inhibited Na+-K+-ATPase activity in a dose-dependent manner, suggesting that the effect of T3 on synaptosomal Na+-K+-ATPase activity was independent of β-adrenergic receptor activation. The effect of T3 on synaptosomal Na+-K+-ATPase activity was inhibited by the α2-adrenergic agonist clonidine and by glutamate. Notably, both clonidine and glutamate activate Gi-proteins of the membrane second messenger system, suggesting a potential mechanism for the inhibition of the effects of TH. In this paper, we provide support for a nongenomic mechanism of action of TH in a neuronal membrane-related energy-linked process for signal transduction in the adult condition. PMID:24307963

[Effect of glycine and strychnine on Cl(-)-activated Mg2+-ATPase from bream brain microsomes (Abamis brama L.)].

PubMed

Menzikov, S A; Menzikova, O V

2001-01-01

The effect of glycine and strychnine on Mg2+-ATPase from the microsomal fraction of the bream (Abramis brama L.) brain was studied. The glycine in the concentration range 10(-7)-10(-4) M activates the enzyme. The effect of glycine on Mg2+-ATPase is obviated by 100 microM strychnine. The strychnine in the concentration range 5-90 microM activates the basal Mg2+-ATPase but decreases the effect of the enzyme activation by 10(-4) M glycine. The effect of Cl- on Mg2+-ATPase depends on the substrate concentration (Mg2+-ATP) and is not observed in the presence of 100 microM strychnine. A receptor-dependent pathway of glycine and strychnine action on Cl(-)-activated Mg2+-ATPase from bream brain microsomes is proposed.

Purification and Properties of an ATPase from Sulfolobus solfataricus

NASA Technical Reports Server (NTRS)

Hochstein, Lawrence I.; Stan-Lotter, Helga

1992-01-01

A sulfite-activated ATPase isolated from Sulfolobus solfataricus had a relative molecular mass of 370,000. It was composed of three subunits whose relative molecular masses were 63,000, 48,000, and 24,000. The enzyme was inhibited by the vacuolar ATPase inhibitors nitrate and N-ethylmaleimide; 4-chloro-7-nitrobenzo-furazan (NBD-Cl) was also inhibitory. N-Ethylmaleimide was predominately bound to the largest subunit while NBD-CL was bound to both subunits. ATPase activity was inhibited by low concentrations of p-chloromercuri-phenyl sulfonate and the inhibition was reversed by cysteine which suggested that thiol groups were essential for activity. While the ATPase from S. solfataricus shared several properties with the ATPase from S. acidocaldarius there were significant differences. The latter enzyme was activated by sulfate and chloride and was unaffected by N-ethylmaleimide, whereas the S. solfataricus ATPase was inhibited by these anions as well as N-ethyimaleimide. These differences as well as differences that occur in other vacuolar-like ATPases isolated from the methanogenic and the extremely halophilic bacteria suggest the existence of several types of archaeal ATPases, none of which have been demonstrated to synthesize ATP.

Effect of endurance swimming on rat cardiac myofibrillar ATPase with experimental diabetes.

PubMed

Belcastro, A N; Maybank, P; Rossiter, M; Secord, D

1985-09-01

Diabetes is characterized by depressed cardiac functional properties attributed to Ca2+-activated ATPase activity. In contrast, endurance swimming enhances the cardiac functional properties and Ca2+-activated myofibril ATPase. Thus, the purpose of this study was to observe if the changes associated with experimental diabetes can be ameliorated with training. Diabetes was induced with a single i.v. injection of streptozotocin (60 mg/kg). Blood and urine glucose concentrations were 802 +/- 44 and 6965 +/- 617 mg/dL, respectively. The training control and training diabetic animals were made to swim (+/- 2% body weight) 4 days/week for 8 weeks. Cardiac myofibril, at 10 microM free Ca2+ concentration was reduced by 54% in the sedentary diabetics compared with sedentary control animals (p less than 0.05). Swim training enhanced the Ca2+-activated myofibril ATPase activities for the normal animals. The diabetic animals, which swam for 8 weeks, had further reduced their Ca2+-activated myofibril ATPase activity when compared with sedentary diabetics (p less than 0.05). Similarly, the Mg2+-stimulated myofibril ATPase activity was depressed by 31% in diabetics following endurance swimming. It is concluded that the depressed Ca2+-activated myofibril ATPase activity of diabetic hearts is not reversible with endurance swimming.

Inhibitory Effect of Fluoride on Na+,K+ ATPase Activity in Human Erythrocyte Membrane.

PubMed

A, Shashi; G, Meenakshi

2015-12-01

The present study was performed to evaluate the role of long-term consumption of excessive fluoride on electrolyte homeostasis and their transporting mechanisms in erythrocytes of subjects afflicted with dental and skeletal fluorosis. A total of 620 adult (20-50 years) Indian residents participated in this study: 258 men and 242 women exposed to high concentrations of fluoride and 120 age and gender-matched control subjects. Erythrocytes were isolated from blood samples, washed, and used for the estimation of intraerythrocyte sodium and potassium concentrations. Na+,K+ ATPase activity was determined spectrophotometrically from a ghost erythrocyte membrane prepared by osmotic lysis. Erythrocyte analytes were correlated with the water and serum fluoride concentrations by Pearson's bivariate correlation and regression analysis. Results indicated a significant increase in intraerythrocyte sodium (F=14306.265, P<0.0001) in subjects from endemic fluorosis study groups as compared to controls. A significant (P<0.05) positive correlation of intracellular sodium was found with water and serum fluoride concentrations. Mean concentration of intraerythrocytic potassium ions showed significant reduction (F=9136.318, P<0.0001) in subjects exposed to fluoride. A significant (P<0.05) negative correlation of potassium ions was noted with water and serum fluoride concentrations. Na+,K+ ATPase activity was significantly declined (F=1572.763, P<0.0001) in subjects exposed to fluoride. A significant (P<0.05) inverse relationship of Na+,K+ ATPase activity was revealed with water and serum fluoride concentrations.

Role of Na+/K+-ATPase in Natriuretic Effect of Prolactin in a Model of Cholestasis of Pregnancy.

PubMed

Abramicheva, P A; Balakina, T A; Bulaeva, O A; Guseva, A A; Lopina, O D; Smirnova, O V

2017-05-01

Participation of Na+/K+-ATPase in the natriuretic effect of prolactin in a cholestasis of pregnancy model was investigated. The Na+/K+-ATPase activity in rat kidney medulla, where active sodium reabsorption occurs, decreased in the model of cholestasis of pregnancy and other hyperprolactinemia types compared with intact animals. This effect was not connected with the protein level of α1- and β-subunits of Na+/K+-ATPase measured by Western blotting in the kidney medulla. Decrease in Na+/K+-ATPase activity in the kidney cortex was not significant, as well as decrease in the quantity of mRNA and proteins of the α1- and β-subunits of Na+/K+-ATPase. There were no correlations between the Na+/K+-ATPase activity and sodium clearance, although sodium clearance increased significantly in the model of cholestasis of pregnancy and other hyperprolactinemia groups under conditions of stable glomerular filtration rate measured by creatinine clearance. We conclude that the Na+/K+-ATPase is not the only mediator of the natriuretic effect of prolactin in the model of cholestasis of pregnancy.

Glutathione S-transferase π complexes with and stimulates Na⁺,K⁺-ATPase.

PubMed

Ochiai, Hideo; Eguchi, Hiroshi; Noguchi, Shunsuke; Hayashi, Yutaro; Nishino, Hideaki; Kawamura, Masaru; Wu, Chau H

2013-01-01

Glutathione S-transferase (GST) was found to complex with the Na⁺,K⁺-ATPase as shown by binding assay using quartz crystal microbalance. The complexation was obstructed by the addition of antiserum to the α-subunit of the Na⁺,K⁺-ATPase, suggesting the specificity of complexation between GST and the Na⁺,K⁺-ATPase. Co-immunoprecipitation experiments, using the anti-α-subunit antiserum to precipitate the GST-Na⁺,K⁺-ATPase complex and then using antibodies specific to an isoform of GST to identify the co-precipitated proteins, revealed that GSTπ was complexed with the Na⁺,K⁺-ATPase. GST stimulated the Na⁺,K⁺-ATPase activity up to 1.4-fold. The level of stimulation exhibited a saturable dose-response relationship with the amount of GST added, although the level of stimulation varied depending on the content of GSTπ in the lots of GST received from supplier. The stimulation was also obtained when recombinant GSTπ was used, confirming the results. When GST was treated with reduced glutathione, GST activity was greatly stimulated, whereas the level of stimulation of the Na⁺,K⁺-ATPase activity was similar to that when untreated GST was added. When GST was treated with H₂O₂, GST activity was greatly diminished while the stimulation of the Na⁺,K⁺-ATPase activity was preserved. The results suggest that GSTπ complexes with the Na⁺,K⁺-ATPase and stimulates the latter independent of its GST activity. Copyright © 2012 John Wiley & Sons, Ltd.

Single-molecule Analysis of Inhibitory Pausing States of V1-ATPase*

PubMed Central

Uner, Naciye Esma; Nishikawa, Yoshihiro; Okuno, Daichi; Nakano, Masahiro; Yokoyama, Ken; Noji, Hiroyuki

2012-01-01

V1-ATPase, the hydrophilic V-ATPase domain, is a rotary motor fueled by ATP hydrolysis. Here, we found that Thermus thermophilus V1-ATPase shows two types of inhibitory pauses interrupting continuous rotation: a short pause (SP, 4.2 s) that occurred frequently during rotation, and a long inhibitory pause (LP, >30 min) that terminated all active rotations. Both pauses occurred at the same angle for ATP binding and hydrolysis. Kinetic analysis revealed that the time constants of inactivation into and activation from the SP were too short to represent biochemically predicted ADP inhibition, suggesting that SP is a newly identified inhibitory state of V1-ATPase. The time constant of inactivation into LP was 17 min, consistent with one of the two time constants governing the inactivation process observed in bulk ATPase assay. When forcibly rotated in the forward direction, V1 in LP resumed active rotation. Solution ADP suppressed the probability of mechanical activation, suggesting that mechanical rotation enhanced inhibitory ADP release. These features were highly consistent with mechanical activation of ADP-inhibited F1, suggesting that LP represents the ADP-inhibited state of V1-ATPase. Mechanical activation largely depended on the direction and angular displacement of forced rotation, implying that V1-ATPase rotation modulates the off rate of ADP. PMID:22736762

Direct interaction of beta-amyloid with Na,K-ATPase as a putative regulator of the enzyme function

NASA Astrophysics Data System (ADS)

Petrushanko, Irina Yu.; Mitkevich, Vladimir A.; Anashkina, Anastasia A.; Adzhubei, Alexei A.; Burnysheva, Ksenia M.; Lakunina, Valentina A.; Kamanina, Yulia V.; Dergousova, Elena A.; Lopina, Olga D.; Ogunshola, Omolara O.; Bogdanova, Anna Yu.; Makarov, Alexander A.

2016-06-01

By maintaining the Na+ and K+ transmembrane gradient mammalian Na,K-ATPase acts as a key regulator of neuronal electrotonic properties. Na,K-ATPase has an important role in synaptic transmission and memory formation. Accumulation of beta-amyloid (Aβ) at the early stages of Alzheimer’s disease is accompanied by reduction of Na,K-ATPase functional activity. The molecular mechanism behind this phenomenon is not known. Here we show that the monomeric Aβ(1-42) forms a tight (Kd of 3 μM), enthalpy-driven equimolar complex with α1β1 Na,K-ATPase. The complex formation results in dose-dependent inhibition of the enzyme hydrolytic activity. The binding site of Aβ(1-42) is localized in the “gap” between the alpha- and beta-subunits of Na,K-ATPase, disrupting the enzyme functionality by preventing the subunits from shifting towards each other. Interaction of Na,K-ATPase with exogenous Aβ(1-42) leads to a pronounced decrease of the enzyme transport and hydrolytic activity and Src-kinase activation in neuroblastoma cells SH-SY5Y. This interaction allows regulation of Na,K-ATPase activity by short-term increase of the Aβ(1-42) level. However prolonged increase of Aβ(1-42) level under pathological conditions could lead to chronical inhibition of Na,K-ATPase and disruption of neuronal function. Taken together, our data suggest the role of beta-amyloid as a novel physiological regulator of Na,K-ATPase.

Na,K-ATPase is a target of cigarette smoke and reduced expression predicts poor patient outcome of smokers with lung cancer

PubMed Central

Huynh, Thu P.; Mah, Vei; Sampson, Valerie B.; Chia, David; Fishbein, Michael C.; Horvath, Steve; Alavi, Mohammad; Wu, Debbie C.; Harper, Jeffrey; Sarafian, Ted; Dubinett, Steven M.; Langhans, Sigrid A.; Goodglick, Lee

2012-01-01

Diminished Na,K-ATPase expression has been reported in several carcinomas and has been linked to tumor progression. However, few studies have determined whether Na,K-ATPase function and expression are altered in lung malignancies. Because cigarette smoke (CS) is a major factor underlying lung carcinogenesis and progression, we investigated whether CS affects Na,K-ATPase activity and expression in lung cell lines. Cells exposed to CS in vitro showed a reduction of Na,K-ATPase activity. We detected the presence of reactive oxygen species (ROS) in cells exposed to CS before Na,K-ATPase inhibition, and neutralization of ROS restored Na,K-ATPase activity. We further determined whether Na,K-ATPase expression correlated with increasing grades of lung adenocarcinoma and survival of patients with smoking history. Immunohistochemical analysis of lung adenocarcinoma tissues revealed reduced Na,K-ATPase expression with increasing tumor grade. Using tissue microarray containing lung adenocarcinomas of patients with known smoking status, we found that high expression of Na,K-ATPase correlated with better survival. For the first time, these data demonstrate that CS is associated with loss of Na,K-ATPase function and expression in lung carcinogenesis, which might contribute to disease progression. PMID:22345575

The role or non-role of ATPase activation by phenytoin in the stabilization of excitable membranes.

PubMed

Deupree, J D

1977-09-01

The role or non-role of NaK ATPase, Mg ATPase, and CaMg ATPase involvement in stabilization of excitable membranes by phenytoin is critically evaluated. There is no substantial evidence to indicate that the membrane-stabilizing effect of phenytoin is due to activation of the NaK ATPase. Previous reports of activation of the NaK ATPase at low potassium and high sodium are probably not due to phenytoin but to a potassium contamination in the phenytoin solution. In vitro experiments do not provide any clear evidence of any alterations of NaK ATPase properties by phenytoin. However, one cannot rule out the possibility that phenytoin alters the efficiency of the sodium-potassium pump. Likewise, the Ca ATPase is not inhibited by phenytoin. However, there is some evidence that the Mg ATPase in synaptic vesicles is substantially inhibited by phenytoin. There is substantial evidence indicating that phenytoin partially blocks passive diffusion of sodium into stimulated nerves. The mechanism by which phenytoin blocks sodium influx and the relationship of this effect to the drug's anticonvulsant action remain to be determined.

Carnitine modulates crucial myocardial adenosine triphosphatases and acetylcholinesterase enzyme activities in choline-deprived rats.

PubMed

Strilakou, Athina A; Tsakiris, Stylianos T; Kalafatakis, Konstantinos G; Stylianaki, Aikaterini T; Karkalousos, Petros L; Koulouris, Andreas V; Mourouzis, Iordanis S; Liapi, Charis A

2014-01-01

Choline is an essential nutrient, and choline deficiency has been associated with cardiovascular morbidity. Choline is also the precursor of acetylcholine (cholinergic component of the heart's autonomic nervous system), whose levels are regulated by acetylcholinesterase (AChE). Cardiac contraction-relaxation cycles depend on ion gradients established by pumps like the adenosine triphosphatases (ATPases) Na(+)/K(+)-ATPase and Mg(2+)-ATPase. This study aimed to investigate the impact of dietary choline deprivation on the activity of rat myocardial AChE (cholinergic marker), Na(+)/K(+)-ATPase, and Mg(2+)-ATPase, and the possible effects of carnitine supplementation (carnitine, structurally relevant to choline, is used as an adjunct in treating cardiac diseases). Adult male albino Wistar rats were distributed among 4 groups, and were fed a standard or choline-deficient diet for one month with or without carnitine in their drinking water (0.15% w/v). The enzyme activities were determined spectrophotometrically in the myocardium homogenate. Choline deficiency seems to affect the activity of the aforementioned parameters, but only the combination of choline deprivation and carnitine supplementation increased myocardial Na(+)/K(+)-ATPase activity along with a concomitant decrease in the activities of Mg(2+)-ATPase and AChE. The results suggest that carnitine, in the setting of choline deficiency, modulates cholinergic myocardial neurotransmission and the ATPase activity in favour of cardiac work efficiency.

Selective inhibition of K(+)-stimulation of Na,K-ATPase by bretylium.

PubMed Central

Tiku, P. E.; Nowell, P. T.

1991-01-01

1. The effects of bretylium were investigated on purified Na,K-ATPase from guinea-pig heart and on the Na/K pump in trout erythrocytes, with a view to further identifying the mechanism(s) associated with its antiarrhythmic effects. 2. Na,K-ATPase activity of the thiocyanate-dispersed enzyme was determined by the measurement of inorganic phosphate produced by ATP hydrolysis. 3. When the concentrations of each of the Na,K-ATPase activating components were varied in turn, bretylium (1-5 mmol l-1) exhibited competitive-type effects against K+ with a Ki of 1.4 mmol l-1 and noncompetitive-type effects against Na+, Mg2+ and ATP. 4. In K+ influx studies in trout erythrocytes with 86Rb+ used as the marker, the inhibition of total influx observed with bretylium (5 and 10 mmol l-1) was attributable to the bretylium cation selectively inhibiting the Na/K pump-mediated influx with the associated tosylate anion inhibiting Na/K cotransport. 5. The observed inhibition kinetics indicated that the bretylium cation (2-15 mmol l-1) competitively inhibited K+ stimulation of the Na/K pump at 6 and 1.25 mmol l-1 external K+ with a mean K1 of 2.3 mmol l-1. 6. The effects demonstrated on the functioning Na/K pump in erythrocytes confirmed the Na,K-ATPase findings, with bretylium selectively inhibiting K+ stimulation of the pump mechanism in both cases. 7. It is suggested that Na,K-ATPase inhibition may contribute to the antiarrhythmic and positive inotropic effects of bretylium with the cardiac accumulation of bretylium also possibly being a further important factor. PMID:1667290

Lack of thyroid hormone effect on activation energy of NaK-ATPase.

PubMed

Rahimifar, M; Ismail-Beigi

1977-02-01

In order to differentiate whether activation of NaK-ATPase in thyroid thermogenesis is due to increased numbers of active 'sodium pump' units or due to a change in the kinetics of the enzyme, the effect of T3 on activation energy (Ea) of NaK-ATPase was determined in rat liver, kidney and brain. Injection of T3 produced significant increases in the specific activity of NaK-ATPase in liver and kidney but not in brain homogenates. T3 injections produced no significant change in the Ea of NaK-ATPase in any of the three tissues. The data are compatible with the hypothesis that thyroid stimulation of the sodium pump is brought about by an increase in the number of active pump units.

The erythrocyte calcium pump is inhibited by non-enzymic glycation: studies in situ and with the purified enzyme.

PubMed Central

González Flecha, F L; Castello, P R; Caride, A J; Gagliardino, J J; Rossi, J P

1993-01-01

In a previous paper we demonstrated that incubation of either intact erythrocytes or erythrocytes membranes with glucose decreases the activity of the membrane Ca(2+)-ATPase [González Flecha, Bermúdez, Cédola, Gagliardino and Rossi (1990) Diabetes 39, 707-711]. The aim of the present work was to obtain information about the mechanism of this inhibition. For this purpose, experiments were carried out with purified Ca(2+)-ATPase, inside-out vesicles and membranes from human erythrocytes. Incubation of the purified Ca(2+)-ATPase with glucose led to a decay in the enzyme activity of up to 50% of the control activity under the conditions used. The decrease in ATPase activity was concomitant with labelling by [6-3H]glucose of the purified Ca2+ pump; the kinetic properties of both processes were almost identical, suggesting that inhibition is a consequence of the incorporation of glucose into the Ca(2+)-ATPase molecule. In inside-out vesicles, glucose also promoted inhibition of Ca(2+)-ATPase activity as well as of active Ca2+ transport. Arabinose, xylose, mannose, ribose, fructose and glucose 6-phosphate (but not mannitol) were also able to inactive the ATPase. The activation energy for both the decrease in ATPase activity by glucose and the labelling of the pump with [6-3H]glucose was about 65 kJ/mol. Furthermore, inorganic phosphate enhanced the inactivation of the Ca(2+)-ATPase by glucose. This evidence strongly suggests that inhibition is a non-enzymically catalysed process. Inactivation of the Ca(2+)-ATPase by glucose was enhanced by reductive alkylation with sodium borohydride. Aminoguanidine, an inhibitor of the formation of the advanced end products of glycosylation, did not prevent the deleterious effect of glucose on the enzyme activity. Therefore it is concluded that inactivation of the Ca2+ pump is a consequence of the glycation of this protein. PMID:8393658

[Adenosine triphosphatase activity in the organs of the crab Hemigrapsus sanguineus, acclimated to sea water of different salinity].

PubMed

Busev, V M

1977-01-01

In crabs acclimated to low salinity, the activity of Na, K-ATPase from the gills increases; the activity also increases in the antennal glands after acclimation of the animals to high salinity. The activity of Na, K-ATPase in the abdominal ganglion and in the heart does not depend on the salinity to which crabs had been acclimated. Changes in the activity of Mg-ATPase in the gills and antennal glands associated with acclimation of crabs to sea water with different salinity correspond to those in the activity of Na, K-ATPase.

ADPase activity of recombinantly expressed thermotolerant ATPases may be caused by copurification of adenylate kinase of Escherichia coli

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chen, Baoyu; Sysoeva, Tatyana A.; Chowdhury, Saikat

2009-10-06

Except for apyrases, ATPases generally target only the {gamma}-phosphate of a nucleotide. Some non-apyrase ATPases from thermophilic microorganisms are reported to hydrolyze ADP as well as ATP, which has been described as a novel property of the ATPases from extreme thermophiles. Here, we describe an apparent ADP hydrolysis by highly purified preparations of the AAA+ ATPase NtrC1 from an extremely thermophilic bacterium, Aquifex aeolicus. This activity is actually a combination of the activities of the ATPase and contaminating adenylate kinase (AK) from Escherichia coli, which is present at 1/10 000 of the level of the ATPase. AK catalyzes conversion ofmore » two molecules of ADP into AMP and ATP, the latter being a substrate for the ATPase. We raise concern that the observed thermotolerance of E. coli AK and its copurification with thermostable proteins by commonly used methods may confound studies of enzymes that specifically catalyze hydrolysis of nucleoside diphosphates or triphosphates. For example, contamination with E. coli AK may be responsible for reported ADPase activities of the ATPase chaperonins from Pyrococcus furiosus, Pyrococcus horikoshii, Methanococcus jannaschii and Thermoplasma acidophilum; the ATP/ADP-dependent DNA ligases from Aeropyrum pernix K1 and Staphylothermus marinus; or the reported ATP-dependent activities of ADP-dependent phosphofructokinase of P. furiosus. Purification methods developed to separate NtrC1 ATPase from AK also revealed two distinct forms of the ATPase. One is tightly bound to ADP or GDP and able to bind to Q but not S ion exchange matrixes. The other is nucleotide-free and binds to both Q and S ion exchange matrixes.« less

A marked animal-vegetal polarity in the localization of Na(+),K(+) -ATPase activity and its down-regulation following progesterone-induced maturation.

PubMed

Mohanty, Basant Kumar; Gupta, Brij L

2012-02-01

The stage-VI Xenopus oocyte has a very distinct animal-vegetal polarity with structural and functional asymmetry. In this study, we show the expression and distribution pattern of Na(+),K(+) -ATPase in stage-VI oocytes, and its changes following progesterone-induced maturation. Using enzyme-specific electron microscopy phosphatase histochemistry, [(3) H]-ouabain autoradiography, and immunofluorescence cytochemistry at light microscopic level, we find that Na(+),K(+) -ATPase activity is mainly confined to the animal hemisphere. Electron microscopy histochemical results also suggest that polarized distribution of Na(+),K(+) -ATPase activity persists following progesterone-induced maturation, and it becomes gradually more polarized towards the animal pole. The time course following progesterone-induced maturation suggests that there is an initial up-regulation and then gradual down-regulation of Na(+),K(+) -ATPase activity leading to germinal vesicle breakdown (GVBD). By GVBD, the Na(+),K(+) -ATPase activity is completely down-regulated due to endocytotic removal of pump molecules from the plasma membrane into the sub-cortical region of the oocyte. This study provides the first direct evidence for a marked asymmetric localization of Na(+),K(+) -ATPase activity in any vertebrate oocyte. Here, we propose that such asymmetry in Na(+),K(+) -ATPase activity in stage-VI oocytes, and their down-regulation following progesterone-induced maturation, is likely to have a role in the active state of the germinal vesicle in stage-VI oocytes and chromosomal condensation after GVBD. Copyright © 2011 Wiley Periodicals, Inc.

Effects of plasmalemmal V-ATPase activity on plasma membrane potential of resident alveolar macrophages.

PubMed

Heming, T A; Bidani, A

2003-01-01

The acid-base status and functional responses of alveolar macrophages (mphi) are influenced by the activity of plasmalemmal V-type H+-pump (V-ATPase), an electrogenic H+ extruder that provides a possible link between intracellular pH (pHi) and plasma membrane potential (Em). This study examined the relationships among Em, pHi, and plasmalemmal V-ATPase activity in resident alveolar mphi from rabbits. Em and pHi were measured using fluorescent probes. Em was -46 mV and pHi was 7.14 at an extracellular pH (pHo) of 7.4. The pHi declined progressively at lower pHo values. Decrements in pHo, also caused depolarization of the plasma membrane, independent of V-ATPase activity. The pH effects on Em were sensitive to external K+, and hence, probably involved pH-sensitive K+ conductance. H+ were not distributed at equilibrium across the plasma membrane. V-ATPase activity was a major determinant of the transmembrane H+ disequilibrium. Pump inhibition with bafilomycin A1 caused cytosolic acidification, due most likely to the retention of metabolically generated H+. V-ATPase inhibition also caused depolarization of the plasma membrane, but the effects were mediated indirectly via the accompanying pHi changes. V-ATPase activity was sensitive to Em. Em hyperpolarization (valinomycin-clamp) reduced V-ATPase activity, causing an acidic shift in baseline pHi under steady-state conditions and slowing pHi recovery from NH4Cl prepulse acid-loads. The findings indicate that a complex relationship exists among Em, pHi, and pHo that was partially mediated by plasmalemmal V-ATPase activity. This relationship could have important consequences for the expression of pH- and/or voltage-sensitive functions in alveolar mphi.

Role of Lactobacillus plantarum MTCC1325 in membrane-bound transport ATPases system in Alzheimer’s disease-induced rat brain

PubMed Central

Mallikarjuna, Nimgampalle; Praveen, Kukkarasapalli; Yellamma, Kuna

2016-01-01

Introduction: Alzheimer’s disease (AD) is a neurodegenerative disorder, clinically characterized by memory dysfunction and progressive loss of cognition. No curative therapeutic or drug is available for the complete cure of this disease. The present study was aimed to evaluate the efficacy of Lactobacillus plantarum MTCC1325 in ATPases activity in the selected brain regions of rats induced with Alzheimer’s. Methods: For the study, 48 healthy Wistar rats were divided into four groups: group I as control group, group II as AD model (AD induced by intraperitoneal injection of D-Galactose, 120 mg/kg body weight for 6 weeks), group III as normal control rats which were orally administered only with L. plantarum MTCC1325 for 60 days, and group IV where the AD-induced rats simultaneously received oral treatment of L. plantarum MTCC1325 (10ml/kg body weight, 12×108 CFU/mL) for 60 days. The well known membrane bound transport enzymes including Na+, K+-ATPases, Ca2+-ATPases, and Mg2+-ATPases were assayed in the selected brain regions of hippocampus and cerebral cortex in all four groups of rats at selected time intervals. Results: Chronic injection of D-Galactose caused lipid peroxidation, oxidative stress, and mitochondrial dysfunction leading to the damage of neurons in the brain, finally bringing a significant decrease (-20%) in the brain total membrane bound ATPases over the controls. Contrary to this, treatment of AD-induced rats with L. plantarum MTCC1325 reverted all the constituents of ATPase enzymes to near normal levels within 30 days. Conclusion: Lactobacillus plantarum MTCC1325 exerted a beneficial action on the entire ATPases system in AD-induced rat brain by delaying neurodegeneration. PMID:28265536

Congruence between PM H+-ATPase and NADPH oxidase during root growth: a necessary probability.

PubMed

Majumdar, Arkajo; Kar, Rup Kumar

2018-07-01

Plasma membrane (PM) H + -ATPase and NADPH oxidase (NOX) are two key enzymes responsible for cell wall relaxation during elongation growth through apoplastic acidification and production of ˙OH radical via O 2 ˙ - , respectively. Our experiments revealed a putative feed-forward loop between these enzymes in growing roots of Vigna radiata (L.) Wilczek seedlings. Thus, NOX activity was found to be dependent on proton gradient generated across PM by H + -ATPase as evident from pharmacological experiments using carbonyl cyanide m-chlorophenylhydrazone (CCCP; protonophore) and sodium ortho-vanadate (PM H + -ATPase inhibitor). Conversely, H + -ATPase activity retarded in response to different ROS scavengers [CuCl 2 , N, N' -dimethylthiourea (DMTU) and catalase] and NOX inhibitors [ZnCl 2 and diphenyleneiodonium (DPI)], while H 2 O 2 promoted PM H + -ATPase activity at lower concentrations. Repressing effects of Ca +2 antagonists (La +3 and EGTA) on the activity of both the enzymes indicate its possible mediation. Since, unlike animal NOX, the plant versions do not possess proton channel activity, harmonized functioning of PM H + -ATPase and NOX appears to be justified. Plasma membrane NADPH oxidase and H + -ATPase are functionally synchronized and they work cooperatively to maintain the membrane electrical balance while mediating plant cell growth through wall relaxation.

The TF1-ATPase and ATPase activities of assembled alpha 3 beta 3 gamma, alpha 3 beta 3 gamma delta, and alpha 3 beta 3 gamma epsilon complexes are stimulated by low and inhibited by high concentrations of rhodamine 6G whereas the dye only inhibits the alpha 3 beta 3, and alpha 3 beta 3 delta complexes.

PubMed

Paik, S R; Yokoyama, K; Yoshida, M; Ohta, T; Kagawa, Y; Allison, W S

1993-12-01

The ATPase activity of the F1-ATPase from the thermophilic bacterium PS3 is stimulated at concentrations of rhodamine 6G up to about 10 microM where 70% stimulation is observed at 36 degrees C. Half maximal stimulation is observed at about 3 microM dye. At rhodamine 6G concentrations greater than 10 microM, ATPase activity declines with 50% inhibition observed at about 75 microM dye. The ATPase activities of the alpha 3 beta 3 gamma and alpha 3 beta 3 gamma delta complexes assembled from isolated subunits of TF1 expressed in E. coli deleted of the unc operon respond to increasing concentrations of rhodamine 6G nearly identically to the response of TF1. In contrast, the ATPase activities of the alpha 3 beta 3 and alpha 3 beta 3 delta complexes are only inhibited by rhodamine 6G with 50% inhibition observed, respectively, at 35 and 75 microM dye at 36 degrees C. The ATPase activity of TF1 is stimulated up to 4-fold by the neutral detergent, LDAO. In the presence of stimulating concentrations of LDAO, the ATPase activity of TF1 is no longer stimulated by rhodamine 6G, but rather, it is inhibited with 50% inhibition observed at about 30 microM dye at 30 degrees C. One interpretation of these results is that binding of rhodamine 6G to a high-affinity site on TF1 stimulates ATPase activity and unmasks a low-affinity, inhibitory site for the dye which is also exposed by LDAO.

Effects of high temperature and noise on erythrocyte membrane ATPase activity in pilots during flight.

PubMed

Qin, S Z; Yu, Q F; Ma, G X; Hao, W W; Li, M G; Zhao, H

1999-12-01

Objective. To determine the effect of heat and noise on erythrocyte membrane ATPase activities in pilots during flying. Method. Twenty-four pilots performing bombing for 3 h (45-53 degrees C, 122-97 dB in the cabin) served as the subjects. 21 ground personnel served as control (27 degrees C in the room). Blood samples were taken from both groups before flying (6:00 a.m.), and immediately (12:00 a.m.) and 8 h (8:00 p.m.) after flying. Na(+)-K+ ATPase, and Ca2(+)-Mg2+ ATPase activities in erythrocyte membrane were determined with colorimetry. Result. The Na(+)-K+ ATPase activity in erythrocyte membrane at 6:00 a.m. in pilots was higher than that in control group at the same time (P<0.01). The Ca2(+)-Mg2+ ATPase activities in erythrocyte membrane at 12:00 a.m. and 8:00 p.m. in pilots were significantly higher, compared with those in control group at the same time (P<0.01). Conclusion. The ATPase values obtained in our study were all within normal range, and the daytime variation of both groups are the same. Exposure of human body to heat and noise for long time may be harmful, the higher ATPase activity is, the more catabolism of ATP will be. ATP exhaustion will lead to Ca2+ overload in erythrocyte thus stiffen the red cell membrane.

Salinity dependent Na+-K+ATPase activity in gills of the euryhaline crab Chasmagnathus granulata.

PubMed

Schleich, C E; Goldemberg, L A; López Mañanes, A A

2001-09-01

The occurrence and response of Na+-K+ATPase specific activity to environmental salinity changes were studied in gill extracts of all of the gills of the euryhaline crab Chasmagnathus granulata from Mar Chiquita coastal lagoon (Buenos Aires Province, Argentina). All of the gills exhibited a salinity dependent Na+-K+ATPase activity, although the pattern of response to environmental salinity was different among gills. As described in other euryhaline crabs highest Na+-K+ATPase specific activity was found in posterior gills (6 to 8), which, with exception of gill 6, increased upon acclimation to reduced salinity. However, a high increase of activity also occurred in anterior gills (1 to 5) in diluted media. Furthermore, both short and long term differential changes of Na+-K+ATPase activity occurred among the gills after the transfer of crabs to reduced salinity. The fact that variations of Na+-K+ATPase activity in the gills were concomitant with the transition from osmoconformity to ionoregulation suggests that this enzyme is a component of the branchial ionoregulatory mechanisms at the biochemical level in this crab.

Na+, K+-activated-ATPase inhibition in rainbow trout: A site for organochlorine pesticide toxicity?

USGS Publications Warehouse

Davis, Paul W.; Wedemeyer, Gary A.

1971-01-01

1. The Na+, K+-activated, Mg2+-dependent-ATPase enzyme system in a heavy microsomal fraction of rainbow trout (Salmo gairdneri) brain was inhibited in vitro by chlorinated hydrocarbon pesticides.2. T50 (concentration at 50 per cent inhibition) values for dicofol, endosulfan and DDT were 5 × 10−6, 3 × 10−5 and 1 × 10−4 M respectively. Similar inhibition by these pesticides occurred in kidney and gill ATPase preparations.3. An unexpected finding was a failure of the classic inhibitor, ouabain, to block the Na+, K+-activated component of ATPase activity in the gill.4. It is suggested that inhibition of ATPase activity may be a causal factor in the toxic effects of organochlorine pesticides in fishes.

Trichoderma asperellum Induces Maize Seedling Growth by Activating the Plasma Membrane H+-ATPase.

PubMed

López-Coria, M; J L Hernández-Mendoza; Sánchez-Nieto, S

2016-10-01

Although Trichoderma spp. have beneficial effects on numerous plants, there is not enough knowledge about the mechanism by which they improves plant growth. In this study, we evaluated the participation of plasma membrane (PM) H + -ATPase, a key enzyme involved in promoting cell growth, in the elongation induced by T. asperellum and compared it with the effect of 10 μM indol acetic acid (IAA) because IAA promotes elongation and PM H + -ATPase activation. Two seed treatments were tested: biopriming and noncontact. In neither were the tissues colonized by T. asperellum; however, the seedlings were longer than the control seedlings, which also accumulated IAA and increased root acidification. An auxin transport inhibitor (2,3,5 triiodobenzoic acid) reduced the plant elongation induced by Trichoderma spp. T. asperellum seed treatment increased the PM H + -ATPase activity in plant roots and shoots. Additionally, the T. asperellum extracellular extract (TE) activated the PM H + -ATPase activity of microsomal fractions of control plants, although it contained 0.3 μM IAA. Furthermore, the mechanism of activation of PM H + -ATPase was different for IAA and TE; in the latter, the activation depends on the phosphorylation state of the enzyme, suggesting that, in addition to IAA, T. asperellum excretes other molecules that stimulate PM H + -ATPase to induce plant growth.

Activation of proteolytic enzymes and depression of the sarcolemmal Na+/K+-ATPase in ischemia-reperfused heart may be mediated through oxidative stress.

PubMed

Singh, Raja B; Hryshko, Larry; Freed, Darren; Dhalla, Naranjan S

2012-02-01

We tested whether the activation of proteolytic enzymes, calpain, and matrix metalloproteinases (MMPs) during ischemia-reperfusion (I/R) is mediated through oxidative stress. For this purpose, isolated rat hearts were subjected to a 30 min global ischemia followed by a 30 min reperfusion. Cardiac function was monitored and the activities of Na(+)/K(+)-ATPase, Mg(2+)-ATPase, calpain, and MMP were measured. Depression of cardiac function and Na(+)/K(+)-ATPase activity in I/R hearts was associated with increased calpain and MMP activities. These alterations owing to I/R were similar to those observed in hearts perfused with hypoxic medium, H(2)O(2) and xanthine plus xanthine oxidase. The I/R-induced changes were attenuated by ischemic preconditioning as well as by perfusing the hearts with N-acetylcysteine or mercaptopropionylglycine. Inhibition of MMP activity in hearts treated with doxycycline depressed the I/R-induced changes in cardiac function and Na(+)/K(+)-ATPase activity without affecting the calpain activation. On the other hand, inhibition of calpain activity upon treatment with leupeptin or MDL 28170 significantly reduced the MMP activity in addition to attenuating the I/R-induced alterations in cardiac function and Na(+)/K(+)-ATPase activity. These results suggest that the I/R-induced depression in Na(+)/K(+)-ATPase and cardiac function may be a consequence of the increased activities of both calpain and MMP because of oxidative stress in the heart.

A Loss in the Plasma Membrane ATPase Activity and Its Recovery Coincides with Incipient Freeze-Thaw Injury and Postthaw Recovery in Onion Bulb Scale Tissue 1

PubMed Central

Arora, Rajeev; Palta, Jiwan P.

1991-01-01

Plasma membrane ATPase has been proposed to be functionally altered during early stages of injury caused by a freeze-thaw stress. Complete recovery from freezing injury in onion cells during the postthaw period provided evidence in support of this proposal. During recovery, a simultaneous decrease in ion leakage and disappearance of water soaking (symptoms of freeze-thaw injury) has been noted. Since reabsorption of ions during recovery must be an active process, recovery of plasma membrane ATPase (active transport system) functions has been implicated. In the present study, onion (Allium cepa L. cv Downing Yellow Globe) bulbs were subjected to a freeze-thaw stress which resulted in a reversible (recoverable) injury. Plasma membrane ATPase activity in the microsomes (isolated from the bulb scales) and ion leakage rate (efflux/hour) from the same scale tissue were measured immediately following thawing and after complete recovery. In injured tissue (30-40% water soaking), plasma membrane ATPase activity was reduced by about 30% and this was paralleled by about 25% higher ion leakage rate. As water soaking disappeared during recovery, the plasma membrane ATPase activity and the ion leakage rate returned to about the same level as the respective controls. Treatment of freeze-thaw injured tissue with vanadate, a specific inhibitor of plasma membrane ATPase, during postthaw prevented the recovery process. These results indicate that recovery of freeze-injured tissue depends on the functional activity of plasma membrane ATPase. PMID:16668063

The AAA protein spastin possesses two levels of basal ATPase activity.

PubMed

Fan, Xiangyu; Lin, Zhijie; Fan, Guanghui; Lu, Jing; Hou, Yongfei; Habai, Gulijiazi; Sun, Linyue; Yu, Pengpeng; Shen, Yuequan; Wen, Maorong; Wang, Chunguang

2018-05-01

The AAA ATPase spastin is a microtubule-severing enzyme that plays important roles in various cellular events including axon regeneration. Herein, we found that the basal ATPase activity of spastin is negatively regulated by spastin concentration. By determining a spastin crystal structure, we demonstrate the necessity of intersubunit interactions between spastin AAA domains. Neutralization of the positive charges in the microtubule-binding domain (MTBD) of spastin dramatically decreases the ATPase activity at low concentration, although the ATP-hydrolyzing potential is not affected. These results demonstrate that, in addition to the AAA domain, the MTBD region of spastin is also involved in regulating ATPase activity, making interactions between spastin protomers more complicated than expected. © 2018 Federation of European Biochemical Societies.

Mechanical loading stimulates ecto-ATPase activity in human tendon cells.

PubMed

Tsuzaki, M; Bynum, D; Almekinders, L; Faber, J; Banes, A J

2005-09-01

Response to external stimuli such as mechanical signals is critical for normal function of cells, especially when subjected to repetitive motion. Tenocytes receive mechanical stimuli from the load-bearing matrix as tension, compression, and shear stress during tendon gliding. Overloading a tendon by high strain, shear, or repetitive motion can cause matrix damage. Injury may induce cytokine expression, matrix metalloproteinase (MMP) expression and activation resulting in loss of biomechanical properties. These changes may result in tendinosis or tendinopathy. Alternatively, an immediate effector molecule may exist that acts in a signal-dampening pathway. Adenosine 5'-triphosphate (ATP) is a candidate signal blocker of mechanical stimuli. ATP suppresses load-inducible inflammatory genes in human tendon cells in vitro. ATP and other extracellular nucleotide signaling are regulated efficiently by two distinct mechanisms: purinoceptors via specific receptor-ligand binding and ecto-nucleotidases via the hydrolysis of specific nucleotide substrates. ATP is released from tendon cells by mechanical loading or by uridine 5'-triphosphate (UTP) stimulation. We hypothesized that mechanical loading might stimulate ecto-ATPase activity. Human tendon cells of surface epitenon (TSC) and internal compartment (TIF) were cyclically stretched (1 Hz, 0.035 strain, 2 h) with or without ATP. Aliquots of the supernatant fluids were collected at various time points, and ATP concentration (ATP) was determined by a luciferin-luciferase bioluminescence assay. Total RNA was isolated from TSC and TIF (three patients) and mRNA expression for ecto-nucleotidase was analyzed by RT-PCR. Human tendon cells secreted ATP in vitro (0.5-1 nM). Exogenous ATP was hydrolyzed within minutes. Mechanical load stimulated ATPase activity. ATP was hydrolyzed in mechanically loaded cultures at a significantly greater rate compared to no load controls. Tenocytes (TSC and TIF) expressed ecto-nucleotidase mRNA (ENTPD3 and ENPP1, ENPP2). These data suggest that motion may release ATP from tendon cells in vivo, where ecto-ATPase may also be activated to hydrolyze ATP quickly. Ecto-ATPase may act as a co-modulator in ATP load-signal modulation by regulating the half-life of extracellular purine nucleotides. The extracellular ATP/ATPase system may be important for tendon homeostasis by protecting tendon cells from responding to excessive load signals and activating injurious pathways. Copyright 2005 Wiley-Liss, Inc

Na, K-ATPase activity regulates AMPA receptor turnover through proteasome-mediated proteolysis

PubMed Central

Zhang, Dawei; Hou, Qingming; Wang, Min; Lin, Amy; Jarzylo, Larissa; Navis, Allison; Raissi, Aram; Liu, Fang; Man, Heng-Ye

2009-01-01

Neuronal activity largely depends on two key components on the membrane: the Na, K-ATPase (NKA) that maintains the ion gradients and sets the foundation of excitability, and the ionotropic glutamatergic AMPA receptors (AMPARs) through which sodium influx forms the driving force for excitation. Because the frequent sodium transients from glutamate receptor activity need to be efficiently extruded, a functional coupling between NKA and AMPARs should be a necessary cellular device for synapse physiology. We show that NKA is enriched at synapses and associates with AMPARs. NKA dysfunction induces a rapid reduction in AMPAR cell-surface expression as well as total protein abundance, leading to a long-lasting depression in synaptic transmission. AMPAR proteolysis requires sodium influx, proteasomal activity and receptor internalization. These data elucidate a novel mechanism by which NKA regulates AMPAR turnover and thereby synaptic strength and brain function. PMID:19357275

Mutant Analysis Reveals Allosteric Regulation of ClpB Disaggregase

PubMed Central

Franke, Kamila B.; Bukau, Bernd; Mogk, Axel

2017-01-01

The members of the hexameric AAA+ disaggregase of E. coli and S. cerevisiae, ClpB, and Hsp104, cooperate with the Hsp70 chaperone system in the solubilization of aggregated proteins. Aggregate solubilization relies on a substrate threading activity of ClpB/Hsp104 fueled by ATP hydrolysis in both ATPase rings (AAA-1, AAA-2). ClpB/Hsp104 ATPase activity is controlled by the M-domains, which associate to the AAA-1 ring to downregulate ATP hydrolysis. Keeping M-domains displaced from the AAA-1 ring by association with Hsp70 increases ATPase activity due to enhanced communication between protomers. This communication involves conserved arginine fingers. The control of ClpB/Hsp104 activity is crucial, as hyperactive mutants with permanently dissociated M-domains exhibit cellular toxicity. Here, we analyzed AAA-1 inter-ring communication in relation to the M-domain mediated ATPase regulation, by subjecting a conserved residue of the AAA-1 domain subunit interface of ClpB (A328) to mutational analysis. While all A328X mutants have reduced disaggregation activities, their ATPase activities strongly differed. ClpB-A328I/L mutants have reduced ATPase activity and when combined with the hyperactive ClpB-K476C M-domain mutation, suppress cellular toxicity. This underlines that ClpB ATPase activation by M-domain dissociation relies on increased subunit communication. The ClpB-A328V mutant in contrast has very high ATPase activity and exhibits cellular toxicity on its own, qualifying it as novel hyperactive ClpB mutant. ClpB-A328V hyperactivity is however, different from that of M-domain mutants as M-domains stay associated with the AAA-1 ring. The high ATPase activity of ClpB-A328V primarily relies on the AAA-2 ring and correlates with distinct conformational changes in the AAA-2 catalytic site. These findings characterize the subunit interface residue A328 as crucial regulatory element to control ATP hydrolysis in both AAA rings. PMID:28275610

Tetrahydrocarbazoles are a novel class of potent P-type ATPase inhibitors with antifungal activity

PubMed Central

Bublitz, Maike; Kjellerup, Lasse; Cohrt, Karen O’Hanlon; Gordon, Sandra; Mortensen, Anne Louise; Clausen, Johannes D.; Pallin, Thomas David; Hansen, John Bondo; Fuglsang, Anja Thoe; Dalby-Brown, William

2018-01-01

We have identified a series of tetrahydrocarbazoles as novel P-type ATPase inhibitors. Using a set of rationally designed analogues, we have analyzed their structure-activity relationship using functional assays, crystallographic data and computational modeling. We found that tetrahydrocarbazoles inhibit adenosine triphosphate (ATP) hydrolysis of the fungal H+-ATPase, depolarize the fungal plasma membrane and exhibit broad-spectrum antifungal activity. Comparative inhibition studies indicate that many tetrahydrocarbazoles also inhibit the mammalian Ca2+-ATPase (SERCA) and Na+,K+-ATPase with an even higher potency than Pma1. We have located the binding site for this compound class by crystallographic structure determination of a SERCA-tetrahydrocarbazole complex to 3.0 Å resolution, finding that the compound binds to a region above the ion inlet channel of the ATPase. A homology model of the Candida albicans H+-ATPase based on this crystal structure, indicates that the compounds could bind to the same pocket and identifies pocket extensions that could be exploited for selectivity enhancement. The results of this study will aid further optimization towards selective H+-ATPase inhibitors as a new class of antifungal agents. PMID:29293507

Effects of obesity and estradiol on Na+/K+-ATPase and their relevance to cardiovascular diseases.

PubMed

Obradovic, Milan; Bjelogrlic, Predrag; Rizzo, Manfredi; Katsiki, Niki; Haidara, Mohamed; Stewart, Alan J; Jovanovic, Aleksandra; Isenovic, Esma R

2013-09-01

Obesity is associated with aberrant sodium/potassium-ATPase (Na(+)/K(+)-ATPase) activity, apparently linked to hyperglycemic hyperinsulinemia, which may repress or inactivate the enzyme. The reduction of Na(+)/K(+)-ATPase activity in cardiac tissue induces myocyte death and cardiac dysfunction, leading to the development of myocardial dilation in animal models; this has also been documented in patients with heart failure (HF). During several pathological situations (cardiac insufficiency and HF) and in experimental models (obesity), the heart becomes more sensitive to the effect of cardiac glycosides, due to a decrease in Na(+)/K(+)-ATPase levels. The primary female sex steroid estradiol has long been recognized to be important in a wide variety of physiological processes. Numerous studies, including ours, have shown that estradiol is one of the major factors controlling the activity and expression of Na(+)/K(+)-ATPase in the cardiovascular (CV) system. However, the effects of estradiol on Na(+)/K(+)-ATPase in both normal and pathological conditions, such as obesity, remain unclear. Increasing our understanding of the molecular mechanisms by which estradiol mediates its effects on Na(+)/K(+)-ATPase function may help to develop new strategies for the treatment of CV diseases. Herein, we discuss the latest data from animal and clinical studies that have examined how pathophysiological conditions such as obesity and the action of estradiol regulate Na(+)/K(+)-ATPase activity.

Myofibril ATPase activity of cardiac and skeletal muscle of exhaustively exercised rats.

PubMed

Belcastro, A N; Turcotte, R; Rossiter, M; Secord, D; Maybank, P E

1984-01-01

The activation characteristics of Mg-ATP and Ca2+ on cardiac and skeletal muscle myofibril ATPase activity were studied in rats following a run to exhaustion. In addition, the effect of varying ionic strength was determined on skeletal muscle from exhausted animals. The exhausted group (E) ran at a speed of 25 m min-1 with an 8% incline. Myofibril ATPase activities for control (C) and E were determined with 1, 3 and 5 mM Mg-ATP and 1 and 10 microM Ca2+ at pH 7.0 and 30 degrees C. For control skeletal muscle, at 1 and 10 microM Ca2+, there was an increase in ATPase activity from 1 to 5 mM Mg-ATP (P less than 0.05). For E animals the myofibril ATPase activities at 10 microM Ca2+ and all Mg-ATP concentrations were similar to C (P greater than 0.05). At 1.0 microM Ca2+ and all Mg-ATP concentrations were similar to C (P greater than 0.05). At 1.0 microM Ca2+ the activities at 3 and 5 mM Mg-ATP were greater for the E animals (P less than 0.05). Increasing KCl concentrations resulted in greater inhibition for E animals. With cardiac muscle, the myofibril ATPase activities at 1.0 microM free Ca2+ were lower for E at all Mg-ATP levels (P less than 0.05). In contrast, at 10 microM Ca2+, the E group exhibited an elevated myofibril ATPase activity. The results indicate that Mg-ATP and Ca2+ activation of cardiac and skeletal muscle myofibril ATPase is altered with exhaustive exercise.

Ginsenoside Rb1 improves energy metabolism in the skeletal muscle of an animal model of postoperative fatigue syndrome.

PubMed

Tan, Shan-Jun; Li, Ning; Zhou, Feng; Dong, Qian-Tong; Zhang, Xiao-Dong; Chen, Bi-Cheng; Yu, Zhen

2014-10-01

Postoperative fatigue syndrome (POFS) is a common clinical complication followed by almost every major abdominal surgery. Ginsenoside Rb1 (GRb1), a principle ginsenoside in ginseng, could exert a potent anti-fatigue effect on POFS. However, the mechanism is still unknown. Previous studies revealed that alterations in the energy metabolism in the skeletal muscle may play a vital role in the development and progression of fatigue. In the present study, we investigate the effect of GRb1 on energy metabolism in the skeletal muscle of a rat model of POFS induced by major small intestinal resection. GRb1 (10 mg/kg) was intraperitoneally administrated once daily for 1, 3, 7, and 10 d from the operation day, respectively. The locomotor activity was recorded every day, and total food intake was calculated starting from 24 h after surgery. After GRb1 treatment was completed, blood and skeletal muscle were sampled. The level of blood glucose was determined by an automatic biochemical analyzer. The content of adenosine triphosphate (ATP) in skeletal muscle was determined by high-performance liquid chromatography. The activity of energy metabolic enzymes Na(+)-K(+)-ATPase, pyruvate kinase, and succinate dehydrogenase (SDH) was assessed by commercially available kits. The results revealed that GRb1 could increase locomotor activity of POFS rats and significantly increase their total food intake postoperatively (P < 0.05). Furthermore, GRb1 also significantly increased ATP content in the skeletal muscle of POFS rats (P < 0.05). Meanwhile, the activity of Na(+)-K(+)-ATPase and SDH in the skeletal muscle of POFS rats was enhanced by GRb1 (P < 0.05). However, no significant differences in blood glucose and pyruvate kinase were found between the POFS and GRb1 treatment rats (P > 0.05). These results suggest that GRb1 may improve skeletal muscle energy metabolism in POFS, and the underlying mechanism may be associated with an increase in the content of ATP and an enhancement in the activity of energy metabolic enzymes such as Na(+)-K(+)-ATPase ATPase and SDH in the skeletal muscle. Copyright © 2014 Elsevier Inc. All rights reserved.

Disruption of the vacuolar-type H+-ATPase complex in liver causes MTORC1-independent accumulation of autophagic vacuoles and lysosomes.

PubMed

Kissing, Sandra; Rudnik, Sönke; Damme, Markus; Lüllmann-Rauch, Renate; Ichihara, Atsuhiro; Kornak, Uwe; Eskelinen, Eeva-Liisa; Jabs, Sabrina; Heeren, Jörg; De Brabander, Jef K; Haas, Albert; Saftig, Paul

2017-04-03

The vacuolar-type H + -translocating ATPase (v-H + -ATPase) has been implicated in the amino acid-dependent activation of the mechanistic target of rapamycin complex 1 (MTORC1), an important regulator of macroautophagy. To reveal the mechanistic links between the v-H + -ATPase and MTORC1, we destablilized v-H + -ATPase complexes in mouse liver cells by induced deletion of the essential chaperone ATP6AP2. ATP6AP2-mutants are characterized by massive accumulation of endocytic and autophagic vacuoles in hepatocytes. This cellular phenotype was not caused by a block in endocytic maturation or an impaired acidification. However, the degradation of LC3-II in the knockout hepatocytes appeared to be reduced. When v-H + -ATPase levels were decreased, we observed lysosome association of MTOR and normal signaling of MTORC1 despite an increase in autophagic marker proteins. To better understand why MTORC1 can be active when v-H + -ATPase is depleted, the activation of MTORC1 was analyzed in ATP6AP2-deficient fibroblasts. In these cells, very little amino acid-elicited activation of MTORC1 was observed. In contrast, insulin did induce MTORC1 activation, which still required intracellular amino acid stores. These results suggest that in vivo the regulation of macroautophagy depends not only on v-H + -ATPase-mediated regulation of MTORC1.

Protective Effect of Eburicoic Acid of the Chicken of the Woods Mushroom, Laetiporus sulphureus (Higher Basidiomycetes), Against Gastric Ulcers in Mice.

PubMed

Wang, Junzhi; Sun, Wenjun; Luo, Huajun; He, Haibo; Deng, Weiqiao; Zou, Kun; Liu, Can; Song, Jing; Huang, Wenfeng

2015-01-01

In this study, we investigated the anti-inflammatory and tumor-inhibiting effects of eburicoic acid, the main bioactive component in the Laetiporus sulphureus, on gastric ulcers. A total of 48 Kunming mice were randomly divided into six groups: control, model, OL (omeprazole, 20 mg/kg/day, orally), EA-L (eburicoic acid, 10 mg/kg/day, orally), EA-M (eburicoic acid, 20 mg/kg/day, orally), and EA-H (eburicoic acid, 40 mg/kg/day, orally). Gastric ulcers were induced in mice by administering 80% ethanol containing 15 mg/mL aspirin (10.0 mL/kg, i.g.) 4 hours after drug administration on day 5. The ulcer index and H+/K+-ATPase activity were evaluated in vivo. Computer-aided molecular docking simulated the interaction between eburicoic acid and H+/K+-ATPase. The results showed that the oral administration of eburicoic acid protected the gastric mucosa from gastric lesions morphologically and especially attenuated H+/K+-ATPase activity. The results of this study indicate that the gastric protective effect of eburicoic acid might inhibit gastric acid.

Molecular basis for the binding and modulation of V-ATPase by a bacterial effector protein

PubMed Central

Alvarez, Claudia P.; Bueler, Stephanie A.; Xu, Caishuang; Boniecki, Michal T.; Kanelis, Voula; Rubinstein, John L.

2017-01-01

Intracellular pathogenic bacteria evade the immune response by replicating within host cells. Legionella pneumophila, the causative agent of Legionnaires’ Disease, makes use of numerous effector proteins to construct a niche supportive of its replication within phagocytic cells. The L. pneumophila effector SidK was identified in a screen for proteins that reduce the activity of the proton pumping vacuolar-type ATPases (V-ATPases) when expressed in the yeast Saccharomyces cerevisae. SidK is secreted by L. pneumophila in the early stages of infection and by binding to and inhibiting the V-ATPase, SidK reduces phagosomal acidification and promotes survival of the bacterium inside macrophages. We determined crystal structures of the N-terminal region of SidK at 2.3 Å resolution and used single particle electron cryomicroscopy (cryo-EM) to determine structures of V-ATPase:SidK complexes at ~6.8 Å resolution. SidK is a flexible and elongated protein composed of an α-helical region that interacts with subunit A of the V-ATPase and a second region of unknown function that is flexibly-tethered to the first. SidK binds V-ATPase strongly by interacting via two α-helical bundles at its N terminus with subunit A. In vitro activity assays show that SidK does not inhibit the V-ATPase completely, but reduces its activity by ~40%, consistent with the partial V-ATPase deficiency phenotype its expression causes in yeast. The cryo-EM analysis shows that SidK reduces the flexibility of the A-subunit that is in the ‘open’ conformation. Fluorescence experiments indicate that SidK binding decreases the affinity of V-ATPase for a fluorescent analogue of ATP. Together, these results reveal the structural basis for the fine-tuning of V-ATPase activity by SidK. PMID:28570695

[Role of mesenteric lymph drainage improving the metabolism of red blood cell in hemorrhagic shock rats following fluid resuscitation].

PubMed

Han, Rui; Du, Hui-bo; Lu, Bei; Si, Yong-hua; Zhang, Li-min; Zhang, Yu-pin; Zhao, Zi-gang; Niu, Chun-yu

2012-08-01

To observe the effects of mesenteric lymph drainage on the metabolism of red blood cell (RBC) in hemorrhagic shock (HS) rats following fluid resuscitation. Eighteen male Wistar rats were randomly divided into sham group (n=6), HS group (n=6), HS + drainage group (n=6). After 1.5 hours of HS model prepared, the animals were given fluid resuscitation by lost blood plus equal volume of Ringer solution within 30 minutes in HS and HS + drainage groups, and mesenteric lymph drainage was performed after 1 hour of hypotension in HS + drainage group. At 3 hours after resuscitation or corresponding time, blood samples were obtained from abdominal aorta. Membrane suspensions of RBC prepared from part of whole blood samples were used to measure the activities of adenosine triphosphate ase (ATPase) and contents of ATP and lactic acid (LA), the intracellular fluid of RBC prepared from part of whole blood samples was used to determine the concentration of 2,3-diphosphoglyceric acid (2,3-DPG), Na(+) and K(+), plasma samples isolated from blood by centrifugation were used to determine the concentration of Na(+), K(+), Cl(-) and total Ca. Compared with sham group, the content of ATP (μmol/g), activity of Na(+)-K(+)-ATPase (μmol×mg(-1)×h(-1)) and Ca(2+)-ATPase (μmol×mg(-1)×h(-1)) in RBC membrane and total Ca (mmol/L) in plasma were decreased markedly (ATP: 6.698±0.938 vs. 10.670±1.466, Na(+)-K(+)-ATPase: 0.042±0.010 vs. 0.066±0.019, Ca(2+)-ATPase: 0.054±0.015 vs. 0.081±0.017, total Ca: 2.27±0.18 vs. 2.66±0.21, P<0.05 or P<0.01) in HS group, and the content of LA (mmol/g) in RBC and K(+) (mmol/L), Cl(-) (mmol/L) in plasma were increased observably in HS group (LA: 3.472±0.853 vs. 1.743±0.395, K(+): 5.83±0.34 vs. 5.23±0.37, Cl(-): 113.37±3.63 vs. 106.35±4.99, P<0.05 or P<0.01), there was no significant difference in term of the contents of 2,3-DPG (mmol/L: 2.196±0.609 vs. 2.590±0.574, P>0.05). Compared with HS group, the contents of 2,3-DPG (4.459±0.900) and ATP (8.859±1.189), the activities of Na(+)-K(+)-ATPase (0.089±0.022), Ca(2+)-ATPase (0.082±0.020) of RBC were increased in HS + drainage group, and the level of LA (2.060±0.810) was decreased observably (P<0.05 or P<0.01), there were no significant differences in the other indices. The results indicate that shock mesenteric lymph drainage plays an important role in improving the metabolism of RBC in HS rats following fluid resuscitation, subsequently, may preserve the structure and function of RBC.

Methylphenidate treatment increases Na(+), K (+)-ATPase activity in the cerebrum of young and adult rats.

PubMed

Scherer, Emilene B S; Matté, Cristiane; Ferreira, Andréa G K; Gomes, Karin M; Comim, Clarissa M; Mattos, Cristiane; Quevedo, João; Streck, Emilio L; Wyse, Angela T S

2009-12-01

Methylphenidate is a central nervous system stimulant used for the treatment of attention-deficit hyperactivity disorder. Na(+), K(+)-ATPase is a membrane-bound enzyme necessary to maintain neuronal excitability. Considering that methylphenidate effects on central nervous system metabolism are poorly known and that Na(+), K(+)-ATPase is essential to normal brain function, the purpose of this study was to evaluate the effect of this drug on Na(+), K(+)-ATPase activity in the cerebrum of young and adult rats. For acute administration, a single injection of methylphenidate (1.0, 2.0, or 10.0 mg/Kg) or saline was given to rats on postnatal day 25 or postnatal day 60, in the young and adult groups, respectively. For chronic administration, methylphenidate (1.0, 2.0, or 10.0 mg/Kg) or saline injections were given to young rats starting at postnatal day 25 once daily for 28 days. In adult rats, the same regimen was performed starting at postnatal day 60. Our results showed that acute methylphenidate administration increased Na(+), K(+)-ATPase activity in hippocampus, prefrontal cortex, and striatum of young and adult rats. In young rats, chronic administration of methylphenidate also enhanced Na(+), K(+)-ATPase activity in hippocampus and prefrontal cortex, but not in striatum. When tested in adult rats, Na(+), K(+)-ATPase activity was increased in all cerebral structures studied. The present findings suggest that increased Na(+), K(+)-ATPase activity may be associated with neuronal excitability caused by methylphenidate.

Chromium-induced membrane damage: protective role of ascorbic acid.

PubMed

Dey, S K; Nayak, P; Roy, S

2001-07-01

Importance of chromium as environmental toxicant is largely due to impact on the body to produce cellular toxicity. The impact of chromium and their supplementation with ascorbic acid was studied on plasma membrane of liver and kidney in male Wistar rats (80-100 g body weight). It has been observed that the intoxication with chromium (i.p.) at the dose of 0.8 mg/100 g body weight per day for a period of 28 days causes significant increase in the level of cholesterol and decrease in the level of phospholipid of both liver and kidney. The alkaline phosphatase, total ATPase and Na(+)-K(+)-ATPase activities were significantly decreased in both liver and kidney after chromium treatment, except total ATPase activity of kidney. It is suggested that chromium exposure at the present dose and duration induce for the alterations of structure and function of both liver and kidney plasma membrane. Ascorbic acid (i.p. at the dose of 0.5 mg/100 g body weight per day for period of 28 days) supplementation can reduce these structural changes in the plasma membrane of liver and kidney. But the functional changes can not be completely replenished by the ascorbic acid supplementation in response to chromium exposure. So it is also suggested that ascorbic acid (nutritional antioxidant) is useful free radical scavenger to restrain the chromium-induced membrane damage.

A bioassay-guided fractionation system to identify endogenous small molecules that activate plasma membrane H+-ATPase activity in Arabidopsis.

PubMed

Han, Xiuli; Yang, Yongqing; Wu, Yujiao; Liu, Xiaohui; Lei, Xiaoguang; Guo, Yan

2017-05-17

Plasma membrane (PM) H+-ATPase is essential for plant growth and development. Various environmental stimuli regulate its activity, a process that involves many protein cofactors. However, whether endogenous small molecules play a role in this regulation remains unknown. Here, we describe a bio-guided isolation method to identify endogenous small molecules that regulate PM H+-ATPase activity. We obtained crude extracts from Arabidopsis seedlings with or without salt treatment and then purified them into fractions based on polarity and molecular mass by repeated column chromatography. By evaluating the effect of each fraction on PM H+-ATPase activity, we found that fractions containing the endogenous, free unsaturated fatty acids oleic acid (C18:1), linoleic acid (C18:2), and linolenic acid (C18:3) extracted from salt-treated seedlings stimulate PM H+-ATPase activity. These results were further confirmed by the addition of exogenous C18:1, C18:2, or C18:3 in the activity assay. The ssi2 mutant, with reduced levels of C18:1, C18:2, and C18:3, displayed reduced PM H+-ATPase activity. Furthermore, C18:1, C18:2, and C18:3 directly bound to the C-terminus of the PM H+-ATPase AHA2. Collectively, our results demonstrate that the binding of free unsaturated fatty acids to the C-terminus of PM H+-ATPase is required for its activation under salt stress. The bio-guided isolation model described in this study could enable the identification of new endogenous small molecules that modulate essential protein functions, as well as signal transduction, in plants. © The Author 2017. Published by Oxford University Press on behalf of the Society for Experimental Biology.

A phytochemical study of the Cuphea glutinosa from Southern Brazil: Na+,K+-ATPase activity inhibition and antioxidant properties.

PubMed

Zago, Adriana M; Carvalho, Fabiano B; Gutierres, Jessié Martins; Bohnert, Crystiani; Fernandes, Marilda da Cruz; Morandini, Liziane M; Coelho, Helena S; Fogaça, Aline O; Andrade, Cinthia M; Mostardeiro, Marco A; Dalcol, Ionara I; Morel, Ademir F

2018-05-21

This study investigated the antioxidant activity of Cuphea glutinosa (CG) and its effect on Na + , K + -ATPase from cardiac muscle. The ethanolic extract showed higher antioxidant capacity compared to aqueous and ethyl acetate fraction. Ethyl acetate fraction showed β-sitosterol-3-O-β-glucoside, kaempferol, quercetin, isoquercetin, gallic acid methyl ester, and gallic acid. The ethanolic extract also reduced the Na + ,K + -ATPase activity. CG presented a promising antioxidant activity and inhibitory effect on the Na + , K + -ATPase activity, supporting biochemical evidences the popular use of this plant in the treatment of heart failure.

Glucose-independent inhibition of yeast plasma-membrane H+-ATPase by calmodulin antagonists.

PubMed

Romero, I; Maldonado, A M; Eraso, P

1997-03-15

Glucose metabolism causes activation of the yeast plasma-membrane H+-ATPase. The molecular mechanism of this regulation is not known, but it is probably mediated by phosphorylation of the enzyme. The involvement in this process of several kinases has been suggested but their actual role has not been proved. The physiological role of a calmodulin-dependent protein kinase in glucose-induced activation was investigated by studying the effect of specific calmodulin antagonists on the glucose-induced ATPase kinetic changes in wild-type and two mutant strains affected in the glucose regulation of the enzyme. Preincubation of the cells with calmidazolium or compound 48/80 impeded the increase in ATPase activity by reducing the Vmax of the enzyme without modifying the apparent affinity for ATP in the three strains. In one mutant, pma1-T912A, the putative calmodulin-dependent protein kinase-phosphorylatable Thr-912 was eliminated, and in the other, pma1-P536L, H+-ATPase was constitutively activated, suggesting that the antagonistic effect was not mediated by a calmodulin-dependent protein kinase and not related to glucose regulation. This was corroborated when the in vitro effect of the calmodulin antagonists on H+-ATPase activity was tested. Purified plasma membranes from glucose-starved or glucose-fermenting cells from both pma1-P890X, another constitutively activated ATPase mutant, and wild-type strains were preincubated with calmidazolium or melittin. In all cases, ATP hydrolysis was inhibited with an IC50 of approximately 1 microM. This inhibition was reversed by calmodulin. Analysis of the calmodulin-binding protein pattern in the plasma-membrane fraction eliminates ATPase as the calmodulin target protein. We conclude that H+-ATPase inhibition by calmodulin antagonists is mediated by an as yet unidentified calmodulin-dependent membrane protein.

Phospholemman is not required for the acute stimulation of Na⁺-K⁺-ATPase α₂-activity during skeletal muscle fatigue.

PubMed

Manoharan, Palanikumar; Radzyukevich, Tatiana L; Hakim Javadi, Hesamedin; Stiner, Cory A; Landero Figueroa, Julio A; Lingrel, Jerry B; Heiny, Judith A

2015-12-15

The Na(+)-K(+)-ATPase α2-isoform in skeletal muscle is rapidly stimulated during muscle use and plays a critical role in fatigue resistance. The acute mechanisms that stimulate α2-activity are not completely known. This study examines whether phosphorylation of phospholemman (PLM/FXYD1), a regulatory subunit of Na(+)-K(+)-ATPase, plays a role in the acute stimulation of α2 in working muscles. Mice lacking PLM (PLM KO) have a normal content of the α2-subunit and show normal exercise capacity, in contrast to the greatly reduced exercise capacity of mice that lack α2 in the skeletal muscles. Nerve-evoked contractions in vivo did not induce a change in total PLM or PLM phosphorylated at Ser63 or Ser68, in either WT or PLM KO. Isolated muscles of PLM KO mice maintain contraction and resist fatigue as well as wild type (WT). Rb(+) transport by the α2-Na(+)-K(+)-ATPase is stimulated to the same extent in contracting WT and contracting PLM KO muscles. Phosphorylation of sarcolemmal membranes prepared from WT but not PLM KO skeletal muscles stimulates the activity of both α1 and α2 in a PLM-dependent manner. The stimulation occurs by an increase in Na(+) affinity without significant change in Vmax and is more effective for α1 than α2. These results demonstrate that phosphorylation of PLM is capable of stimulating the activity of both isozymes in skeletal muscle; however, contractile activity alone is not sufficient to induce PLM phosphorylation. Importantly, acute stimulation of α2, sufficient to support exercise and oppose fatigue, does not require PLM or its phosphorylation. Copyright © 2015 the American Physiological Society.

Photosynthesis Activates Plasma Membrane H+-ATPase via Sugar Accumulation.

PubMed

Okumura, Masaki; Inoue, Shin-Ichiro; Kuwata, Keiko; Kinoshita, Toshinori

2016-05-01

Plant plasma membrane H(+)-ATPase acts as a primary transporter via proton pumping and regulates diverse physiological responses by controlling secondary solute transport, pH homeostasis, and membrane potential. Phosphorylation of the penultimate threonine and the subsequent binding of 14-3-3 proteins in the carboxyl terminus of the enzyme are required for H(+)-ATPase activation. We showed previously that photosynthesis induces phosphorylation of the penultimate threonine in the nonvascular bryophyte Marchantia polymorpha However, (1) whether this response is conserved in vascular plants and (2) the process by which photosynthesis regulates H(+)-ATPase phosphorylation at the plasma membrane remain unresolved issues. Here, we report that photosynthesis induced the phosphorylation and activation of H(+)-ATPase in Arabidopsis (Arabidopsis thaliana) leaves via sugar accumulation. Light reversibly phosphorylated leaf H(+)-ATPase, and this process was inhibited by pharmacological and genetic suppression of photosynthesis. Immunohistochemical and biochemical analyses indicated that light-induced phosphorylation of H(+)-ATPase occurred autonomously in mesophyll cells. We also show that the phosphorylation status of H(+)-ATPase and photosynthetic sugar accumulation in leaves were positively correlated and that sugar treatment promoted phosphorylation. Furthermore, light-induced phosphorylation of H(+)-ATPase was strongly suppressed in a double mutant defective in ADP-glucose pyrophosphorylase and triose phosphate/phosphate translocator (adg1-1 tpt-2); these mutations strongly inhibited endogenous sugar accumulation. Overall, we show that photosynthesis activated H(+)-ATPase via sugar production in the mesophyll cells of vascular plants. Our work provides new insight into signaling from chloroplasts to the plasma membrane ion transport mechanism. © 2016 American Society of Plant Biologists. All Rights Reserved.

Comparative effects of chlordecone and mirex on rat cardiac ATPases and binding of 3H-catecholamines.

PubMed

Desaiah, D

1980-08-01

The effects of chlordecone and mirex on the rat myocardial ATPases and binding of 3H-dopamine and 3H-norepinephrine to the NAK-fraction were determined both by in vitro and in vivo treatment. The in vitro data showed that chlordecone significantly inhibited mitochondrial Mg2+ ATPase and Na+--K+ ATPase in a concentration dependent manner with ID50 values of 5 x 10(-8) and 2 x 10(-6) M, respectively. Mitrex, a close structural analog of chlordecone did not inhibit mitochondrial Mg2+ ATPase but inhibited about 15% of N+--K+ ATPase activity. Rats treated with symptomatogenic doses of chlordecone showed a marked and significant decrease of myocardial Na+--K+ ATPase and the residual Mg2+ ATPase activities. The decrease in the enzyme activities was dose dependent and significant. However, mirex treated rats showed a slight decrease in the myocardial Na+--K+ ATPase. The potency of chlordecone to inhibit the ATPase system was parallel to its ability to decrease the dopamine and norepinephrine binding of the myocardial NAK-fraction. Preincubation of the NAK-fraction with various concentrations of chlordecone resulted in a decreased binding of dopamine and norepinephrine. The decrease was significant and concentration dependent. Similar findings were observed in rats pretreated with chlordecone. Mirex did not show any effect, either in vitro or in vivo treatment, on the binding of dopamine or norepinephrine to the myocardial NAK-fraction. These results suggest that chlordecone may be altering the sodium pump activity by inhibiting both ATP hydrolysis and ATP synthesis and thus reducing other cellular events such as catecholamine uptake.

Photosynthesis Activates Plasma Membrane H+-ATPase via Sugar Accumulation1[OPEN

PubMed Central

Okumura, Masaki; Inoue, Shin-ichiro; Kuwata, Keiko

2016-01-01

Plant plasma membrane H+-ATPase acts as a primary transporter via proton pumping and regulates diverse physiological responses by controlling secondary solute transport, pH homeostasis, and membrane potential. Phosphorylation of the penultimate threonine and the subsequent binding of 14-3-3 proteins in the carboxyl terminus of the enzyme are required for H+-ATPase activation. We showed previously that photosynthesis induces phosphorylation of the penultimate threonine in the nonvascular bryophyte Marchantia polymorpha. However, (1) whether this response is conserved in vascular plants and (2) the process by which photosynthesis regulates H+-ATPase phosphorylation at the plasma membrane remain unresolved issues. Here, we report that photosynthesis induced the phosphorylation and activation of H+-ATPase in Arabidopsis (Arabidopsis thaliana) leaves via sugar accumulation. Light reversibly phosphorylated leaf H+-ATPase, and this process was inhibited by pharmacological and genetic suppression of photosynthesis. Immunohistochemical and biochemical analyses indicated that light-induced phosphorylation of H+-ATPase occurred autonomously in mesophyll cells. We also show that the phosphorylation status of H+-ATPase and photosynthetic sugar accumulation in leaves were positively correlated and that sugar treatment promoted phosphorylation. Furthermore, light-induced phosphorylation of H+-ATPase was strongly suppressed in a double mutant defective in ADP-glucose pyrophosphorylase and triose phosphate/phosphate translocator (adg1-1 tpt-2); these mutations strongly inhibited endogenous sugar accumulation. Overall, we show that photosynthesis activated H+-ATPase via sugar production in the mesophyll cells of vascular plants. Our work provides new insight into signaling from chloroplasts to the plasma membrane ion transport mechanism. PMID:27016447

Sodium plus Potassium-Activated, Ouabain-Inhibited AdenosineTriphosphatase from a Fraction of Rat Skeletal Muscle, and Lack of Insulin Effect on It

PubMed Central

Rogus, Ellen; Price, Thomas; Zierler, Kenneth L.

1969-01-01

An ATPase, activated by Na+ plus K+ in the presence of Mg++ and inhibited by ouabain, has been obtained from rat skeletal muscle. Unlike ATPase's with similar properties obtained from other preparations, this ATPase was found only in the fraction containing fragmented sarcoplasmic reticulum. It is suggested that in rat skeletal muscle this ATPase may reside in sarcoplasmic reticulum and not in sarcolemma. This ATPase differed in its pH optimum and in its cation sensitivity from that of rat brain and from that of human muscle reported by Samaha and Gergely (1965, 1966). Because insulin accelerates Na+ efflux from muscle, efforts were made to determine whether or not this effect of insulin could be attributed to increased Na+ + K+-activated ATPase activity. Insulin, administered either in vivo or in vitro, had no demonstrable effect on the enzyme system, nor did it protect against inhibition by ouabain. PMID:4240329

Neurotensin effect on Na+, K+-ATPase is CNS area- and membrane-dependent and involves high affinity NT1 receptor.

PubMed

López Ordieres, María Graciela; Rodríguez de Lores Arnaiz, Georgina

2002-11-01

We have previously shown that peptide neurotensin inhibits cerebral cortex synaptosomal membrane Na+, K+-ATPase, an effect fully prevented by blockade of neurotensin NT1 receptor by antagonist SR 48692. The work was extended to analyze neurotensin effect on Na+, K+-ATPase activity present in other synaptosomal membranes and in CNS myelin and mitochondrial fractions. Results indicated that, besides inhibiting cerebral cortex synaptosomal membrane Na+, K+-ATPase, neurotensin likewise decreased enzyme activity in homologous striatal membranes as well as in a commercial preparation obtained from porcine cerebral cortex. However, the peptide failed to alter either Na+, K+-ATPase activity in cerebellar synaptosomal and myelin membranes or ATPase activity in mitochondrial preparations. Whenever an effect was recorded with the peptide, it was blocked by antagonist SR 48692, indicating the involvement of the high affinity neurotensin receptor (NT1), as well as supporting the contention that, through inhibition of ion transport at synaptic membrane level, neurotensin plays a regulatory role in neurotransmission.

[Effects of ifenprodil on the adenosine triphosphatase of guinea pig liver mitochondria].

PubMed

Yamashita, Y; Miyake, Y; Mitsuhiro, S; Furukawa, T

1992-07-01

The effects of ifenprodil on adenosine triphosphatase (ATPase) activity were examined using guinea pig liver mitochondria. 1) Intact mitochondrial ATPase activity was stimulated by ifenprodil in a concentration-dependent manner, this effect being further potentiated with dinitrophenol. The stimulation by ifenprodil appeared with only ATP among four nucleotides as substrate. Mg2+ and Ca2+ attenuated the effect of ifenprodil. Ifenprodil abolished the KCN-induced inhibition. 2) Heat-treated mitochondrial ATPase activity, kept for 60 min at 50 degrees C, was decreased in a concentration-dependent manner by ifenprodil. The inhibitory effect of ifenprodil was abolished by Mg2+ and Ca2+. These results indicate that ifenprodil has two behaviors, acceleration of a latent ATPase and inhibition of an activated ATPase. These findings, together with our previous data, suggest that ifenprodil seems to affect the actions of Mg2+ and Ca2+ on mitochondrial ATPase by directly affecting the membrane, and these mechanisms may be involved in its anti-cyanide effect.

Calcium Modulation of Plant Plasma Membrane-Bound Atpase Activities

NASA Technical Reports Server (NTRS)

Caldwell, C.

1983-01-01

The kinetic properties of barley enzyme are discussed and compared with those of other plants. Possibilities for calcium transport in the plasma membrane by proton pump and ATPase-dependent calcium pumps are explored. Topics covered include the ph phase of the enzyme; high affinity of barley for calcium; temperature dependence, activation enthalpy, and the types of ATPase catalytic sites. Attention is given to lipids which are both screened and bound by calcium. Studies show that barley has a calmodulin activated ATPase that is found in the presence of magnesium and calcium.

The Role of the N-Domain in the ATPase Activity of the Mammalian AAA ATPase p97/VCP*

PubMed Central

Niwa, Hajime; Ewens, Caroline A.; Tsang, Chun; Yeung, Heidi O.; Zhang, Xiaodong; Freemont, Paul S.

2012-01-01

p97/valosin-containing protein (VCP) is a type II ATPase associated with various cellular activities that forms a homohexamer with each protomer containing an N-terminal domain (N-domain); two ATPase domains, D1 and D2; and a disordered C-terminal region. Little is known about the role of the N-domain or the C-terminal region in the p97 ATPase cycle. In the p97-associated human disease inclusion body myopathy associated with Paget disease of bone and frontotemporal dementia, the majority of missense mutations are located at the N-domain D1 interface. Structure-based predictions suggest that such mutations affect the interaction of the N-domain with D1. Here we have tested ten major inclusion body myopathy associated with Paget disease of bone and frontotemporal dementia-linked mutants for ATPase activity and found that all have increased activity over the wild type, with one mutant, p97A232E, having three times higher activity. Further mutagenesis of p97A232E shows that the increase in ATPase activity is mediated through D2 and requires both the N-domain and a flexible ND1 linker. A disulfide mutation that locks the N-domain to D1 in a coplanar position reversibly abrogates ATPase activity. A cryo-EM reconstruction of p97A232E suggests that the N-domains are flexible. Removal of the C-terminal region also reduces ATPase activity. Taken together, our data suggest that the conformation of the N-domain in relation to the D1-D2 hexamer is directly linked to ATP hydrolysis and that the C-terminal region is required for hexamer stability. This leads us to propose a model where the N-domain adopts either of two conformations: a flexible conformation compatible with ATP hydrolysis or a coplanar conformation that is inactive. PMID:22270372

A Mutation within the Extended X Loop Abolished Substrate-induced ATPase Activity of the Human Liver ATP-binding Cassette (ABC) Transporter MDR3*

PubMed Central

Kluth, Marianne; Stindt, Jan; Dröge, Carola; Linnemann, Doris; Kubitz, Ralf; Schmitt, Lutz

2015-01-01

The human multidrug resistance protein 3 (MDR3/ABCB4) belongs to the ubiquitous family of ATP-binding cassette (ABC) transporters and is located in the canalicular membrane of hepatocytes. There it flops the phospholipids of the phosphatidylcholine (PC) family from the inner to the outer leaflet. Here, we report the characterization of wild type MDR3 and the Q1174E mutant, which was identified previously in a patient with progressive familial intrahepatic cholestasis type 3 (PFIC-3). We expressed different variants of MDR3 in the yeast Pichia pastoris, purified the proteins via tandem affinity chromatography, and determined MDR3-specific ATPase activity in the presence or absence of phospholipids. The ATPase activity of wild type MDR3 was stimulated 2-fold by liver PC or 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine lipids. Furthermore, the cross-linking of MDR3 with a thiol-reactive fluorophore blocked ATP hydrolysis and exhibited no PC stimulation. Similarly, phosphatidylethanolamine, phosphatidylserine, and sphingomyelin lipids did not induce an increase of wild type MDR3 ATPase activity. The phosphate analogues beryllium fluoride and aluminum fluoride led to complete inhibition of ATPase activity, whereas orthovanadate inhibited exclusively the PC-stimulated ATPase activity of MDR3. The Q1174E mutation is located in the nucleotide-binding domain in direct proximity of the leucine of the ABC signature motif and extended the X loop, which is found in ABC exporters. Our data on the Q1174E mutant demonstrated basal ATPase activity, but PC lipids were incapable of stimulating ATPase activity highlighting the role of the extended X loop in the cross-talk of the nucleotide-binding domain and the transmembrane domain. PMID:25533467

SAUR Inhibition of PP2C-D Phosphatases Activates Plasma Membrane H+-ATPases to Promote Cell Expansion in Arabidopsis[C][W

PubMed Central

Spartz, Angela K.; Ren, Hong; Park, Mee Yeon; Grandt, Kristin N.; Lee, Sang Ho; Murphy, Angus S.; Sussman, Michael R.; Overvoorde, Paul J.; Gray, William M.

2014-01-01

The plant hormone auxin promotes cell expansion. Forty years ago, the acid growth theory was proposed, whereby auxin promotes proton efflux to acidify the apoplast and facilitate the uptake of solutes and water to drive plant cell expansion. However, the underlying molecular and genetic bases of this process remain unclear. We have previously shown that the SAUR19-24 subfamily of auxin-induced SMALL AUXIN UP-RNA (SAUR) genes promotes cell expansion. Here, we demonstrate that SAUR proteins provide a mechanistic link between auxin and plasma membrane H+-ATPases (PM H+-ATPases) in Arabidopsis thaliana. Plants overexpressing stabilized SAUR19 fusion proteins exhibit increased PM H+-ATPase activity, and the increased growth phenotypes conferred by SAUR19 overexpression are dependent upon normal PM H+-ATPase function. We find that SAUR19 stimulates PM H+-ATPase activity by promoting phosphorylation of the C-terminal autoinhibitory domain. Additionally, we identify a regulatory mechanism by which SAUR19 modulates PM H+-ATPase phosphorylation status. SAUR19 as well as additional SAUR proteins interact with the PP2C-D subfamily of type 2C protein phosphatases. We demonstrate that these phosphatases are inhibited upon SAUR binding, act antagonistically to SAURs in vivo, can physically interact with PM H+-ATPases, and negatively regulate PM H+-ATPase activity. Our findings provide a molecular framework for elucidating auxin-mediated control of plant cell expansion. PMID:24858935

Influence of lorcainide on microsomal Na+, K(+)-ATPase in guinea-pig isolated heart preparations.

PubMed Central

Almotrefi, A. A.; Dzimiri, N.

1991-01-01

1. The effects of lorcainide on the myocardial Mg2(+)-dependent, Na+ and K(+)-activated adenosine triphosphatase (Na+, K(+)-ATPase) were compared in guinea-pig heart preparations with those of ouabain, a specific inhibitor of the enzyme activity. 2. Both ouabain and lorcainide inhibited the microsomal Na+, K(+)-ATPase activity in a concentration-dependent fashion. Their inhibitory effective ranges were 0.05-100 microM and 0.15-125 microM, respectively, and the concentrations for half maximal inhibition (IC50 values) were 2.1 +/- 0.3 and 33.5 +/- 7.3 microM, respectively. 3. In a second series of experiments, the combined effects of the two drugs on the enzyme activity were studied. In these experiments, lorcainide produced a concentration-dependent potentiation of the inhibitory effects of ouabain on Na+, K(+)-ATPase activity. 4. The present study demonstrates that lorcainide is a potent inhibitor of myocardial Na+, K(+)-ATPase. PMID:1849773

Multimeric species in equilibrium in detergent-solubilized Na,K-ATPase.

PubMed

Yoneda, Juliana Sakamoto; Scanavachi, Gustavo; Sebinelli, Heitor Gobbi; Borges, Júlio Cesar; Barbosa, Leandro R S; Ciancaglini, Pietro; Itri, Rosangela

2016-08-01

In this work, we find an equilibrium between different Na,K-ATPase (NKA) oligomeric species solubilized in a non-ionic detergent C12E8 by means of Dynamic Light Scattering (DLS), Analytical Ultracentrifugation (AUC), Small Angle X-ray Scattering (SAXS), Spectrophotometry (absorption at 280/350nm) and enzymatic activity assay. The NKA sample after chromatography purification presented seven different populations as identified by AUC, with monomers and tetramers amounting to ∼55% of the total protein mass in solution. These two species constituted less than 40% of the total protein mass after increasing the NKA concentration. Removal of higher-order oligomer/aggregate species from the NKA solution using 220nm-pore filter resulted in an increase of the specific enzymatic activity. Nevertheless, the enzyme forms new large aggregates over an elapsed time of 20h. The results thus point out that C12E8-solubilized NKA is in a dynamic equilibrium of monomers, tetramers and high-order oligomers/subunit aggregates. These latter have low or null activity. High amount of detergent leads to the dissociation of NKA into smaller aggregates with no enzymatic activity. Copyright © 2016. Published by Elsevier B.V.

Significance of the glutamate-139 residue of the V-type Na+-ATPase NtpK subunit in catalytic turnover linked with salt tolerance of Enterococcus hirae.

PubMed

Kawano-Kawada, Miyuki; Takahashi, Hiroko; Igarashi, Kazuei; Murata, Takeshi; Yamato, Ichiro; Homma, Michio; Kakinuma, Yoshimi

2011-07-01

A Glu139Asp mutant of the NtpK subunit (kE139D) of Enterococcus hirae vacuolar-type ATPase (V-ATPase) lost tolerance to sodium but not to lithium at pH 10. Purified kE139D V-ATPase retained relatively high specific activity and affinity for the lithium ion compared to the sodium ion. The kE139 residue of V-ATPase is indispensable for its enzymatic activity that is linked with the salt tolerance of enterococci.

The actin-activated ATPase of co-polymer filaments of myosin and myosin-rod.

PubMed Central

Stepkowski, D; Orlova, A A; Moos, C

1994-01-01

The actin activated ATPase of myosin at low ionic strength shows a complex dependence on actin concentration, in contrast with the simple hyperbolic actin activation kinetics of heavy meromyosin and subfragment-1. To investigate how the aggregation of myosin influences the actomyosin ATPase kinetics, we have studied the actin-activated ATPase of mixed filaments in which the myosin molecules are separated from each other by copolymerization with myosin rod. Electron microscopy of copolymer filaments, alone and bound to actin, indicates that the myosin heads are distributed randomly along the co-polymer filaments. The actin-activated ATPase of myosin decreases with increasing rod, approaching a plateau of about 30% of the control at a rod/myosin molar ratio of 4:1. The decrease in ATPase persists even at Vmax, the extrapolated limit at infinite actin, indicating that it is not due merely to the loss of cooperative actin binding. Furthermore, the actin dependence of the ATPase still shows a biphasic character like that of control myosin, even at rod/myosin ratio of 12:1, so this complexity is not probably due solely to the structural proximity of myosin molecules, but may involve a non-equivalence of myosin heads or myosin molecules in the filament environment. Images Figure 1 Figure 2 PMID:8198528

The V-ATPase accessory protein Atp6ap1b mediates dorsal forerunner cell proliferation and left-right asymmetry in zebrafish.

PubMed

Gokey, Jason J; Dasgupta, Agnik; Amack, Jeffrey D

2015-11-01

Asymmetric fluid flows generated by motile cilia in a transient 'organ of asymmetry' are involved in establishing the left-right (LR) body axis during embryonic development. The vacuolar-type H(+)-ATPase (V-ATPase) proton pump has been identified as an early factor in the LR pathway that functions prior to cilia, but the role(s) for V-ATPase activity are not fully understood. In the zebrafish embryo, the V-ATPase accessory protein Atp6ap1b is maternally supplied and expressed in dorsal forerunner cells (DFCs) that give rise to the ciliated organ of asymmetry called Kupffer's vesicle (KV). V-ATPase accessory proteins modulate V-ATPase activity, but little is known about their functions in development. We investigated Atp6ap1b and V-ATPase in KV development using morpholinos, mutants and pharmacological inhibitors. Depletion of both maternal and zygotic atp6ap1b expression reduced KV organ size, altered cilia length and disrupted LR patterning of the embryo. Defects in other ciliated structures-neuromasts and olfactory placodes-suggested a broad role for Atp6ap1b during development of ciliated organs. V-ATPase inhibitor treatments reduced KV size and identified a window of development in which V-ATPase activity is required for proper LR asymmetry. Interfering with Atp6ap1b or V-ATPase function reduced the rate of DFC proliferation, which resulted in fewer ciliated cells incorporating into the KV organ. Analyses of pH and subcellular V-ATPase localizations suggested Atp6ap1b functions to localize the V-ATPase to the plasma membrane where it regulates proton flux and cytoplasmic pH. These results uncover a new role for the V-ATPase accessory protein Atp6ap1b in early development to maintain the proliferation rate of precursor cells needed to construct a ciliated KV organ capable of generating LR asymmetry. Copyright © 2015 Elsevier Inc. All rights reserved.

The V-ATPase accessory protein Atp6ap1b mediates dorsal forerunner cell proliferation and left-right asymmetry in zebrafish

PubMed Central

Gokey, Jason J.; Dasgupta, Agnik; Amack, Jeffrey D.

2015-01-01

Asymmetric fluid flows generated by motile cilia in a transient ‘organ of asymmetry’ are involved in establishing the left-right (LR) body axis during embryonic development. The vacuolar-type H+-ATPase (V-ATPase) proton pump has been identified as an early factor in the LR pathway that functions prior to cilia, but the role(s) for V-ATPase activity are not fully understood. In the zebrafish embryo, the V-ATPase accessory protein Atp6ap1b is maternally supplied and expressed in dorsal forerunner cells (DFCs) that give rise to the ciliated organ of asymmetry called Kupffer’s vesicle (KV). V-ATPase accessory proteins modulate V-ATPase activity, but little is known about their functions in development. We investigated Atp6ap1b and V-ATPase in KV development using morpholinos, mutants and pharmacological inhibitors. Depletion of both maternal and zygotic atp6ap1b expression reduced KV organ size, altered cilia length and disrupted LR patterning of the embryo. Defects in other ciliated structures—neuromasts and olfactory placodes—suggested a broad role for Atp6ap1b during development of ciliated organs. V-ATPase inhibitor treatments reduced KV size and identified a window of development in which V-ATPase activity is required for proper LR asymmetry. Interfering with Atp6ap1b or V-ATPase function reduced the rate of DFC proliferation, which resulted in fewer ciliated cells incorporating into the KV organ. Analyses of pH and subcellular V-ATPase localizations suggested Atp6ap1b functions to localize the V-ATPase to the plasma membrane where it regulates proton flux and cytoplasmic pH. These results uncover a new role for the V-ATPase accessory protein Atp6ap1b in early development to maintain the proliferation rate of precursor cells needed to construct a ciliated KV organ capable of generating LR asymmetry. PMID:26254189

Effect of synthetic detergents on the swelling and the ATPase of mitochondria isolated from rat liver.

PubMed

WITTER, R F; MINK, W

1958-01-25

A study was made of the effects of various types of detergents on the swelling of isolated mitochondria and on mitochondrial ATPases which are activated by Mg or DNP respectively. The rate of swelling was measured in the Beckman spectrophotometer by following the decrease in turbidity of dilute suspensions of these organelles. It was found that non-ionic detergents containing a nonyl phenoxy side chain or anionic detergents caused swelling of the mitochondria and activation of Mg-ATPase. On the other hand, cationic detergents promoted the clumping of mitochondria and did not activate Mg-ATPase. DNP-ATPase was inhibited by all of the detergents tested. It would appear from these observations that the inhibition of DNP-ATPase is not related to a gross change in the morphology of the organelles; in contrast, the activation of Mg-ATPase definitely is correlated with swelling of the isolated mitochondria. These data also suggest that the ionic detergents combine with charged sites on the protein moiety of the lipoprotein in the mitochondrial surface, whereas the non-ionic detergents form inclusion compounds with the lipide moiety, thereby altering the mitochondrial structure and permeability.

Cytochrome b5 Reductase 1 Triggers Serial Reactions that Lead to Iron Uptake in Plants.

PubMed

Oh, Young Jun; Kim, Hanul; Seo, Sung Hee; Hwang, Bae Geun; Chang, Yoon Seok; Lee, Junho; Lee, Dong Wook; Sohn, Eun Ju; Lee, Sang Joon; Lee, Youngsook; Hwang, Inhwan

2016-04-04

Rhizosphere acidification is essential for iron (Fe) uptake into plant roots. Plasma membrane (PM) H(+)-ATPases play key roles in rhizosphere acidification. However, it is not fully understood how PM H(+)-ATPase activity is regulated to enhance root Fe uptake under Fe-deficient conditions. Here, we present evidence that cytochrome b5 reductase 1 (CBR1) increases the levels of unsaturated fatty acids, which stimulate PM H(+)-ATPase activity and thus lead to rhizosphere acidification. CBR1-overexpressing (CBR1-OX) Arabidopsis thaliana plants had higher levels of unsaturated fatty acids (18:2 and 18:3), higher PM H(+)-ATPase activity, and lower rhizosphere pH than wild-type plants. By contrast, cbr1 loss-of-function mutant plants showed lower levels of unsaturated fatty acids and lower PM H(+)-ATPase activity but higher rhizosphere pH. Reduced PM H(+)-ATPase activity in cbr1 could be restored in vitro by addition of unsaturated fatty acids. Transcript levels of CBR1, fatty acids desaturase2 (FAD2), and fatty acids desaturase3 (FAD3) were increased under Fe-deficient conditions. We propose that CBR1 has a crucial role in increasing the levels of unsaturated fatty acids, which activate the PM H(+)-ATPase and thus reduce rhizosphere pH. This reaction cascade ultimately promotes root Fe uptake. Copyright © 2016 The Author. Published by Elsevier Inc. All rights reserved.

Purification and properties of an ATPase from Sulfolobus solfataricus

NASA Technical Reports Server (NTRS)

Hochstein, Lawrence I.; Stan-Lotter, Helga

1992-01-01

The paper reports properties of a sulfite-activated ATPase from Sulfolobus solfataricus, purified using ammonium sulfate precipitation, column chromatography on UltraGel and Sepharose 6B, and SDS-PAGE. The 92-fold purified enzyme had a relative molecular mass of 370,000. It could be dissociated into three subunits with respective molecular masses of 63,000, 48,000, and 24,000. The ATPase activity was found to be inhibitable by nitrate, N-ethylmaleimide (which bound predominantly to the largest subunit), and 4-chloro 7-nitrobenzofurazan, but not by azide, quercetin, or vanadate. While the ATPase from S. solfataricus shared a number of properties with the S. acidocaldarius ATPase, there were also significant differences suggesting the existence of several types of archaeal ATPases.

Response of plasma membrane H+-ATPase in rice (Oryza sativa) seedlings to simulated acid rain.

PubMed

Liang, Chanjuan; Ge, Yuqing; Su, Lei; Bu, Jinjin

2015-01-01

Understanding the adaptation of plants to acid rain is important to find feasible approaches to alleviate such damage to plants. We studied effects of acid rain on plasma membrane H(+)-ATPase activity and transcription, intracellular H(+), membrane permeability, photosynthetic efficiency, and relative growth rate during stress and recovery periods. Simulated acid rain at pH 5.5 did not affect plasma membrane H(+)-ATPase activity, intracellular H(+), membrane permeability, photosynthetic efficiency, and relative growth rate. Plasma membrane H(+)-ATPase activity and transcription in leaves treated with acid rain at pH 3.5 was increased to maintain ion homeostasis by transporting excessive H(+) out of cells. Then intracellular H(+) was close to the control after a 5-day recovery, alleviating damage on membrane and sustaining photosynthetic efficiency and growth. Simulated acid rain at pH 2.5 inhibited plasma membrane H(+)-ATPase activity by decreasing the expression of H(+)-ATPase at transcription level, resulting in membrane damage and abnormal intracellular H(+), and reduction in photosynthetic efficiency and relative growth rate. After a 5-day recovery, all parameters in leaves treated with pH 2.5 acid rain show alleviated damage, implying that the increased plasma membrane H(+)-ATPase activity and its high expression were involved in repairing process in acid rain-stressed plants. Our study suggests that plasma membrane H(+)-ATPase can play a role in adaptation to acid rain for rice seedlings.

Virtual prototyping study shows increased ATPase activity of Hsp90 to be the key determinant of cancer phenotype.

PubMed

Vali, Shireen; Pallavi, Rani; Kapoor, Shweta; Tatu, Utpal

2010-03-01

Hsp90 is an ATP-dependent molecular chaperone that regulates key signaling proteins and thereby impacts cell growth and development. Chaperone cycle of Hsp90 is regulated by ATP binding and hydrolysis through its intrinsic ATPase activities, which is in turn modulated by interaction with its co-chaperones. Hsp90 ATPase activity varies in different organisms and is known to be increased in tumor cells. In this study we have quantitatively analyzed the impact of increasing Hsp90 ATPase activity on the activities of its clients through a virtual prototyping technology, which comprises a dynamic model of Hsp90 interaction with clients involved in proliferation pathways. Our studies highlight the importance of increased ATPase activity of Hsp90 in cancer cells as the key modulator for increased proliferation and survival. A tenfold increase in ATPase activity of Hsp90 often seen in cancer cells increases the levels of active client proteins such as Akt-1, Raf-1 and Cyclin D1 amongst others to about 12-, 8- and 186-folds respectively. Additionally we studied the effect of a competitive inhibitor of Hsp90 activity on the reduction in the client protein levels. Virtual prototyping experiments corroborate with findings that the drug has almost 10- to 100-fold higher affinity as indicated by a lower IC(50) value (30-100 nM) in tumor cells with higher ATPase activity. The results also indicate a 15- to 25-fold higher efficacy of the inhibitor in reducing client levels in tumor cells. This analysis provides mechanistic insights into the links between increased Hsp90 ATPase activity, tumor phenotype and the hypersensitivity of tumor Hsp90 to inhibition by ATP analogs. The online version of this article (doi:10.1007/s11693-009-9046-3) contains supplementary material, which is available to authorized users.

The role of endomembrane-localized VHA-c in plant growth.

PubMed

Zhou, Aimin; Takano, Tetsuo; Liu, Shenkui

2018-01-02

In plant cells, the vacuolar-type H + -ATPase (V-ATPase), a large multis`ubunit endomembrane proton pump, plays an important role in acidification of subcellular organelles, pH and ion homeostasis, and endocytic and secretory trafficking. V-ATPase subunit c (VHA-c) is essential for V-ATPase assembly, and is directly responsible for binding and transmembrane transport of protons. In previous studies, we identified a PutVHA-c gene from Puccinellia tenuiflora, and investigated its function in plant growth. Subcellular localization revealed that PutVHA-c is mainly localized in endosomal compartments. Overexpression of PutVHA-c enhanced V-ATPase activity and promoted plant growth in transgenic Arabidopsis. Furthermore, the activity of V-ATPase affected intracellular transport of the Golgi-derived endosomes. Our results showed that endomembrane localized-VHA-c contributes to plant growth by influencing V-ATPase-dependent endosomal trafficking. Here, we discuss these recent findings and speculate on the VHA-c mediated molecular mechanisms involved in plant growth, providing a better understanding of the functions of VHA-c and V-ATPase.

Identification of NaK-ATPase inhibitors in human plasma as nonesterified fatty acids and lysophospholipids.

PubMed

Kelly, R A; O'Hara, D S; Mitch, W E; Smith, T W

1986-09-05

Elevated plasma levels of factors with cardiac glycoside-like activity have been implicated in the response to volume expansion in animals and in the pathogenesis of certain human diseases. We recently described four fractions (IR1, EI1, EI2, EI3) from normal human plasma that inhibit NaK-ATPase, displace ouabain from the enzyme, and exhibit digoxin-like immunoreactivity (Kelly, R. A., O'Hara, D. S., Canessa, M. L., Mitch, W. E., and Smith, T. W. (1985) J. Biol. Chem. 260, 11396-11405). In this report, we identify the active component of these plasma fractions as long-chain nonesterified fatty acids (NEFA) and lysophospholipids. These lipids were present in fractions EI1, EI2, and EI3 in quantities sufficient to account for all of the NaK-ATPase inhibitory activity. The digoxin-like immunoreactivity in fraction IR1 could be attributed to hydrocortisone and other endogenous steroids. To explore the nature of the lipid-NaK-ATPase interactions, we examined the effects of various ATP or sodium concentrations on the NaK-ATPase activity measured in the presence of NEFA. Varying sodium did not affect the inhibition of NaK-ATPase by linoleic acid. At less than 0.15 mM ATP, linoleic acid stimulated NaK-ATPase, but at higher ATP concentrations, the enzyme was progressively inhibited. In summary, NEFA and lysophospholipids, at levels similar to those occurring in human plasma, may account for all of the NaK-ATPase inhibitory activity observed in human plasma fractions. These lipids probably do not directly regulate NaK-ATPase in vivo under normal physiologic conditions, but may alter the sodium pump in disease states characterized by abnormalities in lipid metabolism or plasma protein binding.

The vacuolar-ATPase complex and assembly factors, TMEM199 and CCDC115, control HIF1α prolyl hydroxylation by regulating cellular iron levels.

PubMed

Miles, Anna L; Burr, Stephen P; Grice, Guinevere L; Nathan, James A

2017-03-15

Hypoxia Inducible transcription Factors (HIFs) are principally regulated by the 2-oxoglutarate and Iron(II) prolyl hydroxylase (PHD) enzymes, which hydroxylate the HIFα subunit, facilitating its proteasome-mediated degradation. Observations that HIFα hydroxylation can be impaired even when oxygen is sufficient emphasise the importance of understanding the complex nature of PHD regulation. Here, we use an unbiased genome-wide genetic screen in near-haploid human cells to uncover cellular processes that regulate HIF1α. We identify that genetic disruption of the Vacuolar H+ ATPase (V-ATPase), the key proton pump for endo-lysosomal acidification, and two previously uncharacterised V-ATPase assembly factors, TMEM199 and CCDC115, stabilise HIF1α in aerobic conditions. Rather than preventing the lysosomal degradation of HIF1α, disrupting the V-ATPase results in intracellular iron depletion, thereby impairing PHD activity and leading to HIF activation. Iron supplementation directly restores PHD catalytic activity following V-ATPase inhibition, revealing important links between the V-ATPase, iron metabolism and HIFs.

The role of the plasma membrane H+-ATPase in plant-microbe interactions.

PubMed

Elmore, James Mitch; Coaker, Gitta

2011-05-01

Plasma membrane (PM) H+-ATPases are the primary pumps responsible for the establishment of cellular membrane potential in plants. In addition to regulating basic aspects of plant cell function, these enzymes contribute to signaling events in response to diverse environmental stimuli. Here, we focus on the roles of the PM H+-ATPase during plant-pathogen interactions. PM H+-ATPases are dynamically regulated during plant immune responses and recent quantitative proteomics studies suggest complex spatial and temporal modulation of PM H+-ATPase activity during early pathogen recognition events. Additional data indicate that PM H+-ATPases cooperate with the plant immune signaling protein RIN4 to regulate stomatal apertures during bacterial invasion of leaf tissue. Furthermore, pathogens have evolved mechanisms to manipulate PM H+-ATPase activity during infection. Thus, these ubiquitous plant enzymes contribute to plant immune responses and are targeted by pathogens to increase plant susceptibility.

Quantification of Na+,K+ pumps and their transport rate in skeletal muscle: Functional significance

PubMed Central

2013-01-01

During excitation, muscle cells gain Na+ and lose K+, leading to a rise in extracellular K+ ([K+]o), depolarization, and loss of excitability. Recent studies support the idea that these events are important causes of muscle fatigue and that full use of the Na+,K+-ATPase (also known as the Na+,K+ pump) is often essential for adequate clearance of extracellular K+. As a result of their electrogenic action, Na+,K+ pumps also help reverse depolarization arising during excitation, hyperkalemia, and anoxia, or from cell damage resulting from exercise, rhabdomyolysis, or muscle diseases. The ability to evaluate Na+,K+-pump function and the capacity of the Na+,K+ pumps to fill these needs require quantification of the total content of Na+,K+ pumps in skeletal muscle. Inhibition of Na+,K+-pump activity, or a decrease in their content, reduces muscle contractility. Conversely, stimulation of the Na+,K+-pump transport rate or increasing the content of Na+,K+ pumps enhances muscle excitability and contractility. Measurements of [3H]ouabain binding to skeletal muscle in vivo or in vitro have enabled the reproducible quantification of the total content of Na+,K+ pumps in molar units in various animal species, and in both healthy people and individuals with various diseases. In contrast, measurements of 3-O-methylfluorescein phosphatase activity associated with the Na+,K+-ATPase may show inconsistent results. Measurements of Na+ and K+ fluxes in intact isolated muscles show that, after Na+ loading or intense excitation, all the Na+,K+ pumps are functional, allowing calculation of the maximum Na+,K+-pumping capacity, expressed in molar units/g muscle/min. The activity and content of Na+,K+ pumps are regulated by exercise, inactivity, K+ deficiency, fasting, age, and several hormones and pharmaceuticals. Studies on the α-subunit isoforms of the Na+,K+-ATPase have detected a relative increase in their number in response to exercise and the glucocorticoid dexamethasone but have not involved their quantification in molar units. Determination of ATPase activity in homogenates and plasma membranes obtained from muscle has shown ouabain-suppressible stimulatory effects of Na+ and K+. PMID:24081980

Estrogen Modulation of MgATPase Activity of Nonmuscle Myosin-II-B Filaments

PubMed Central

Gorodeski, George I.

2008-01-01

The study tested the hypothesis that estrogen controls epithelial paracellular resistance through modulation of myosin. The objective was to understand how estrogen modulates non-muscle myosin-II-B (NMM-II-B), the main component of the cortical actomyosin in human epithelial cervical cells. Experiments used human cervical epithelial cells CaSki as a model, and end points were NMM-II-B phosphorylation, filamentation, and MgATPase activity. The results were as follows: 1) treatment with estrogen increased phosphorylation and MgATPase activity and decreased NMM-II-B filamentation; 2) estrogen effects could be blocked by antisense nucleotides for the estrogen receptor-α and by ICI-182,780, tamoxifen, and the casein kinase-II (CK2) inhibitor, 5,6-dichloro-1-β-(D)-ribofuranosylbenzimidazole and attenuated by AG1478 and PD98059 (inhibitors of epithelial growth factor receptor and ERK/MAPK) but not staurosporine [blocker of protein kinase C (PKC)]; 3) treatments with the PKC activator sn-1,2-di-octanoyl diglyceride induced biphasic effect on NMM-II-B MgATPase activity: an increase at 1 nM to 1 μM and a decrease in activity at more than 1 μM; 4) sn-1,2-dioctanoyl diglyceride also decreased NMM-II-B filamentation in a monophasic and saturable dose dependence (EC50 1–10 μM); 5) when coincubated directly with purified NMM-II-B filaments, both CK2 and PKC decreased filamentation and increased MgATPase activity; 6) assays done on disassembled NMM-II-B filaments showed MgATPase activity in filaments obtained from estrogen-treated cells but not estrogen-depleted cells; and 7) incubations in vitro with CK2, but not PKC, facilitated MgATPase activity, even in disassembled NMM-II-B filaments. The results suggest that estrogen, in an effect mediated by estrogen receptor-α and CK2 and involving the epithelial growth factor receptor and ERK/MAPK cascades, increases NMM-II-B MgATPase activity independent of NMM-II-B filamentation status. PMID:17023528

Effects of hyper- and hypothyroidism on acetylcholinesterase, (Na(+), K (+))- and Mg ( 2+ )-ATPase activities of adult rat hypothalamus and cerebellum.

PubMed

Carageorgiou, Haris; Pantos, Constantinos; Zarros, Apostolos; Stolakis, Vasileios; Mourouzis, Iordanis; Cokkinos, Dennis; Tsakiris, Stylianos

2007-03-01

Thyroid hormones (THs) are recognized as key metabolic hormones, and the metabolic rate increases in hyperthyroidism, while it decreases in hypothyroidism. The aim of this work was to investigate how changes in metabolism induced by THs could affect the activities of acetylcholinesterase (AChE), (Na(+), K(+))- and Mg(2+)-ATPase in the hypothalamus and the cerebellum of adult rats. Hyperthyroidism was induced by subcutaneous administration of thyroxine (25 microg/100 g body weight) once daily for 14 days, while hypothyroidism was induced by oral administration of propylthiouracil (0.05%) for 21 days. All enzyme activities were evaluated spectrophotometrically in the homogenated brain regions of 10 three-animal pools. Neither hyper-, nor hypothyroidism had any effect on the examined hypothalamic enzyme activities. In the cerebellum, hyperthyroidism provoked a significant decrease in both the AChE (-23%, p < 0.001) and the Na(+), K(+)-ATPase activities (-26%, p < 0.001). Moreover, hypothyroidism had a similar effect on the examined enzyme activities: AChE (-17%, p < 0.001) and Na(+), K(+)-ATPase (-27%, p < 0.001). Mg(2+)-ATPase activity was found unaltered in both the hyper- and the hypothyroid brain regions. neither hyper-, nor hypothyroidism had any effect on the examined hypothalamic enzyme activities. In the cerebellum, hyperthyroidism provoked a significant decrease in both the AChE and the Na(+), K(+)-ATPase activities. The decreased (by the THs) Na(+), K(+)-ATPase activities may increase the synaptic acetylcholine release, and thus, could result in a decrease in the cerebellar AChE activity. Moreover, the above TH-induced changes may affect the monoamine neurotransmitter systems.

Immunochemical analysis of Micrococcus lysodeikticus (luteus) F1-ATPase and its subunits.

PubMed

Urban, C; Salton, M R

1983-08-31

The F1-ATPase from Micrococcus lysodeikticus has been purified to 95% protein homogeneity in this laboratory and as all other bacterial F1S, possesses five distinct subunits with molecular weights ranging from 60 000 to 10 000 (Huberman, M. and Salton, M.R.J. (1979) Biochim. Biophys. Acta 547, 230-240). In this communication, we demonstrate the immunochemical reactivities of antibodies to native and SDS-dissociated subunits with the native and dissociated F1-ATPase and show that: (1) the antibodies generated to the native or SDS-dissociated subunits react with the native molecule; (2) all of the subunits comprising the F1 are antigenically unique as determined by crossed immunoelectrophoresis and the Ouchterlony double-diffusion techniques; (3) antibodies to the SDS-denatured individual delta- and epsilon-subunits can be used to destabilize the interaction of these specific subunits with the rest of the native F1; and (4) all subunit antibodies as well as anti-native F1 were found to inhibit ATPase activity to varying degrees, the strongest inhibition being seen with antibodies to the total F1 and anti-alpha- and anti-beta-subunit antibodies. The interaction of specific subunit antibodies may provide a new and novel way to study further and characterize the catalytic portions of F1-ATPases and in general may offer an additional method for the examination of multimeric proteins.

The inhibition of the mitochondrial F1FO-ATPase activity when activated by Ca2+ opens new regulatory roles for NAD.

PubMed

Nesci, Salvatore; Trombetti, Fabiana; Ventrella, Vittoria; Pirini, Maurizio; Pagliarani, Alessandra

2018-01-26

The mitochondrial F1FO-ATPase is uncompetitively inhibited by NAD+ only when the natural cofactor Mg2+ is replaced by Ca2+, a mode putatively involved in cell death. The Ca2+-dependent F1FO-ATPase is also inhibited when NAD+ concentration in mitochondria is raised by acetoacetate. The enzyme inhibition by NAD+ cannot be ascribed to any de-ac(et)ylation or ADP-ribosylation by sirtuines, as it is not reversed by nicotinamide. Moreover, the addition of acetyl-CoA or palmitate, which would favor the enzyme ac(et)ylation, does not affect the F1FO-ATPase activity. Consistently, NAD+ may play a new role, not associated with redox and non-redox enzymatic reactions, in the Ca2+-dependent regulation of the F1FO-ATPase activity.

Job Sharing in the Endomembrane System: Vacuolar Acidification Requires the Combined Activity of V-ATPase and V-PPase.

PubMed

Kriegel, Anne; Andrés, Zaida; Medzihradszky, Anna; Krüger, Falco; Scholl, Stefan; Delang, Simon; Patir-Nebioglu, M Görkem; Gute, Gezahegn; Yang, Haibing; Murphy, Angus S; Peer, Wendy Ann; Pfeiffer, Anne; Krebs, Melanie; Lohmann, Jan U; Schumacher, Karin

2015-12-01

The presence of a large central vacuole is one of the hallmarks of a prototypical plant cell, and the multiple functions of this compartment require massive fluxes of molecules across its limiting membrane, the tonoplast. Transport is assumed to be energized by the membrane potential and the proton gradient established by the combined activity of two proton pumps, the vacuolar H(+)-pyrophosphatase (V-PPase) and the vacuolar H(+)-ATPase (V-ATPase). Exactly how labor is divided between these two enzymes has remained elusive. Here, we provide evidence using gain- and loss-of-function approaches that lack of the V-ATPase cannot be compensated for by increased V-PPase activity. Moreover, we show that increased V-ATPase activity during cold acclimation requires the presence of the V-PPase. Most importantly, we demonstrate that a mutant lacking both of these proton pumps is conditionally viable and retains significant vacuolar acidification, pointing to a so far undetected contribution of the trans-Golgi network/early endosome-localized V-ATPase to vacuolar pH. © 2015 American Society of Plant Biologists. All rights reserved.

Metal Fluoride Inhibition of a P-type H+ Pump

PubMed Central

Pedersen, Jesper Torbøl; Falhof, Janus; Ekberg, Kira; Buch-Pedersen, Morten Jeppe; Palmgren, Michael

2015-01-01

The plasma membrane H+-ATPase is a P-type ATPase responsible for establishing electrochemical gradients across the plasma membrane in fungi and plants. This essential proton pump exists in two activity states: an autoinhibited basal state with a low turnover rate and a low H+/ATP coupling ratio and an activated state in which ATP hydrolysis is tightly coupled to proton transport. Here we characterize metal fluorides as inhibitors of the fungal enzyme in both states. In contrast to findings for other P-type ATPases, inhibition of the plasma membrane H+-ATPase by metal fluorides was partly reversible, and the stability of the inhibition varied with the activation state. Thus, the stability of the ATPase inhibitor complex decreased significantly when the pump transitioned from the activated to the basal state, particularly when using beryllium fluoride, which mimics the bound phosphate in the E2P conformational state. Taken together, our results indicate that the phosphate bond of the phosphoenzyme intermediate of H+-ATPases is labile in the basal state, which may provide an explanation for the low H+/ATP coupling ratio of these pumps in the basal state. PMID:26134563

Phosphatidylinositol 3-Kinase Promotes V-ATPase Activation and Vacuolar Acidification and Delays Methyl Jasmonate-Induced Leaf Senescence1

PubMed Central

Liu, Jian; Ji, Yingbin; Zhou, Jun; Xing, Da

2016-01-01

PI3K and its product PI3P are both involved in plant development and stress responses. In this study, the down-regulation of PI3K activity accelerated leaf senescence induced by methyl jasmonate (MeJA) and suppressed the activation of vacuolar H+-ATPase (V-ATPase). Yeast two-hybrid analyses indicated that PI3K bound to the V-ATPase B subunit (VHA-B). Analysis of bimolecular fluorescence complementation in tobacco guard cells showed that PI3K interacted with VHA-B2 in the tonoplasts. Through the use of pharmacological and genetic tools, we found that PI3K and V-ATPase promoted vacuolar acidification and stomatal closure during leaf senescence. Vacuolar acidification was suppressed by the PIKfyve inhibitor in 35S:AtVPS34-YFP Arabidopsis during MeJA-induced leaf senescence, but the decrease was lower than that in YFP-labeled Arabidopsis. These results suggest that PI3K promotes V-ATPase activation and consequently induces vacuolar acidification and stomatal closure, thereby delaying MeJA-induced leaf senescence. PMID:26739232

Changes in acetylcholinesterase, Na+,K+-ATPase, and Mg2+-ATPase activities in the frontal cortex and the hippocampus of hyper- and hypothyroid adult rats.

PubMed

Carageorgiou, Haris; Pantos, Constantinos; Zarros, Apostolos; Stolakis, Vasileios; Mourouzis, Iordanis; Cokkinos, Dennis; Tsakiris, Stylianos

2007-08-01

The thyroid hormones (THs) are crucial determinants of normal development and metabolism, especially in the central nervous system. The metabolic rate is known to increase in hyperthyroidism and decrease in hypothyroidism. The aim of this work was to investigate how changes in metabolism induced by THs could affect the activities of acetylcholinesterase (AChE), (Na+,K+)- and Mg2+-adenosinetriphosphatase (ATPase) in the frontal cortex and the hippocampus of adult rats. Hyperthyroidism was induced by subcutaneous administration of thyroxine (25 microg/100 g body weight) once daily for 14 days, and hypothyroidism was induced by oral administration of propylthiouracil (0.05%) for 21 days. All enzyme activities were evaluated spectrophotometrically in the homogenated brain regions of 10 three-animal pools. A region-specific behavior was observed concerning the examined enzyme activities in hyper- and hypothyroidism. In hyperthyroidism, AChE activity was significantly increased only in the hippocampus (+22%), whereas Na+,K+-ATPase activity was significantly decreased in the hyperthyroid rat hippocampus (-47%) and remained unchanged in the frontal cortex. In hypothyroidism, AChE activity was significantly decreased in the frontal cortex (-23%) and increased in the hippocampus (+21%). Na+,K+-ATPase activity was significantly decreased in both the frontal cortex (-35%) and the hippocampus (-43%) of hypothyroid rats. Mg2+-ATPase remained unchanged in the regions of both hyper- and hypothyroid rat brains. Our data revealed that THs affect the examined adult rat brain parameters in a region- and state-specific way. The TH-reduced Na+,K+-ATPase activity may increase the synaptic acetylcholine release and, thus, modulate AChE activity. Moreover, the above TH-induced changes may affect the monoamine neurotransmitter systems in the examined brain regions.

Is the Paracoccus halodenitrificans ATPase a chimeric enzyme?

NASA Technical Reports Server (NTRS)

Hochstein, L. I.

1996-01-01

Membranes from Paracoccus halodenitrificans contain an ATPase that is most active in the absence of NaCl. The most unusual characteristic of the enzyme is its pattern of sensitivity to various inhibitors. Azide and rhodamine 6G, inhibitors of F1F0-ATPases, inhibit ATP hydrolysis as do bafilomycin A1, concanamycin A (folimycin), N-ethylmaleimide, and p-chloromercuriphenylsulfonate which are inhibitors of vacuolar ATPases. This indiscriminate sensitivity suggests that this ATPase may be a hybrid and that caution should be exercised when using inhibition as a diagnostic for distinguishing between F1F0-ATPases and vacuolar ATPases.

D-Methionine attenuated cisplatin-induced vestibulotoxicity through altering ATPase activities and oxidative stress in guinea pigs

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cheng, P.-W.; Department of Otolaryngology, Far Eastern Memorial Hospital, Taipei, Taiwan; Liu, S.-H.

2006-09-01

Cisplatin has been used as a chemotherapeutic agent to treat many kinds of malignancies. Its damage to the vestibulo-ocular reflex (VOR) system has been reported. However, the underlying biochemical change in the inner ear or central vestibular nervous system is not fully understood. In this study, we attempted to examine whether cisplatin-induced vestibulotoxicity and D-methionine protection were correlated with the changes of ATPase activities and oxidative stress of ampullary tissue of vestibules as well as cerebellar cortex (the inhibitory center of VOR system) of guinea pigs. By means of a caloric test coupled with electronystagmographic recordings, we found that cisplatinmore » exposure caused a dose-dependent (1, 3, or 5 mg/kg) vestibular dysfunction as revealed by a decrease of slow phase velocity (SPV). In addition, cisplatin significantly inhibited the Na{sup +}, K{sup +}-ATPase and Ca{sup 2+}-ATPase activities in the ampullary tissue with a good dose-response relationship but not those of cerebellar cortex. Regression analysis indicated that a decrease of SPV was well correlated with the reduction of Na{sup +}, K{sup +}-ATPase and Ca{sup 2+}-ATPase activities of the ampullary tissue. D-Methionine (300 mg/kg) reduced both abnormalities of SPV and ATPase activities in a correlated manner. Moreover, cisplatin exposure led to a significant dose-dependent increase of lipid peroxidation and nitric oxide concentrations of the vestibules, which could be significantly suppressed by D-methionine. However, cisplatin did not alter the levels of lipid peroxidation and nitric oxide of the cerebellum. In conclusion, cisplatin inhibited ATPase activities and increased oxidative stress in guinea pig vestibular labyrinths. D-Methionine attenuated cisplatin-induced vestibulotoxicity associated with ionic disturbance through its antioxidative property.« less

Regulation of expression of Na+,K+-ATPase in androgen-dependent and androgen-independent prostate cancer

PubMed Central

Blok, L J; Chang, G T G; Steenbeek-Slotboom, M; Weerden, W M van; Swarts, H G P; Pont, J J H H M De; Steenbrugge, G J van; Brinkmann, A O

1999-01-01

The β1-subunit of Na+,K+-ATPase was isolated and identified as an androgen down-regulated gene. Expression was observed at high levels in androgen-independent as compared to androgen-dependent (responsive) human prostate cancer cell lines and xenografts when grown in the presence of androgens. Down-regulation of the β1-subunit was initiated at concentrations between 0.01 nM and 0.03 nM of the synthetic androgen R1881 after relatively long incubation times (> 24 h). Using polyclonal antibodies, the concentration of β1-subunit protein, but not of the α1-subunit protein, was markedly reduced in androgen-dependent human prostate cancer cells (LNCaP-FGC) cultured in the presence of androgens. In line with these observations it was found that the protein expression of total Na+,K+-ATPase in the membrane (measured by 3H-ouabain binding) was also markedly decreased. The main function of Na+,K+-ATPase is to maintain sodium and potassium homeostasis in animal cells. The resulting electrochemical gradient is facilitative for transport of several compounds over the cell membrane (for example cisplatin, a chemotherapeutic agent experimentally used in the treatment of hormone-refractory prostate cancer). Here we observed that a ouabain-induced decrease of Na+,K+-ATPase activity in LNCaP-FGC cells results in reduced sensitivity of these cells to cisplatin-treatment. Surprisingly, androgen-induced decrease of Na+,K+-ATPase expression, did not result in significant protection against the chemotherapeutic agent. © 1999 Cancer Research Campaign PMID:10487609

Phenylethynyl-butyltellurium inhibits the sulfhydryl enzyme Na+, K+ -ATPase: an effect dependent on the tellurium atom.

PubMed

Quines, Caroline B; Rosa, Suzan G; Neto, José S S; Zeni, Gilson; Nogueira, Cristina W

2013-11-01

Organotellurium compounds are known for their toxicological effects. These effects may be associated with the chemical structure of these compounds and the oxidation state of the tellurium atom. In this context, 2-phenylethynyl-butyltellurium (PEBT) inhibits the activity of the sulfhydryl enzyme, δ-aminolevulinate dehydratase. The present study investigated on the importance of the tellurium atom in the PEBT ability to oxidize mono- and dithiols of low molecular weight and sulfhydryl enzymes in vitro. PEBT, at high micromolar concentrations, oxidized dithiothreitol (DTT) and inhibited cerebral Na(+), K(+)-ATPase activity, but did not alter the lactate dehydrogenase activity. The inhibition of cerebral Na(+), K(+)-ATPase activity was completely restored by DTT. By contrast, 2-phenylethynyl-butyl, a molecule without the tellurium atom, neither oxidized DTT nor altered the Na(+), K(+)-ATPase activity. In conclusion, the tellurium atom of PEBT is crucial for the catalytic oxidation of sulfhydryl groups from thiols of low molecular weight and from Na(+), K(+)-ATPase.

A Global Survey of ATPase Activity in Plasmodium falciparum Asexual Blood Stages and Gametocytes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ortega, Corrie; Frando, Andrew; Webb-Robertson, Bobbie-Jo

Effective malaria control and elimination in hyperendemic areas of the world will require treatment of disease-causing Plasmodium falciparum (Pf) blood stage infection but also blocking parasite transmission from humans to mosquito to prevent disease spread. Numerous antimalarial drugs have become ineffective due to parasite drug resistance and many currently used therapies do not kill gametocytes, highly specialized sexual parasite stages with distinct physiology that are necessary for transmission from the human host to the mosquito vector. Further confounding next generation drug development against Pf is the lack of known biochemical activity for most parasite gene products as well as themore » unknown metabolic needs of non-replicating gametocyte. Here, we take a systematic activity-based proteomics approach to survey the large and druggable ATPase family that is associated with replicating blood stage asexual parasites and transmissible gametocytes. We experimentally confirm existing annotation and predict ATPase function for 38 uncharacterized proteins. ATPase activity broadly changes during the transition from asexual schizonts to gametocytes, indicating altered metabolism and regulatory roles of ATPases specific for each lifecycle stage. By mapping the activity of ATPases associated with gametocytogenesis, we assign biochemical activity to a large number of uncharacterized proteins and identify new candidate transmission blocking targets.« less

Increased oxidative stress and decreased activities of Ca2+/Mg2+-ATPase and Na+/K+-ATPase in the red blood cells of the hibernating black bear

USGS Publications Warehouse

Chauhan, V.P.S.; Tsiouris, J.A.; Chauhan, A.; Sheikh, A.M.; Brown, W. Ted; Vaughan, M.

2002-01-01

During hibernation, animals undergo metabolic changes that result in reduced utilization of glucose and oxygen. Fat is known to be the preferential source of energy for hibernating animals. Malonyldialdehyde (MDA) is an end product of fatty acid oxidation, and is generally used as an index of lipid peroxidation. We report here that peroxidation of lipids is increased in the plasma and in the membranes of red blood cells in black bears during hibernation. The plasma MDA content was about four fold higher during hibernation as compared to that during the active, non-hibernating state (P < 0.0001). Similarly, MDA content of erythrocyte membranes was significantly increased during hibernation (P < 0.025). The activity of Ca2+/Mg2+-ATPase in the erythrocyte membrane was significantly decreased in the hibernating state as compared to the active state. Na+/K+-ATPase activity was also decreased, though not significant, during hibernation. These results suggest that during hibernation, the bears are under increased oxidative stress, and have reduced activities of membrane-bound enzymes such as Ca2+/Mg2+-ATPase and Na+/K+-ATPase. These changes can be considered part of the adaptive for survival process of metabolic depression. ?? 2002 Elsevier Science Inc. All rights reserved.

Salinity fluctuation influencing biological adaptation: growth dynamics and Na+ /K+ -ATPase activity in a euryhaline bacterium.

PubMed

Yang, Hao; Meng, Yang; Song, Youxin; Tan, Yalin; Warren, Alan; Li, Jiqiu; Lin, Xiaofeng

2017-07-01

Although salinity fluctuation is a prominent characteristic of many coastal ecosystems, its effects on biological adaptation have not yet been fully recognized. To test the salinity fluctuations on biological adaptation, population growth dynamics and Na + /K + -ATPase activity were investigated in the euryhaline bacterium Idiomarina sp. DYB, which was acclimated at different salinity exposure levels, exposure times, and shifts in direction of salinity. Results showed: (1) bacterial population growth dynamics and Na + /K + -ATPase activity changed significantly in response to salinity fluctuation; (2) patterns of variation in bacterial growth dynamics were related to exposure times, levels of salinity, and shifts in direction of salinity change; (3) significant tradeoffs were detected between growth rate (r) and carrying capacity (K) on the one hand, and Na + /K + -ATPase activity on the other; and (4) beneficial acclimation was confirmed in Idiomarina sp. DYB. In brief, this study demonstrated that salinity fluctuation can change the population growth dynamics, Na + /K + -ATPase activity, and tradeoffs between r, K, and Na + /K + -ATPase activity, thus facilitating bacterial adaption in a changing environment. These findings provide constructive information for determining biological response patterns to environmental change. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Increased pressure during retrograde cerebral perfusion provides better preservation of the Na+, K+-ATPase activity.

PubMed

Yang, Luojia; Li, Zhijun; Yang, Yanmin; Zhu, Raymound; Summers, Randy; Deslauriers, Roxanne; Ye, Jian

2006-11-01

This study was carried out to determine if increased perfusion pressure during retrograde cerebral perfusion (RCP) provides better preservation of the brain Na+, K+-ATPase activity. Twenty pigs were subjected to anesthesia alone (control group, n=5), hypothermic circulatory arrest (HCA) (HCA group, n = 5), HCA+RCP at perfusion pressures of 24-29 mmHg (Low-pressure group, n=5), or HCA+RCP at perfusion pressures of 34-40 mmHg (High-pressure group, n = 5). The brain was harvested for the measurement of tissue Na+, K+-ATPase activity. Relative to the control pigs (67.2 +/- 2.1%), significant impairment of Na+, K+-ATPase activity was observed in all three experimental groups (29.8 +/- 7.4% in HCA group, 33.5 +/- 2.9% in the Low-pressure group, and 52.0 +/- 1.8% in the High-pressure group, p < 0.01). The best preservation of the enzyme, particularly in the cortex and cerebellum regions, was observed in the High-pressure group (p < 0.01). In conclusion, HCA causes severe impairment of Na+, K+-ATPase activity, and increasing perfusion pressures from 24-29 to 34-40 mmHg during RCP significantly improves preservation of Na+, K+-ATPase activity, and the improvement of the protection varies in different regions of the brain.

Dependence of renal (Na+ + k+)-adenosine triphosphatase activity on thyroid status.

PubMed

Lo, S C; August, T R; Liberman, U A; Edelman, I S

1976-12-25

In thyroidectomized rats, a single injection of L-2,,5,2'-triiodothyronine (T3) (50mug/100 g body weight) elicited at 45% increase in (Na+ + k+)-dependent adenosine triphosphatase (NaK-ATPase) activity of the membrane-rich fraction of renal cortex at the optimal time of response, 48 h after injection. Three successive doses of T3 (50 mug/100 g body weight), given on alternate days, increased NaK-ATPase by 67% in the renal cortex but had no significant effect on the outer medulla or the papilla. Moreover, T3 had no effect on Mg2+-dependent adenosine trisphatase (MgATPase) in cortex, cedulla, or papilla. Three doses of T3 (50 mug/100 g body weight) given on alternate days to thyroidectomized rats elecited a 134, 79, and 46% increase in Vmax for ATP, Na4, and K+, respectively. There were no changes in the Km for ATP or the K1/2 values for Na+ and K+. Two methods were used to estimate the effect of T3 on the number of NaK-ATPase units (assumed to represent the number of Na+ pump sites); rat renal plasma membrane fractions were incubated with [gamma-32P]ATP, Mg2+, and Na+; the 32P-labeled membrane protein yeild was quantitatively dependent on Na+ and was hydrolyzed on addition of K+. There was a linear correlation between the specific activity of NaK-ATPase (Vmax) and the amount of phosphorylated intermediate formed, in renal cortical membrane fractions from thyroidectomized rats given T3 or the diluent. There was also a linear correlation between the specific activity of NaK-ATPase (Vmax) and the amount of [3H]ouabain specifically bound (Na+-, Mg2+-, APT-dependent) to the NaK-ATPase preparation. Injection of T3 resulted in a 70% increase in NaK-ATPase activity, a 79% increase in formation of the phosphorylated intermediate, and a 65% increase in the [H]ouabain specifically bound to the NaK-ATPase system. The T3-dependent increases in Vmax for ATP, Na+, and K+ and the proportionate increases in the phosphorylated intermediate and in the amount of [3H]ouabain bound indicate that T3 increases the number of NaK-ATPase units and that this increase accounts for the increase in NaK-ATPase activity.

Inositol phosphates influence the membrane bound Ca/sup 2 +//Mg/sup 2 +/ stimulated ATPase from human erythrocyte membranes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kester, M.; Ekholm, J.; Kumar, R.

1986-03-01

The modulation by exogenous inositol phosphates of the membrane Ca/sup 2 +//Mg/sup 2 +/ ATPase from saponin/EGTA lysed human erythrocytes was determined in a buffer (pH 7.6) containing histidine, 80 mM, MgCl/sub 2/, 3.3 mM, NaCl, 74 mM, KCl, 30 mM, Na/sub 2/ATP, 2.3 mM, ouabain, 0.83 mM, with variable amounts of CaCl/sub 2/ and EGTA. The ATPase assay was linear with time at 44/sup 0/C. The inositol phosphates were commercially obtained and were also prepared from /sup 32/P labeled rabbit platelet inositol phospholipids. Inositol triphosphate (IP/sub 3/) elevated the Ca/sup 2 +//Mg/sup 2 +/ ATPase activity over basal levelsmore » in a dose, time, and calcium dependent manner and were increased up to 85% of control values. Activities for the Na/sup +//K/sup +/-ATPase and a Mg/sup 2 +/ ATPase were not effected by IP/sub 3/. Ca/sup 2 +//Mg/sup 2 +/APTase activity with IP/sub 2/ or IP/sub 3/ could be synergistically elevated with calmodulin addition. The activation of the ATPase with IP/sub 3/ was calcium dependent in a range from .001 to .02 mM. The apparent Km and Vmax values were determined for IP/sub 3/ stimulated Ca/sup 2 +//Mg/sup 2 +/ ATPase.« less

Effect of Reduction of Redox Modifications of Cys-Residues in the Na,K-ATPase α1-Subunit on Its Activity

PubMed Central

Dergousova, Elena A.; Petrushanko, Irina Yu.; Klimanova, Elizaveta A.; Mitkevich, Vladimir A.; Ziganshin, Rustam H.; Lopina, Olga D.; Makarov, Alexander A.

2017-01-01

Sodium-potassium adenosine triphosphatase (Na,K-ATPase) creates a gradient of sodium and potassium ions necessary for the viability of animal cells, and it is extremely sensitive to intracellular redox status. Earlier we found that regulatory glutathionylation determines Na,K-ATPase redox sensitivity but the role of basal glutathionylation and other redox modifications of cysteine residues is not clear. The purpose of this study was to detect oxidized, nitrosylated, or glutathionylated cysteine residues in Na,K-ATPase, evaluate the possibility of removing these modifications and assess their influence on the enzyme activity. To this aim, we have detected such modifications in the Na,K-ATPase α1-subunit purified from duck salt glands and tried to eliminate them by chemical reducing agents and the glutaredoxin1/glutathione reductase enzyme system. Detection of cysteine modifications was performed using mass spectrometry and Western blot analysis. We have found that purified Na,K-ATPase α1-subunit contains glutathionylated, nitrosylated, and oxidized cysteines. Chemical reducing agents partially eliminate these modifications that leads to the slight increase of the enzyme activity. Enzyme system glutaredoxin/glutathione reductase, unlike chemical reducing agents, produces significant increase of the enzyme activity. At the same time, the enzyme system deglutathionylates native Na,K-ATPase to a lesser degree than chemical reducing agents. This suggests that the enzymatic reducing system glutaredoxin/glutathione reductase specifically affects glutathionylation of the regulatory cysteine residues of Na,K-ATPase α1-subunit. PMID:28230807

Dual Role of DNA in Regulating ATP Hydrolysis by the SopA Partition Protein*

PubMed Central

Ah-Seng, Yoan; Lopez, Frederic; Pasta, Franck; Lane, David; Bouet, Jean-Yves

2009-01-01

In bacteria, mitotic stability of plasmids and many chromosomes depends on replicon-specific systems, which comprise a centromere, a centromere-binding protein and an ATPase. Dynamic self-assembly of the ATPase appears to enable active partition of replicon copies into cell-halves, but for Walker-box partition ATPases the molecular mechanism is unknown. ATPase activity appears to be essential for this process. DNA and centromere-binding proteins are known to stimulate the ATPase activity but molecular details of the stimulation mechanism have not been reported. We have investigated the interactions which stimulate ATP hydrolysis by the SopA partition ATPase of plasmid F. By using SopA and SopB proteins deficient in DNA binding, we have found that the intrinsic ability of SopA to hydrolyze ATP requires direct DNA binding by SopA but not by SopB. Our results show that two independent interactions of SopA act in synergy to stimulate its ATPase. SopA must interact with (i) DNA, through its ATP-dependent nonspecific DNA binding domain and (ii) SopB, which we show here to provide an arginine-finger motif. In addition, the latter interaction stimulates ATPase maximally when SopB is part of the partition complex. Hence, our data demonstrate that DNA acts on SopA in two ways, directly as nonspecific DNA and through SopB as centromeric DNA, to fully activate SopA ATP hydrolysis. PMID:19740757

The V-ATPase subunit A is essential for salt tolerance through participating in vacuolar Na+ compartmentalization in Salicornia europaea.

PubMed

Lv, Sulian; Jiang, Ping; Tai, Fang; Wang, Duoliya; Feng, Juanjuan; Fan, Pengxiang; Bao, Hexigeduleng; Li, Yinxin

2017-12-01

The V-ATPase subunit A participates in vacuolar Na + compartmentalization in Salicornia europaea regulating V-ATPase and V-PPase activities. Na + sequestration into the vacuole is an efficient strategy in response to salinity in many halophytes. However, it is not yet fully understood how this process is achieved. Particularly, the role of vacuolar H + -ATPase (V-ATPase) in this process is controversial. Our previous proteomic investigation in the euhalophyte Salicornia europaea L. found a significant increase of the abundance of V-ATPase subunit A under salinity. Here, the gene encoding this subunit named SeVHA-A was characterized, and its role in salt tolerance was demonstrated by RNAi directed downregulation in suspension-cultured cells of S. europaea. The transcripts of genes encoding vacuolar H + -PPase (V-PPase) and vacuolar Na + /H + antiporter (SeNHX1) also decreased significantly in the RNAi cells. Knockdown of SeVHA-A resulted in a reduction in both V-ATPase and vacuolar H + -PPase (V-PPase) activities. Accordingly, the SeVHA-A-RNAi cells showed increased vacuolar pH and decreased cell viability under different NaCl concentrations. Further Na + staining showed the reduced vacuolar Na + sequestration in RNAi cells. Taken together, our results evidenced that SeVHA-A participates in vacuolar Na + sequestration regulating V-ATPase and V-PPase activities and thereby vacuolar pH in S. europaea. The possible mechanisms underlying the reduction of vacuolar V-PPase activity in SeVHA-A-RNAi cells were also discussed.

Down-regulated Na+/K+-ATPase activity in ischemic penumbra after focal cerebral ischemia/reperfusion in rats

PubMed Central

Huang, Hao; Chen, Yang-Mei; Zhu, Fei; Tang, Shi-Ting; Xiao, Ji-Dong; Li, Lv-Li; Lin, Xin-Jing

2015-01-01

This study was aimed to examine whether the Na+/K+ adenosine triphosphatase (Na+/K+-ATPase) activity in ischemic penumbra is associated with the pathogenesis of ischemia/reperfusion-induced brain injury. An experimental model of cerebral ischemia/reperfusion was made by transient middle cerebral artery occlusion (tMCAO) in rats and the changes of Na+/K+-ATPase activity in the ischemic penumbra was examined by Enzyme Assay Kit. Extensive infarction was observed in the frontal and parietal cortical and subcortical areas at 6 h, 24 h, 48 h, 3 d and 7 d after tMCAO. Enzyme Assay analyses revealed the activity of Na+/K+-ATPase was decreased in the ischemic penumbra of model rats after focal cerebral ischemia/reperfusion compared with sham-operated rats, and reduced to its minimum at 48 h, while the infarct volume was enlarged gradually. In addition, accompanied by increased brain water content, apoptosis-related bcl-2 and Bax proteins, apoptotic index and neurologic deficits Longa scores, but fluctuated the ratio of bcl-2/Bax. Correlation analysis showed that the infarct volume, apoptotic index, neurologic deficits Longa scores and brain water content were negatively related with Na+/K+-ATPase activity, while the ratio of bcl-2/Bax was positively related with Na+/K+-ATPase activity. Our results suggest that down-regulated Na+/K+-ATPase activity in ischemic penumbra might be involved in the pathogenesis of cerebral ischemia/reperfusion injury presumably through the imbalance ratio of bcl-2/Bax and neuronal apoptosis, and identify novel target for neuroprotective therapeutic intervention in cerebral ischemic disease. PMID:26722460

A mutation within the extended X loop abolished substrate-induced ATPase activity of the human liver ATP-binding cassette (ABC) transporter MDR3.

PubMed

Kluth, Marianne; Stindt, Jan; Dröge, Carola; Linnemann, Doris; Kubitz, Ralf; Schmitt, Lutz

2015-02-20

The human multidrug resistance protein 3 (MDR3/ABCB4) belongs to the ubiquitous family of ATP-binding cassette (ABC) transporters and is located in the canalicular membrane of hepatocytes. There it flops the phospholipids of the phosphatidylcholine (PC) family from the inner to the outer leaflet. Here, we report the characterization of wild type MDR3 and the Q1174E mutant, which was identified previously in a patient with progressive familial intrahepatic cholestasis type 3 (PFIC-3). We expressed different variants of MDR3 in the yeast Pichia pastoris, purified the proteins via tandem affinity chromatography, and determined MDR3-specific ATPase activity in the presence or absence of phospholipids. The ATPase activity of wild type MDR3 was stimulated 2-fold by liver PC or 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine lipids. Furthermore, the cross-linking of MDR3 with a thiol-reactive fluorophore blocked ATP hydrolysis and exhibited no PC stimulation. Similarly, phosphatidylethanolamine, phosphatidylserine, and sphingomyelin lipids did not induce an increase of wild type MDR3 ATPase activity. The phosphate analogues beryllium fluoride and aluminum fluoride led to complete inhibition of ATPase activity, whereas orthovanadate inhibited exclusively the PC-stimulated ATPase activity of MDR3. The Q1174E mutation is located in the nucleotide-binding domain in direct proximity of the leucine of the ABC signature motif and extended the X loop, which is found in ABC exporters. Our data on the Q1174E mutant demonstrated basal ATPase activity, but PC lipids were incapable of stimulating ATPase activity highlighting the role of the extended X loop in the cross-talk of the nucleotide-binding domain and the transmembrane domain. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Multibiomarker assessment of three Brazilian estuaries using oysters as bioindicators

DOE Office of Scientific and Technical Information (OSTI.GOV)

Valdez Domingos, F.X.; Azevedo, M.; Silva, M.D.

2007-11-15

Oysters have been largely employed as bioindicators of environmental quality in biomonitoring studies. Crassostrea rhizophorae was selected to evaluate the health status of three estuarine areas impacted by anthropogenic activities along the Brazilian coast, in three estuarine complexes, ranging in latitude from 7 to 25 deg. S. In each estuary three sites were sampled in Winter and in Summer: a site considered as reference, and two sites next to contamination sources. Condition index was similar at all sites and estuaries, with the highest values found for Itamaraca oysters in Summer. Necrosis, hyperplasia, mucocyte hypertrophy and fusion of ordinary filaments weremore » the main histopathological lesions observed. Muscle cholinesterase activity was overall similar, but with a strong seasonal effect. Inhibition or activation of branchial total ATPase and Na,K-ATPase activities at the contaminated sites was observed. The health status of these estuarine areas is quite similar, and the combined use of biomarkers is recommended.« less

Reduction of oxidative-nitrosative stress underlies anticataract effect of topically applied tocotrienol in streptozotocin-induced diabetic rats

PubMed Central

Abdul Nasir, Nurul Alimah; Agarwal, Renu; Sheikh Abdul Kadir, Siti Hamimah; Vasudevan, Sushil; Tripathy, Minaketan; Iezhitsa, Igor; Mohammad Daher, Aqil; Ibrahim, Mohd Ikraam; Mohd Ismail, Nafeeza

2017-01-01

Cataract, a leading cause of blindness, is of special concern in diabetics as it occurs at earlier onset. Polyol accumulation and increased oxidative-nitrosative stress in cataractogenesis are associated with NFκB activation, iNOS expression, ATP depletion, loss of ATPase functions, calpain activation and proteolysis of soluble to insoluble proteins. Tocotrienol was previously shown to reduce lens oxidative stress and inhibit cataractogenesis in galactose-fed rats. In current study, we investigated anticataract effects of topical tocotrienol and possible mechanisms involved in streptozotocin-induced diabetic rats. Diabetes was induced in Sprague Dawley rats by intraperitoneal injection of streptozotocin. Diabetic rats were treated with vehicle (DV) or tocotrienol (DT). A third group consists of normal, non-diabetic rats were treated with vehicle (NV). All treatments were given topically, bilaterally, twice daily for 8 weeks with weekly slit lamp monitoring. Subsequently, rats were euthanized and lenses were subjected to estimation of polyol accumulation, oxidative-nitrosative stress, NFκB activation, iNOS expression, ATP levels, ATPase activities, calpain activity and total protein levels. Cataract progression was delayed from the fifth week onwards in DT with lower mean of cataract stages compared to DV group (p<0.01) despite persistent hyperglycemia. Reduced cataractogenesis in DT group was accompanied with lower aldose reductase activity and sorbitol level compared to DV group (p<0.01). DT group also showed reduced NFκB activation, lower iNOS expression and reduced oxidative-nitrosative stress compared to DV group. Lenticular ATP and ATPase and calpain 2 activities in DT group were restored to normal. Consequently, soluble to insoluble protein ratio in DT group was higher compared to DV (p<0.05). In conclusion, preventive effect of topical tocotrienol on development of cataract in STZ-induced diabetic rats could be attributed to reduced lens aldose reductase activity, polyol levels and oxidative-nitrosative stress. These effects of tocotrienol invlove reduced NFκB activation, lower iNOS expression, restoration of ATP level, ATPase activities, calpain activity and lens protein levels. PMID:28350848

Does the intercept of the heat-stress relation provide an accurate estimate of cardiac activation heat?

PubMed

Pham, Toan; Tran, Kenneth; Mellor, Kimberley M; Hickey, Anthony; Power, Amelia; Ward, Marie-Louise; Taberner, Andrew; Han, June-Chiew; Loiselle, Denis

2017-07-15

The heat of activation of cardiac muscle reflects the metabolic cost of restoring ionic homeostasis following a contraction. The accuracy of its measurement depends critically on the abolition of crossbridge cycling. We abolished crossbridge activity in isolated rat ventricular trabeculae by use of blebbistatin, an agent that selectively inhibits myosin II ATPase. We found cardiac activation heat to be muscle length independent and to account for 15-20% of total heat production at body temperature. We conclude that it can be accurately estimated at minimal muscle length. Activation heat arises from two sources during the contraction of striated muscle. It reflects the metabolic expenditure associated with Ca 2+ pumping by the sarcoplasmic reticular Ca 2+ -ATPase and Ca 2+ translocation by the Na + /Ca 2+ exchanger coupled to the Na + ,K + -ATPase. In cardiac preparations, investigators are constrained in estimating its magnitude by reducing muscle length to the point where macroscopic twitch force vanishes. But this experimental protocol has been criticised since, at zero force, the observed heat may be contaminated by residual crossbridge cycling activity. To eliminate this concern, the putative thermal contribution from crossbridge cycling activity must be abolished, at least at minimal muscle length. We achieved this using blebbistatin, a selective inhibitor of myosin II ATPase. Using a microcalorimeter, we measured the force production and heat output, as functions of muscle length, of isolated rat trabeculae from both ventricles contracting isometrically at 5 Hz and at 37°C. In the presence of blebbistatin (15 μmol l -1 ), active force was zero but heat output remained constant, at all muscle lengths. Activation heat measured in the presence of blebbistatin was not different from that estimated from the intercept of the heat-stress relation in its absence. We thus reached two conclusions. First, activation heat is independent of muscle length. Second, residual crossbridge heat is negligible at zero active force; hence, the intercept of the cardiac heat-force relation provides an estimate of activation heat uncontaminated by crossbridge cycling. Both results resolve long-standing disputes in the literature. © 2017 The Authors. The Journal of Physiology © 2017 The Physiological Society.

Reduction of oxidative-nitrosative stress underlies anticataract effect of topically applied tocotrienol in streptozotocin-induced diabetic rats.

PubMed

Abdul Nasir, Nurul Alimah; Agarwal, Renu; Sheikh Abdul Kadir, Siti Hamimah; Vasudevan, Sushil; Tripathy, Minaketan; Iezhitsa, Igor; Mohammad Daher, Aqil; Ibrahim, Mohd Ikraam; Mohd Ismail, Nafeeza

2017-01-01

Cataract, a leading cause of blindness, is of special concern in diabetics as it occurs at earlier onset. Polyol accumulation and increased oxidative-nitrosative stress in cataractogenesis are associated with NFκB activation, iNOS expression, ATP depletion, loss of ATPase functions, calpain activation and proteolysis of soluble to insoluble proteins. Tocotrienol was previously shown to reduce lens oxidative stress and inhibit cataractogenesis in galactose-fed rats. In current study, we investigated anticataract effects of topical tocotrienol and possible mechanisms involved in streptozotocin-induced diabetic rats. Diabetes was induced in Sprague Dawley rats by intraperitoneal injection of streptozotocin. Diabetic rats were treated with vehicle (DV) or tocotrienol (DT). A third group consists of normal, non-diabetic rats were treated with vehicle (NV). All treatments were given topically, bilaterally, twice daily for 8 weeks with weekly slit lamp monitoring. Subsequently, rats were euthanized and lenses were subjected to estimation of polyol accumulation, oxidative-nitrosative stress, NFκB activation, iNOS expression, ATP levels, ATPase activities, calpain activity and total protein levels. Cataract progression was delayed from the fifth week onwards in DT with lower mean of cataract stages compared to DV group (p<0.01) despite persistent hyperglycemia. Reduced cataractogenesis in DT group was accompanied with lower aldose reductase activity and sorbitol level compared to DV group (p<0.01). DT group also showed reduced NFκB activation, lower iNOS expression and reduced oxidative-nitrosative stress compared to DV group. Lenticular ATP and ATPase and calpain 2 activities in DT group were restored to normal. Consequently, soluble to insoluble protein ratio in DT group was higher compared to DV (p<0.05). In conclusion, preventive effect of topical tocotrienol on development of cataract in STZ-induced diabetic rats could be attributed to reduced lens aldose reductase activity, polyol levels and oxidative-nitrosative stress. These effects of tocotrienol invlove reduced NFκB activation, lower iNOS expression, restoration of ATP level, ATPase activities, calpain activity and lens protein levels.

Prolonged Exposure of Primary Human Muscle Cells to Plasma Fatty Acids Associated with Obese Phenotype Induces Persistent Suppression of Muscle Mitochondrial ATP Synthase β Subunit.

PubMed

Tran, Lee; Hanavan, Paul D; Campbell, Latoya E; De Filippis, Elena; Lake, Douglas F; Coletta, Dawn K; Roust, Lori R; Mandarino, Lawrence J; Carroll, Chad C; Katsanos, Christos S

2016-01-01

Our previous studies show reduced abundance of the β-subunit of mitochondrial H+-ATP synthase (β-F1-ATPase) in skeletal muscle of obese individuals. The β-F1-ATPase forms the catalytic core of the ATP synthase, and it is critical for ATP production in muscle. The mechanism(s) impairing β-F1-ATPase metabolism in obesity, however, are not completely understood. First, we studied total muscle protein synthesis and the translation efficiency of β-F1-ATPase in obese (BMI, 36±1 kg/m2) and lean (BMI, 22±1 kg/m2) subjects. Both total protein synthesis (0.044±0.006 vs 0.066±0.006%·h-1) and translation efficiency of β-F1-ATPase (0.0031±0.0007 vs 0.0073±0.0004) were lower in muscle from the obese subjects when compared to the lean controls (P<0.05). We then evaluated these same responses in a primary cell culture model, and tested the specific hypothesis that circulating non-esterified fatty acids (NEFA) in obesity play a role in the responses observed in humans. The findings on total protein synthesis and translation efficiency of β-F1-ATPase in primary myotubes cultured from a lean subject, and after exposure to NEFA extracted from serum of an obese subject, were similar to those obtained in humans. Among candidate microRNAs (i.e., non-coding RNAs regulating gene expression), we identified miR-127-5p in preventing the production of β-F1-ATPase. Muscle expression of miR-127-5p negatively correlated with β-F1-ATPase protein translation efficiency in humans (r = - 0.6744; P<0.01), and could be modeled in vitro by prolonged exposure of primary myotubes derived from the lean subject to NEFA extracted from the obese subject. On the other hand, locked nucleic acid inhibitor synthesized to target miR-127-5p significantly increased β-F1-ATPase translation efficiency in myotubes (0.6±0.1 vs 1.3±0.3, in control vs exposure to 50 nM inhibitor; P<0.05). Our experiments implicate circulating NEFA in obesity in suppressing muscle protein metabolism, and establish impaired β-F1-ATPase translation as an important consequence of obesity.

The Role of the Plasma Membrane H+-ATPase in Plant Responses to Aluminum Toxicity.

PubMed

Zhang, Jiarong; Wei, Jian; Li, Dongxu; Kong, Xiangying; Rengel, Zed; Chen, Limei; Yang, Ye; Cui, Xiuming; Chen, Qi

2017-01-01

Aluminum (Al) toxicity is a key factor limiting plant growth and crop production on acid soils. Increasing the plant Al-detoxification capacity and/or breeding Al-resistant cultivars are a cost-effective strategy to support crop growth on acidic soils. The plasma membrane H + -ATPase plays a central role in all plant physiological processes. Changes in the activity of the plasma membrane H + -ATPase through regulating the expression and phosphorylation of this enzyme are also involved in many plant responses to Al toxicity. The plasma membrane H + -ATPase mediated H + influx may be associated with the maintenance of cytosolic pH and the plasma membrane gradients as well as Al-induced citrate efflux mediated by a H + -ATPase-coupled MATE co-transport system. In particular, modulating the activity of plasma membrane H + -ATPase through application of its activators (e.g., magnesium or IAA) or using transgenics has effectively enhanced plant resistance to Al stress in several species. In this review, we critically assess the available knowledge on the role of the plasma membrane H + -ATPase in plant responses to Al stress, incorporating physiological and molecular aspects.

Characterization of digitalis-like factors in human plasma. Interactions with NaK-ATPase and cross-reactivity with cardiac glycoside-specific antibodies.

PubMed

Kelly, R A; O'Hara, D S; Canessa, M L; Mitch, W E; Smith, T W

1985-09-25

Much of the evidence for a physiologically important endogenous inhibitor of the sodium pump has been either contradictory or indirect. We have identified three discrete fractions in desalted deproteinized plasma from normal humans that resemble the digitalis glycosides in that they: are of low molecular weight; are resistant to acid and enzymatic proteolysis; inhibit NaK-ATPase activity; inhibit Na+ pump activity in human erythrocytes; displace [3H]ouabain bound to the enzyme; and cross-react with high-affinity polyclonal and monoclonal digoxin-specific antibodies but not with anti-ouabain or anti-digitoxin antibodies. An additional fraction cross-reacted with digoxin-specific antibodies but had no detectable activity against NaK-ATPase. The three inhibitory fractions differed from cardiac glycosides in that their concentration-effect curves in a NaK-ATPase inhibition and [3H]ouabain radioreceptor assays were steeper than unlabeled ouabain. This suggests that these inhibitors are not simple competitive ligands for binding to NaK-ATPase. In the presence of sodium, no fraction required ATP for binding to NaK-ATPase, and in the presence of potassium, only one fraction had the reduced affinity for the enzyme that is characteristic of cardiac glycosides. Unlike digitalis, all three NaK-ATPase inhibitory fractions stimulated the activity of skeletal muscle sarcoplasmic reticulum Ca-ATPase. The presence of at least three fractions in human plasma that inhibit NaK-ATPase and cross-react to a variable degree with different digoxin-specific antibody populations could explain much of the conflicting evidence for the existence of endogenous digitalis-like compounds in plasma.

Compensatory Internalization of Pma1 in V-ATPase Mutants in Saccharomyces cerevisiae Requires Calcium- and Glucose-Sensitive Phosphatases.

PubMed

Velivela, Swetha Devi; Kane, Patricia M

2018-02-01

Loss of V-ATPase activity in organelles, whether through V-ATPase inhibition or V-ATPase ( vma ) mutations, triggers a compensatory downregulation of the essential plasma membrane proton pump Pma1 in Saccharomyces cerevisiae We have previously determined that the α-arrestin Rim8 and ubiquitin ligase Rsp5 are essential for Pma1 ubiquination and endocytosis in response to loss of V-ATPase activity. Here, we show that Pma1 endocytosis in V-ATPase mutants does not require Rim101 pathway components upstream and downstream of Rim8, indicating that Rim8 is acting independently in Pma1 internalization. We find that two phosphatases, the calcium-responsive phosphatase calcineurin and the glucose-sensitive phosphatase Glc7 (PP1), and one of the Glc7 regulatory subunits Reg1, exhibit negative synthetic genetic interactions with vma mutants, and demonstrate that both phosphatases are essential for ubiquitination and endocytic downregulation of Pma1 in these mutants. Although both acute and chronic loss of V-ATPase activity trigger the internalization of ∼50% of surface Pma1, a comparable reduction in Pma1 expression in a pma1-007 mutant neither compensates for loss of V-ATPase activity nor stops further Pma1 endocytosis. The results indicate that the cell surface level of Pma1 is not directly sensed and that internalized Pma1 may play a role in compensating for loss of V-ATPase-dependent acidification. Taken together, these results provide new insights into cross talk between two major proton pumps central to cellular pH control. Copyright © 2018 by the Genetics Society of America.

DNA polymerase V activity is autoregulated by a novel intrinsic DNA-dependent ATPase

PubMed Central

Erdem, Aysen L; Jaszczur, Malgorzata; Bertram, Jeffrey G; Woodgate, Roger; Cox, Michael M; Goodman, Myron F

2014-01-01

Escherichia coli DNA polymerase V (pol V), a heterotrimeric complex composed of UmuD′2C, is marginally active. ATP and RecA play essential roles in the activation of pol V for DNA synthesis including translesion synthesis (TLS). We have established three features of the roles of ATP and RecA. (1) RecA-activated DNA polymerase V (pol V Mut), is a DNA-dependent ATPase; (2) bound ATP is required for DNA synthesis; (3) pol V Mut function is regulated by ATP, with ATP required to bind primer/template (p/t) DNA and ATP hydrolysis triggering dissociation from the DNA. Pol V Mut formed with an ATPase-deficient RecA E38K/K72R mutant hydrolyzes ATP rapidly, establishing the DNA-dependent ATPase as an intrinsic property of pol V Mut distinct from the ATP hydrolytic activity of RecA when bound to single-stranded (ss)DNA as a nucleoprotein filament (RecA*). No similar ATPase activity or autoregulatory mechanism has previously been found for a DNA polymerase. DOI: http://dx.doi.org/10.7554/eLife.02384.001 PMID:24843026

Effect of alpha-tocopherol supplementation on renal oxidative stress and Na+/K+ -adenosine triphosphatase in ethanol treated Wistar rats.

PubMed

Mailankot, Maneesh; Jayalekshmi, H; Chakrabarti, Amit; Alang, Neha; Vasudevan, D M

2009-07-01

Ethanol intoxication resulted in high extent of lipid peroxidation, and reduction in antioxidant defenses (decreased GSH, GSH/GSSG ratio, and catalase, SOD and GPx activities) and (Na+/K+)-ATPase activity in kidney. Alpha-tocopherol treatment effectively protected kidney from ethanol induced oxidative challenge and improved renal (Na+/K+)-ATPase activity. Ethanol induced oxidative stress in the kidney and decreased (Na+/K+)-ATPase activity could be reversed by treatment with ascorbic acid.

Gill Na+,K+-ATPase of Atlantic salmon smolts in freshwater is not a predictor of long-term growth in seawater

USGS Publications Warehouse

Zydlewski, Gayle B.; Zydlewski, Joseph D.

2012-01-01

Gill Na+,K+-ATPase activity is a widely used measure of osmoregulatory preparedness in salmonid smolts. The degree to which this measure may predict long term performance is uncertain. In order to assess the relationship of this enzyme to long term growth and ion homeostasis, a cohort of Atlantic salmon hatchery smolts was used in a controlled environment with no salinity perturbations. In May 2006, gill Na+,K+-ATPase activity from 940 individually PIT tagged, Penobscot River smolts (USFWS, Green Lake National Fish Hatchery, Maine, United States) was measured immediately prior to isothermal transfer from freshwater to 32 ppt seawater. From the observed range of activities, individuals were classified as having “low”, “middle”, or “high” enzyme activity levels. Individual size (fork length and mass) was recorded on days 0, 1, 3, and 14 and monthly for four months. Growth rates over four time periods were calculated for individual fish maintained until the end of the experiment. Gill Na+,K+-ATPase activities were also measured from a subset of sampled fish. All groups effectively osmoregulated as evidenced by minor perturbations in plasma osmolyte levels. Apart from initial weight loss on transfer, fish grew throughout the experiment, however, there were no differences (fish size, growth rate, and gill Na+,K+-ATPase activity in seawater) among groups with initially different gill Na+,K+-ATPase activities (prior to seawater entry). While gill Na+,K+-ATPase activity may be predictive of performance during the acute phase of acclimation (first few days), typical variation in this enzyme, expressed in freshwater at the peak of smolting, does not appear to be predictive of long-term growth in seawater.

Characterization of sarcoplasmic reticulum Ca(2+) ATPase pumps in muscle of patients with myotonic dystrophy and with hypothyroid myopathy.

PubMed

Guglielmi, V; Oosterhof, A; Voermans, N C; Cardani, R; Molenaar, J P; van Kuppevelt, T H; Meola, G; van Engelen, B G; Tomelleri, G; Vattemi, G

2016-06-01

Sarcoplasmic/endoplasmic reticulum Ca(2+) ATPase (SERCA) pumps play the major role in lowering cytoplasmic calcium concentration in skeletal muscle by catalyzing the ATP-dependent transport of Ca(2+) from the cytosol to the lumen of the sarcoplasmic reticulum (SR). Although SERCA abnormalities have been hypothesized to contribute to the dysregulation of intracellular Ca(2+) homeostasis and signaling in muscle of patients with myotonic dystrophy (DM) and hypothyroid myopathy, the characterization of SERCA pumps remains elusive and their impairment is still unclear. We assessed the activity of SR Ca(2+)-ATPase, expression levels and fiber distribution of SERCA1 and SERCA2, and oligomerization of SERCA1 protein in muscle of patients with DM type 1 and 2, and with hypothyroid myopathy. Our data provide evidence that SR Ca(2+) ATPase activity, protein levels and muscle fiber distribution of total SERCA1 and SERCA2, and SERCA1 oligomerization pattern are similar in patients with both DM1 and DM2, hypothyroid myopathy and in control subjects. We prove that SERCA1b, the neonatal isoform of SERCA1, is expressed at protein level in muscle of patients with DM2 and, in lower amount, of patients with DM1. Our present study demonstrates that SERCA function is not altered in muscle of patients with DM and with hypothyroid myopathy. Copyright © 2016 Elsevier B.V. All rights reserved.

[Signal transudation pathways in parietal cells of the gastric mucosa in experimental stomach ulcer].

PubMed

Ostapchenko, L I; Drobins'ka, O V; Chaĭka, V O; Bohun, L I; Bohdanova, O V; Kot, L I; Haĭda, L M

2009-01-01

The goal of the presented work was the research of signal transduction mechanism in the rat gastric parietal cells under stomach ulcer conditions. In these cells activation of adenylate cyclase (increase of cAMP level and proteinkinase A activity) and phosphoinositide (increases [Ca2+]i; cGMP and phoshatidylinocitole levels; proteinkinase C, proteinkinase G, and calmodulin-dependent-proteinkinase activity) of signals pathway was shown. An increase of plasma membrane phospholipids (PC, PS, PE, PI, LPC) level was shown. Under conditions of influence of the stress factor the membran enzymes activity (H+, K+ -ATPase, 5'-AMPase, Na+, K+ -ATPase, Ca2+, Mg2+ -ATPase and H+, K+ -ATPase) was considerably increased. The intensification of lipid peroxidation processes in rats was demonstrated.

Glycation of myofibrillar proteins and ATPase activity after incubation with eleven sugars.

PubMed

Syrový, I

1994-01-01

Rat skeletal muscle myofibrils were incubated in the presence of D-glucose, D-fructose, D-galactose, D-ribose, D-tagatose, D-arabinose, D-xylose, D-mannose, L-sorbose, L-rhamnose or DL-glyceraldehyde and myofibrillar ATPase activity as well as the extent of glycation was measured. The attachment of sugars to proteins during glycation was generally dependent on the percentage of a given sugar present in the open-chain form. Glycation resulted in the decrease of myofibrillar ATPase activity. This decrease was low after incubation of myofibrillar proteins with slowly glycating sugars (e.g. glucose) and high with fast glycating sugars (e.g. ribose or glyceraldehyde). ATPase activity was less reduced in the presence of beta-mercaptoethanol.

Silencing overexpression of FXYD3 protein in breast cancer cells amplifies effects of doxorubicin and γ-radiation on Na(+)/K(+)-ATPase and cell survival.

PubMed

Liu, Chia-Chi; Teh, Rachel; Mozar, Christine A; Baxter, Robert C; Rasmussen, Helge H

2016-01-01

FXYD3, also known as mammary tumor protein 8, is overexpressed in several common cancers, including in many breast cancers. We examined if such overexpression might protect Na(+)/K(+)-ATPase and cancer cells against the high levels of oxidative stress characteristic of many tumors and often induced by cancer treatments. We measured FXYD3 expression, Na(+)/K(+)-ATPase activity and glutathionylation of the β1 subunit of Na(+)/K(+)-ATPase, a reversible oxidative modification that inhibits the ATPase, in MCF-7 and MDA-MB-468 cells. Expression of FXYD3 was suppressed by transfection with FXYD3 siRNA. A colorimetric end-point assay was used to estimate cell viability. Apoptosis was estimated by caspase 3/7 (DEVDase) activation using a Caspase fluorogenic substrate kit. Expression of FXYD3 in MCF-7 breast cancer cells was ~eightfold and ~twofold higher than in non-cancer MCF-10A cells and MDA-MB-468 cancer cells, respectively. A ~50 % reduction in FXYD3 expression increased glutathionylation of the β1 Na(+)/K(+)-ATPase subunit and reduced Na(+)/K(+)-ATPase activity by ~50 %, consistent with the role of FXYD3 to facilitate reversal of glutathionylation of the β1 subunit of Na(+)/K(+)-ATPase and glutathionylation-induced inhibition of Na(+)/K(+)-ATPase. Treatment of MCF-7 and MDA-MB- 468 cells with doxorubicin or γ-radiation decreased cell viability and induced apoptosis. The treatments upregulated FXYD3 expression in MCF-7 but not in MDA-MB-468 cells and suppression of FXYD3 in MCF-7 but not in MDA-MB-468 cells amplified effects of treatments on Na(+)/K(+)-ATPase activity and treatment-induced cell death and apoptosis. Overexpression of FXYD3 may be a marker of resistance to cancer treatments and a potentially important therapeutic target.

H(+) -ATPase-defective variants of Lactobacillus delbrueckii subsp. bulgaricus contribute to inhibition of postacidification of yogurt during chilled storage.

PubMed

Wang, Xinhui; Ren, Hongyang; Liu, Dayu; Wang, Bing; Zhu, Wenyou; Wang, Wei

2013-02-01

Continued acid production by Lactobacillus delbrueckii subsp. bulgaricus during the chilled storage of yogurt is the major cause of postacidification, resulting in a short shelf life. Two H(+) -ATPase defective variants of L. delbrueckii subsp. bulgaricus were successfully isolated and their H(+) -ATPase activities were reduced by 51.3% and 34.3%, respectively. It was shown that growth and acid production of variants were remarkably inhibited. The variants were more sensitive to acidic condition and had a significant rate for inactivation of H(+) -ATPase by N, N-dicyclohexylcarbodiimide (DCCD), along with a low H(+) -extrusion, suggesting that H(+) -ATPase is direct response for H(+) -extrusion. In addition, the variants were also more sensitive to NaCl, while H(+) -ATPase activities of variants and parent strain were significantly enhanced by NaCl stress. Obviously, H(+) -ATPase might be involved in Na(+) transportation. Furthermore, variants were inoculated in fermented milk to ferment yogurt. There was no significant difference in flavor, whereas the postacidification of yogurt during chilled storage was remarkably inhibited. It is suggested that application of L. delbrueckii subsp. bulgaricus with reduced H(+) -ATPase activity in yogurt fermentation is one of effect, economic and simple avenues of inhibiting postacidification of yogurt during refrigerated storage, giving a longer shelf life. During yogurt fermentation, continued acid production by Lactobacillus delbrueckii subsp. bulgaricus during the chilled storage of yogurt leads to milk fermentation with high postacidification, resulting in a short shelf life. In this work, 2 acid-sensitive variant strains of L. delbrueckii subsp. bulgaricus were isolated. The characteristics related to H(+) -ATPase were compared and it was observed that milk fermented by the variants had lower postacidification, giving a longer shelf life. Application of L. delbrueckii subsp. bulgaricus with reduced H(+) -ATPase activity in yogurt fermentation might be one of effect, economic and simple avenues of inhibiting yogurt postacidification during chilled storage, giving a longer shelf life. © 2013 Institute of Food Technologists®

GammaM23K, gammaM232K, and gammaL77K single substitutions in the TF1-ATPase lower ATPase activity by disrupting a cluster of hydrophobic side chains.

PubMed

Bandyopadhyay, Sanjay; Allison, William S

2004-07-27

In crystal structures of the bovine F(1)-ATPase (MF(1)), the side chains of gammaMet(23), gammaMet(232), and gammaLeu(77) interact in a cluster. Substitution of the corresponding residues in the alpha(3)beta(3)gamma subcomplex of TF(1) with lysine lowers the ATPase activity to 2.3, 11, and 15%, respectively, of that displayed by wild-type. In contrast, TF(1) subcomplexes containing the gammaM(23)C, gammaM(232)C, and gammaL(77)C substitutions display 36, 36, and 130%, respectively, of the wild-type ATPase activity. The ATPase activity of the gammaM(23)C/gammaM(232)C double mutant subcomplex is 36% that of the wild-type subcomplex before and after cross-linking the introduced cysteines, whereas the ATPase activity of the gammaM(23)C/L(77)C double mutant increased from 50 to 85% that of wild-type after cross-linking the introduced cysteines. Only beta-beta cross-links formed when the alpha(3)(betaE(395)C)(3)gammaM(23)C double mutant was inactivated with CuCl(2). The overall results suggest that the attenuated ATPase of the mutant subcomplexes containing the gammaM(23)K, gammaL(77)K, and gammaM(232)K substitutions is caused by disruption of the cluster of hydrophobic amino acid side chains and that the midregion of the coiled-coil comprised of the amino- and carboxyl-terminal alpha helices of the gamma subunit does not undergo unwinding or major displacement from the side chain of gammaLeu(77) during ATP-driven rotation of the gamma subunit.

Comprehensive analog synthesis of (S)-valine thiazole peptidomimetic TTT-28 to understand enigmatic drug-binding sites of P-glycoprotein

NASA Astrophysics Data System (ADS)

Patel, Bhargav A.

P-glycoprotein (P-gp) is considered an important therapeutic target for reversal of multidrug resistance (MDR) in cancer. It recognizes a diverse range of chemically and mechanistically dissimilar drugs. It has been postulated that the efflux by P-gp plays a major role in failure of chemotherapy. Hence, researchers have been trying to obtain a potent inhibitor of P-gp with specificity to tumor sites. In this pursuit, we previously were able to obtain a novel (S)-valine thiazole-derived peptidomimetic compound 1 ( TTT-28), which showed potent reversal of MDR in vitro as well as in vivo compared to verapamil, a well-known MDR modulator. We have also found that compound 1 triggers ATPase stimulation when incubated with P-gp alike verapamil, which implies its mechanism of action as competitive in nature. In this study, we attempted to understand structural requirements of ligands binding to a perplexing drug-binding site of P-gp and affecting its ATPase function. Toward this goal, we prepared a novel set of 64 analogues by fine tuning lead compound 1. These synthesized analogues were tested using ATPase activity assay. During the course of the study, a potent stimulator (1) of ATPase activity was transformed into an ATPase inhibitory leads such as compounds 43 , 57 and 113. The ATPase inhibitory activity of these compounds is predominantly contributed by the presence of a cyclohexyl group in place of the 2-aminobenzophenone moiety of ATPase activity stimulatory lead compound 1. Molecular modeling studies suggested a need for specific interactions with the drug-binding site of P-gp to induce different conformational states of P-gp to produce either stimulation or inhibition of ATPase activity. Collectively, this comprehensive synthesis work will facilitate further research towards P-gp inhibitor development.

Orphan Kinesin NOD Lacks Motile Properties But Does Possess a Microtubule-stimulated ATPase Activity

PubMed Central

Matthies, Heinrich J.G.; Baskin, Ronald J.; Hawley, R. Scott

2001-01-01

NOD is a Drosophila chromosome-associated kinesin-like protein that does not fall into the chromokinesin subfamily. Although NOD lacks residues known to be critical for kinesin function, we show that microtubules activate the ATPase activity of NOD >2000-fold. Biochemical and genetic analysis of two genetically identified mutations of NOD (NODDTW and NOD“DR2”) demonstrates that this allosteric activation is critical for the function of NOD in vivo. However, several lines of evidence indicate that this ATPase activity is not coupled to vectorial transport, including 1) NOD does not produce microtubule gliding; and 2) the substitution of a single amino acid in the Drosophila kinesin heavy chain with the analogous amino acid in NOD results in a drastic inhibition of motility. We suggest that the microtubule-activated ATPase activity of NOD provides transient attachments of chromosomes to microtubules rather than producing vectorial transport. PMID:11739796

Cell Signaling Associated with Na+/K+-ATPase: Activation of Phosphatidylinositide 3-Kinase IA/Akt by Ouabain Is Independent of Src

PubMed Central

2013-01-01

Exposure of intact cells to selective inhibitors of Na+/K+-ATPase such as ouabain activates several growth-related cell signaling pathways. It has been suggested that the initial event of these pathways is the binding of ouabain to a preexisting complex of Src with Na+/K+-ATPase of the plasma membrane. The aim of this work was to evaluate the role of Src in the ouabain-induced activation of phosphatidylinositide 3-kinase 1A (PI3K1A) and its downstream consequences. When fibroblasts devoid of Src (SYF cells) and controls (Src++ cells) were exposed to ouabain, PI3K1A, Akt, and proliferative growth were similarly stimulated in both cell lines. Ouabain-induced activation of Akt was not prevented by the Src inhibitor PP2. In contrast, ERK1/2 were not activated by ouabain in SYF cells but were stimulated in Src++ cells; this was prevented by PP2. In isolated adult mouse cardiac myocytes, where ouabain induces hypertrophic growth, PP2 also did not prevent ouabain-induced activation of Akt and the resulting hypertrophy. Ouabain-induced increases in the levels of co-immunoprecipitation of the α-subunit of Na+/K+-ATPase with the p85 subunit of PI3K1A were noted in SYF cells, Src++ cells, and adult cardiac myocytes. In conjunction with previous findings, the results presented here indicate that (a) if there is a preformed complex of Src and Na+/K+-ATPase, it is irrelevant to ouabain-induced activation of the PI3K1A/Akt pathway through Na+/K+-ATPase and (b) a more likely, but not established, mechanism of linkage of Na+/K+-ATPase to PI3K1A is the ouabain-induced interaction of a proline-rich domain of the α-subunit of Na+/K+-ATPase with the SH3 domain of the p85 subunit of PI3K1A. PMID:24266852

Purification and ATPase activity of human ABCA1.

PubMed

Takahashi, Kei; Kimura, Yasuhisa; Kioka, Noriyuki; Matsuo, Michinori; Ueda, Kazumitsu

2006-04-21

ATP-binding cassette protein A1 (ABCA1) plays a major role in cholesterol homeostasis and high density lipoprotein metabolism. Apolipoprotein A-I binds to ABCA1 and cellular cholesterol and phospholipids, mainly phosphatidylcholine, are loaded onto apoA-I to form pre-beta high density lipoprotein (HDL). It is proposed that ABCA1 translocates phospholipids and cholesterol directly or indirectly to form pre-beta HDL. To explore the mechanism of ABCA1-mediated pre-beta HDL formation, we expressed human ABCA1 in insect Sf9 cells and purified it. Trypsin limited-digestion of purified ABCA1 in the detergent-soluble form suggested that it retained conformation similar to ABCA1 expressed in the membranes of human fibroblast WI-38 cells. Purified ABCA1 showed robust ATPase activity when reconstituted in liposomes made of synthetic phosphatidylcholine. ABCA1 showed lower ATPase activity when reconstituted in liposomes containing phosphatidylserine, phosphatidylethanolamine, or phosphatidylglycerol and also showed weak specificity in acyl chain species. ATPase activity was reduced by the addition of cholesterol and decreased by 25% in the presence of 20% cholesterol. Beta-sitosterol and campesterol showed similar inhibitory effects but stigmasterol did not, suggesting structure-specific interaction between ABCA1 and sterols. Glibenclamide suppressed ABCA1 ATPase, suggesting that it inhibits apoA-I-dependent cellular cholesterol efflux by suppressing ABCA1 ATPase activity. These results suggest that the ATPase activity of ABCA1 is stimulated preferentially by phospholipids with choline head groups, phosphatidylcholine and sphingomyelin. This study with purified human ABCA1 provides the first biochemical basis of the mechanism for HDL formation mediated by ABCA1.

The N Termini of a-Subunit Isoforms Are Involved in Signaling between Vacuolar H+-ATPase (V-ATPase) and Cytohesin-2*

PubMed Central

Hosokawa, Hiroyuki; Dip, Phat Vinh; Merkulova, Maria; Bakulina, Anastasia; Zhuang, Zhenjie; Khatri, Ashok; Jian, Xiaoying; Keating, Shawn M.; Bueler, Stephanie A.; Rubinstein, John L.; Randazzo, Paul A.; Ausiello, Dennis A.; Grüber, Gerhard; Marshansky, Vladimir

2013-01-01

Previously, we reported an acidification-dependent interaction of the endosomal vacuolar H+-ATPase (V-ATPase) with cytohesin-2, a GDP/GTP exchange factor (GEF), suggesting that it functions as a pH-sensing receptor. Here, we have studied the molecular mechanism of signaling between the V-ATPase, cytohesin-2, and Arf GTP-binding proteins. We found that part of the N-terminal cytosolic tail of the V-ATPase a2-subunit (a2N), corresponding to its first 17 amino acids (a2N(1–17)), potently modulates the enzymatic GDP/GTP exchange activity of cytohesin-2. Moreover, this peptide strongly inhibits GEF activity via direct interaction with the Sec7 domain of cytohesin-2. The structure of a2N(1–17) and its amino acids Phe5, Met10, and Gln14 involved in interaction with Sec7 domain were determined by NMR spectroscopy analysis. In silico docking experiments revealed that part of the V-ATPase formed by its a2N(1–17) epitope competes with the switch 2 region of Arf1 and Arf6 for binding to the Sec7 domain of cytohesin-2. The amino acid sequence alignment and GEF activity studies also uncovered the conserved character of signaling between all four (a1–a4) a-subunit isoforms of mammalian V-ATPase and cytohesin-2. Moreover, the conserved character of this phenomenon was also confirmed in experiments showing binding of mammalian cytohesin-2 to the intact yeast V-ATPase holo-complex. Thus, here we have uncovered an evolutionarily conserved function of the V-ATPase as a novel cytohesin-signaling receptor. PMID:23288846

[Function of transport H+-ATPases in plant cell plasma and vacuolar membranes of maize under salt stress conditions and effect of adaptogenic preparations].

PubMed

Rybchenko, Zh I; Palladina, T O

2011-01-01

Participations of electrogenic H+-pumps of plasma and vacuolar membranes represented by E1-E2 and V-type H+-ATPases in plant cell adaptation to salt stress conditions has been studied by determination of their transport activities. Experiments were carried out on corn seedlings exposed during 1 or 10 days at 0.1 M NaCl. Preparations Methyure and Ivine were used by seed soaking at 10(-7) M. Plasma and vacuolar membrane fractions were isolated from corn seedling roots. In variants without NaCl a hydrolytical activity of plasma membrane H+-ATPase was increased with seedling age and its transport one was changed insignificantly, wherease the response of the weaker vacuolar H+-ATPase was opposite. NaCl exposition decreased hydrolytical activities of both H+-ATPases and increased their transport ones. These results demonstrated amplification of H+-pumps function especially represented by vacuolar H+-ATPase. Both preparations, Methyure mainly, caused a further increase of transport activity which was more expressed in NaCl variants. Obtained results showed the important role of these H+-pumps in plant adaptation under salt stress conditions realized by energetical maintenance of the secondary active Na+/H+ -antiporters which remove Na+ from cytoplasm.

Time-measurement-regulating peptide PIN may alter a timer conformation of Time Interval Measuring Enzyme (TIME).

PubMed

Ti, Xiaonan; Tani, Naoki; Isobe, Minoru; Kai, Hidenori

2006-05-01

The TIME (Time Interval Measuring Enzyme) ATPase measures time intervals in accordance with diapause development, which indispensably requires cold for resumption of embryonic development in the silkworm (Bombyx mori). The PIN (Peptidyl Inhibitory Needle) peptide regulates the time measurement function of TIME. In the present study we investigated the interaction between TIME and PIN in order to address the mechanism of diapause development. When TIME was isolated from eggs later than 12 days after oviposition, transient bursts of ATPase activity occurred 18h after isolation of TIME, and the younger the eggs and pupal ovaries from which TIME was isolated, the earlier the bursts of ATPase activity appeared. However, no interval-timer activation of ATPase occurred in ovaries earlier than 6 days after pupation. Similar patterns of ATPase activity occurred in test tubes after mixing TIME with PIN. The shorter the time PIN was mixed with TIME, the earlier the ATPase activity appeared. The timer may be built into the protein conformation of TIME, and PIN (which is present in ovaries beginning 6 days after pupation) appears able to alter this timer conformation through pupal stages to laid eggs. We discuss the possible mechanism of diapause development in relation to the timer mechanism of TIME.

Electromagnetic irradiation of Enterococcus hirae at low-intensity 51.8- and 53.0-GHz frequencies: changes in bacterial cell membrane properties and enhanced antibiotics effects.

PubMed

Torgomyan, Heghine; Ohanyan, Vahe; Blbulyan, Syuzanna; Kalantaryan, Vitaly; Trchounian, Armen

2012-04-01

Exposure to electromagnetic irradiation (EMI) of 51.8 and 53.0 GHz and low intensity (flux capacity of 0.06 mW cm(-2) ) for 1 h markedly decreased the energy-dependent H(+) and K(+) transport across membranes of Enterococcus hirae ATCC 9790. After EMI, there was also a significant decrease of overall and N,N'-dicyclohexylcarbodiimide (DCCD)-sensitive ATPase activity of the membrane vesicles. These measures were considerably lower at 53.0 GHz. EMI in combination with different antibiotics, such as ceftriaxone and kanamycin at their minimal inhibitory concentrations (100 and 200 μM, respectively), enhanced bacterial cell growth and altered their membrane transport properties. Total H(+) efflux was most sensitive to ceftriaxone but DCCD-inhibited H(+) efflux and total K(+) influx were sensitive to kanamycin. The results indicate that cell membrane proteins could be a target in the action of EMI and enhanced antibacterial effects in combination with antibiotics. The DCCD-sensitive F(0) F(1) -ATPase or this ATPase in combination with K(+) uptake protein probably plays a key role in these effects. © 2012 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

Regulation of hepatic Na+/K+-ATPase in obese female and male rats: involvement of ERK1/2, AMPK, and Rho/ROCK.

PubMed

Stanimirovic, Julijana; Obradovic, Milan; Panic, Anastasija; Petrovic, Voin; Alavantic, Dragan; Melih, Irena; Isenovic, Esma R

2018-03-01

In this study, we assessed whether the disturbed regulation of sodium/potassium-adenosine-triphosphatase (Na + /K + -ATPase) occurs as a consequence of obesity-induced IR in sex-specific manner. We also assessed whether alterations of IRS/PI3K/Akt, ERK1/2, AMPKα, and RhoA/ROCK signaling cascades have an important role in this pathology. Female and male Wistar rats (150-200 g, 8 weeks old) were fed a standard laboratory diet or a high-fat (HF) diet (42% fat) for 10 weeks. The activity of hepatic Na + /K + -ATPase and Rho, and the association of IRS-1/p85 were assessed in liver. Furthermore, the protein level of α 1 Na + /K + -ATPase in plasma membrane fractions, and protein levels of IRS-1, PI3K-p85, -p110, RhoA, ROCK1, ROCK2, ERK1/2, AMPKα, ERα, and ERβ in liver lysates were assessed. The expression of hepatic α 1 Na + /K + -ATPase mRNA was also analyzed by qRT-PCR. The results show that HF-fed female rats exhibited an increase in hepatic ERK1/2 (p < 0.05) and AMPKα (p < 0.05) phosphorylation levels, unchanged level of Na + /K + -ATPase α 1 mRNA, decreased level of Na + /K + -ATPase activity (p < 0.05), and decreased α 1 Na + /K + -ATPase protein expression (p < 0.01). In liver of HF-fed male rats, results show decreased levels of Na + /K + -ATPase activity (p < 0.01), both protein and mRNA of α 1 subunit (p < 0.05), but significant increase in Rho activity (p < 0.05). Our results indicate significant sex differences in α 1 Na + /K + -ATPase mRNA expression and activation of ERK1/2, AMPKα, and Rho in the liver. Exploring the sex-specific factors and pathways that promote obesity-related diseases may lead to a better understanding of pathogenesis and discovering new therapeutic targets.

Effect of thyroid status on the development of the different molecular forms of Na+,K+-ATPase in rat brain.

PubMed

Atterwill, C K; Reid, J; Athayde, C M

1985-05-01

The effect of thyroid status on the postnatal development of the two molecular forms of Na+,K+-ATPase, distinguished kinetically on the basis of their ouabain sensitivity, was examined in rat brain. Hypothyroidism induced by PTU from day 1 postnatally significantly reduced the Na+,K+-ATPase activity in cerebellum (22-30 days) but not forebrain, whereas hyperthyroidism (T4 treatment from day 1) had no effect. The hypothyroidism-induced reduction in cerebellum was reflected by a 20-45% reduction in the activity of the alpha(+) form of Na+,K+-ATPase (high ouabain affinity) against control brains compared to a 60-70% reduction in the activity of the alpha form (low ouabain affinity). These results show that neonatally induced hypothyroidism leads to a selectively greater impairment of the ontogenesis of the activity of cerebellar alpha form of Na+,K+-ATPase. This may possibly reflect a retarded development of a selective cerebellar cell population containing predominantly the alpha form of the enzyme.

The Antibiotic Novobiocin Binds and Activates the ATPase That Powers Lipopolysaccharide Transport.

PubMed

May, Janine M; Owens, Tristan W; Mandler, Michael D; Simpson, Brent W; Lazarus, Michael B; Sherman, David J; Davis, Rebecca M; Okuda, Suguru; Massefski, Walter; Ruiz, Natividad; Kahne, Daniel

2017-12-06

Novobiocin is an orally active antibiotic that inhibits DNA gyrase by binding the ATP-binding site in the ATPase subunit. Although effective against Gram-positive pathogens, novobiocin has limited activity against Gram-negative organisms due to the presence of the lipopolysaccharide-containing outer membrane, which acts as a permeability barrier. Using a novobiocin-sensitive Escherichia coli strain with a leaky outer membrane, we identified a mutant with increased resistance to novobiocin. Unexpectedly, the mutation that increases novobiocin resistance was not found to alter gyrase, but the ATPase that powers lipopolysaccharide (LPS) transport. Co-crystal structures, biochemical, and genetic evidence show novobiocin directly binds this ATPase. Novobiocin does not bind the ATP binding site but rather the interface between the ATPase subunits and the transmembrane subunits of the LPS transporter. This interaction increases the activity of the LPS transporter, which in turn alters the permeability of the outer membrane. We propose that novobiocin will be a useful tool for understanding how ATP hydrolysis is coupled to LPS transport.

Ubiquitinated Proteins Activate the Proteasomal ATPases by Binding to Usp14 or Uch37 Homologs*

PubMed Central

Peth, Andreas; Kukushkin, Nikolay; Bossé, Marc; Goldberg, Alfred L.

2013-01-01

Degradation of ubiquitinated proteins by 26 S proteasomes requires ATP hydrolysis, but it is unclear how the proteasomal ATPases are regulated and how proteolysis, substrate deubiquitination, degradation, and ATP hydrolysis are coordinated. Polyubiquitinated proteins were shown to stimulate ATP hydrolysis by purified proteasomes, but only if the proteins contain a loosely folded domain. If they were not ubiquitinated, such proteins did not increase ATPase activity. However, they did so upon addition of ubiquitin aldehyde, which mimics the ubiquitin chain and binds to 26 S-associated deubiquitinating enzymes (DUBs): in yeast to Ubp6, which is essential for the ATPase activation, and in mammalian 26 S to the Ubp6 homolog, Usp14, and Uch37. Occupancy of either DUB by a ubiquitin conjugate leads to ATPase stimulation, thereby coupling deubiquitination and ATP hydrolysis. Thus, ubiquitinated loosely folded proteins, after becoming bound to the 26 S, interact with Ubp6/Usp14 or Uch37 to activate ATP hydrolysis and enhance their own destruction. PMID:23341450

Poliovirus 2C protein forms homo-oligomeric structures required for ATPase activity.

PubMed

Adams, Peter; Kandiah, Eaazhisai; Effantin, Grégory; Steven, Alasdair C; Ehrenfeld, Ellie

2009-08-14

The poliovirus protein 2C plays an essential role in viral RNA replication, although its precise biochemical activities or structural requirements have not been elucidated. The protein has several distinctive properties, including ATPase activity and membrane and RNA binding, that are conserved among orthologs of many positive-strand RNA viruses. Sequence alignments have placed these proteins in the SF3 helicase family, a subset of the AAA+ ATPase superfamily. A feature common to AAA+ proteins is the formation of oligomeric rings that are essential for their catalytic functions. Here we show that a recombinant protein, MBP-2C, in which maltose-binding protein was fused to 2C, formed soluble oligomers and that ATPase activity was restricted to oligomer-containing fractions from gel-filtration chromatography. The active fraction was visualized by negative-staining electron microscopy as ring-like particles composed of 5-8 protomers. This conclusion was confirmed by mass measurements obtained by scanning transmission electron microscopy. Mutation of amino acid residues in the 2C nucleotide-binding domain demonstrated that loss of the ability to bind or hydrolyze ATP did not affect oligomerization. Co-expression of active MBP-2C and inactive mutant proteins generated mixed oligomers that exhibited little ATPase activity, suggesting that incorporation of inactive subunits eliminates the function of the entire particle. Finally, deletion of the N-terminal 38 amino acids blocked oligomerization of the fusion protein and eliminated ATPase activity, despite retention of an unaltered nucleotide-binding domain.

Poliovirus 2C Protein Forms Homo-oligomeric Structures Required for ATPase Activity*

PubMed Central

Adams, Peter; Kandiah, Eaazhisai; Effantin, Grégory; Steven, Alasdair C.; Ehrenfeld, Ellie

2009-01-01

The poliovirus protein 2C plays an essential role in viral RNA replication, although its precise biochemical activities or structural requirements have not been elucidated. The protein has several distinctive properties, including ATPase activity and membrane and RNA binding, that are conserved among orthologs of many positive-strand RNA viruses. Sequence alignments have placed these proteins in the SF3 helicase family, a subset of the AAA+ ATPase superfamily. A feature common to AAA+ proteins is the formation of oligomeric rings that are essential for their catalytic functions. Here we show that a recombinant protein, MBP-2C, in which maltose-binding protein was fused to 2C, formed soluble oligomers and that ATPase activity was restricted to oligomer-containing fractions from gel-filtration chromatography. The active fraction was visualized by negative-staining electron microscopy as ring-like particles composed of 5–8 protomers. This conclusion was confirmed by mass measurements obtained by scanning transmission electron microscopy. Mutation of amino acid residues in the 2C nucleotide-binding domain demonstrated that loss of the ability to bind or hydrolyze ATP did not affect oligomerization. Co-expression of active MBP-2C and inactive mutant proteins generated mixed oligomers that exhibited little ATPase activity, suggesting that incorporation of inactive subunits eliminates the function of the entire particle. Finally, deletion of the N-terminal 38 amino acids blocked oligomerization of the fusion protein and eliminated ATPase activity, despite retention of an unaltered nucleotide-binding domain. PMID:19520852

Modulation by K+ Plus NH4+ of microsomal (Na+, K+)-ATPase activity in selected ontogenetic stages of the diadromous river shrimp Macrobrachium amazonicum (Decapoda, Palaemonidae).

PubMed

Leone, Francisco A; Bezerra, Thais M S; Garçon, Daniela P; Lucena, Malson N; Pinto, Marcelo R; Fontes, Carlos F L; McNamara, John C

2014-01-01

We investigate the synergistic stimulation by K(+) plus NH4 (+) of (Na(+), K(+))-ATPase activity in microsomal preparations of whole zoea I and decapodid III, and in juvenile and adult river shrimp gills. Modulation of (Na(+), K(+))-ATPase activity is ontogenetic stage-specific, and particularly distinct between juveniles and adults. Although both gill enzymes exhibit two different sites for K(+) and NH4 (+) binding, in the juvenile enzyme, these two sites are equivalent: binding by both ions results in slightly stimulated activity compared to that of a single ionic species. In the adult enzyme, the sites are not equivalent: when one ion occupies its specific binding site, (Na(+), K(+))-ATPase activity is stimulated synergistically by ≈ 50% on binding of the complementary ion. Immunolocalization reveals the enzyme to be distributed predominantly throughout the intralamellar septum in the gill lamellae of juveniles and adults. Western blot analyses demonstrate a single immunoreactive band, suggesting a single (Na(+), K(+))-ATPase α-subunit isoform that is distributed into different density membrane fractions, independently of ontogenetic stage. We propose a model for the modulation by K(+) and NH4 (+) of gill (Na(+), K(+))-ATPase activity. These findings suggest that the gill enzyme may be regulated by NH4 (+) during ontogenetic development in M. amazonicum.

Modulation By K+ Plus NH4 + of Microsomal (Na+, K+)-ATPase Activity in Selected Ontogenetic Stages of the Diadromous River Shrimp Macrobrachium amazonicum (Decapoda, Palaemonidae)

PubMed Central

Leone, Francisco A.; Bezerra, Thais M. S.; Garçon, Daniela P.; Lucena, Malson N.; Pinto, Marcelo R.; Fontes, Carlos F. L.; McNamara, John C.

2014-01-01

We investigate the synergistic stimulation by K+ plus NH4 + of (Na+, K+)-ATPase activity in microsomal preparations of whole zoea I and decapodid III, and in juvenile and adult river shrimp gills. Modulation of (Na+, K+)-ATPase activity is ontogenetic stage-specific, and particularly distinct between juveniles and adults. Although both gill enzymes exhibit two different sites for K+ and NH4 + binding, in the juvenile enzyme, these two sites are equivalent: binding by both ions results in slightly stimulated activity compared to that of a single ionic species. In the adult enzyme, the sites are not equivalent: when one ion occupies its specific binding site, (Na+, K+)-ATPase activity is stimulated synergistically by ≈50% on binding of the complementary ion. Immunolocalization reveals the enzyme to be distributed predominantly throughout the intralamellar septum in the gill lamellae of juveniles and adults. Western blot analyses demonstrate a single immunoreactive band, suggesting a single (Na+, K+)-ATPase α-subunit isoform that is distributed into different density membrane fractions, independently of ontogenetic stage. We propose a model for the modulation by K+ and NH4 + of gill (Na+, K+)-ATPase activity. These findings suggest that the gill enzyme may be regulated by NH4 + during ontogenetic development in M. amazonicum. PMID:24586919

Substrates Control Multimerization and Activation of the Multi-Domain ATPase Motor of Type VII Secretion

DOE PAGES

Rosenberg, Oren S.; Dovala, Dustin; Li, Xueming; ...

2015-04-09

We report that Mycobacterium tuberculosis and Staphylococcus aureus secrete virulence factors via type VII protein secretion (T7S), a system that intriguingly requires all of its secretion substrates for activity. To gain insights into T7S function, we used structural approaches to guide studies of the putative translocase EccC, a unique enzyme with three ATPase domains, and its secretion substrate EsxB. The crystal structure of EccC revealed that the ATPase domains are joined by linker/pocket interactions that modulate its enzymatic activity. EsxB binds via its signal sequence to an empty pocket on the C-terminal ATPase domain, which is accompanied by an increasemore » in ATPase activity. Surprisingly, substrate binding does not activate EccC allosterically but, rather, by stimulating its multimerization. Thus, the EsxB substrate is also an integral T7S component, illuminating a mechanism that helps to explain interdependence of substrates, and suggests a model in which binding of substrates modulates their coordinate release from the bacterium.« less

Inhibition of membrane Na(+)-K+ Atpase of the brain, liver and RBC in rats administered di(2-ethyl hexyl) phthalate (DEHP) a plasticizer used in polyvinyl chloride (PVC) blood storage bags.

PubMed

Dhanya, C R; Indu, A R; Deepadevi, K V; Kurup, P A

2003-08-01

Significant amounts of di(2-ethylhexyl) phthalate (DEHP) leach out into blood stored in DEHP plasticized polyvinyl chloride (PVC) bags resulting in the exposure of recipients of blood transfusion to this compound. The aim of this study was to find out whether DEHP at these low levels has any effect on the activity of membrane Na(+)-K+ ATPase, since a decrease in this enzyme activity has been reported to take place in a number of disorders like neurodegenerative and psychiatric disorders, coronary artery disease and stroke, syndrome-X, tumours etc. DEHP was administered (ip) at a low dose of 750 microg/100 g body weight to rats and the activity of membrane Na(+)-K+ ATPase in liver, brain and RBC was estimated. Histopathology of brain, activity of HMG CoA reductase (a major rate limiting enzyme in the isoprenoid pathway of which digoxin, the physiological inhibitor of Na(+)-K+ ATPase is a product), intracellular concentration of Ca2+ and Mg2+ in RBC (which is altered as a result of inhibition of Na(+)-K+ ATPase) were also studied. (In the light of the observation of increase of intracellular Ca2+ load and intracellular depletion of Mg2+ when Na(+)-K+ ATPase is inhibited). Histopathology of brain revealed areas of degeneration in the rats administered DEHP. There was significant inhibition of membrane Na(+)-K+ ATPase in brain, liver and RBC. Intracellular Ca2+ increased in the RBC while intracellular Mg2+ decreased. However activity of hepatic HMG CoA reductase decreased. Activity of Na(+)-K+ ATPase and HMG CoA reductase, however returned to normal levels within 7 days of stopping administration of DEHP. The inhibition of membrane Na(+)-K+ ATPase activity by DEHP may indicate the possibility of predisposing recipients of transfusion of blood or hemodialysis to the various disorders mentioned above. However since this effect is reversed when DEHP administration is stopped, it may not be a serious problem in the case of a few transfusion; but in patients receiving repeated blood transfusion as in thalassemia patients or patients undergoing hemodialysis, possibility of this risk has to be considered. This inhibition is a direct effect of DEHP or its metabolites, since activity of HMG CoA reductase, (an enzyme which catalyses a major rate limiting step in the isoprenoid pathway by which digoxin, the physiological inhibitor of Na(+)-K+ ATPase is synthesized) showed a decrease.

Exploration of flexible phenylpropylurea scaffold as novel cardiac myosin activators for the treatment of systolic heart failure.

PubMed

Manickam, Manoj; Jalani, Hitesh B; Pillaiyar, Thanigaimalai; Sharma, Niti; Boggu, Pulla Reddy; Venkateswararao, Eeda; Lee, You-Jung; Jeon, Eun-Seok; Jung, Sang-Hun

2017-07-07

A series of flexible urea derivatives have been synthesized and demonstrated as selective cardiac myosin ATPase activator. Among them 1-phenethyl-3-(3-phenylpropyl)urea (1, cardiac myosin ATPase activation at 10 μM = 51.1%; FS = 18.90; EF = 12.15) and 1-benzyl-3-(3-phenylpropyl)urea (9, cardiac myosin ATPase activation = 53.3%; FS = 30.04; EF = 18.27) showed significant activity in vitro and in vivo. The change of phenyl ring with tetrahydropyran-4-yl moiety viz., 1-(3-phenylpropyl)-3-((tetrahydro-2H-pyran-4-yl)methyl)urea (14, cardiac myosin ATPase activation = 81.4%; FS = 20.50; EF = 13.10), and morpholine moiety viz., 1-(2-morpholinoethyl)-3-(3-phenylpropyl)urea (21, cardiac myosin ATPase activation = 44.0%; FS = 24.79; EF = 15.65), proved to be efficient to activate the cardiac myosin. The potent compounds 1, 9, 14 and 21 were found to be selective for cardiac myosin over skeletal and smooth myosins. Thus, these urea derivatives are potent scaffold to develop as a newer cardiac myosin activator for the treatment of systolic heart failure. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Protective Effect of Bacoside-A against Morphine-Induced Oxidative Stress in Rats

PubMed Central

Sumathi, T.; Nathiya, V. C.; Sakthikumar, M.

2011-01-01

In the present study, we investigated the protective effect of bacoside-A the active principle isolated from the plant Bacopa monniera against oxidative damage induced by morphine in rat brain. Morphine intoxicated rats received 10-160 mg/kg b.w. of morphine hydrochloride intraperitoneally for 21 days. Bacoside-A pretreated rats were administered with bacoside-A (10 mg/kg b.w/day) orally, 2 h before the injection of morphine for 21 days. Pretreatment with bacoside-A has shown to possess a significant protective role against morphine induced brain oxidative damage in the antioxidant status (total reduced glutathione, superoxide dismutase, catalase, glutathione peroxidase and lipid peroxidation) and membrane bound ATP-ases(Na+/K+ATPase. Ca2+ and Mg2+ ATPases) activities in rat. The results of the present study indicate that bacoside-A protects the brain from oxidative stress induced by morphine. PMID:22707825

Protective Effect of Bacoside-A against Morphine-Induced Oxidative Stress in Rats.

PubMed

Sumathi, T; Nathiya, V C; Sakthikumar, M

2011-07-01

In the present study, we investigated the protective effect of bacoside-A the active principle isolated from the plant Bacopa monniera against oxidative damage induced by morphine in rat brain. Morphine intoxicated rats received 10-160 mg/kg b.w. of morphine hydrochloride intraperitoneally for 21 days. Bacoside-A pretreated rats were administered with bacoside-A (10 mg/kg b.w/day) orally, 2 h before the injection of morphine for 21 days. Pretreatment with bacoside-A has shown to possess a significant protective role against morphine induced brain oxidative damage in the antioxidant status (total reduced glutathione, superoxide dismutase, catalase, glutathione peroxidase and lipid peroxidation) and membrane bound ATP-ases(Na(+)/K(+)ATPase. Ca(2+) and Mg(2+) ATPases) activities in rat. The results of the present study indicate that bacoside-A protects the brain from oxidative stress induced by morphine.

Enzymatic properties of separated isozymes of the Na,K-ATPase. Substrate affinities, kinetic cooperativity, and ion transport stoichiometry.

PubMed

Sweadner, K J

1985-09-25

There are two isozymes of the Na,K-ATPase, which can be purified separately from rat renal medulla and brainstem axolemma. Here the basic kinetic properties of the two Na,K-ATPases have been compared in conditions permitting enzyme turnover. The two isozymes are half-maximally activated at different concentrations of ATP, the axolemma Na,K-ATPase having the higher affinity. They are half-maximally activated by Na+ and K+ at very similar concentrations but show differences in cooperativity toward Na+. The affinities of both isozymes for ATP and Na+ are affected in a qualitatively similar way by variations in the concentration of K+. Both isozymes transport 22Na+ and 42K+ in a ratio close to 3:2 in artificial lipid vesicles. The two isozymes differ most strikingly in the inhibition of ATPase activity by ouabain. The axolemma Na,K-ATPase has a high affinity for ouabain with positive cooperativity, while the renal medulla Na,K-ATPase has a lower affinity with negative cooperativity. It is likely that the cooperativity differences are due to kinetic effects, reflecting different rates of conformation transitions during enzyme turnover. The functional result of the contrasting cooperativities is that the difference in sensitivity to ouabain is amplified.

D1-like dopamine receptors downregulate Na+-K+-ATPase activity and increase cAMP production in the posterior gills of the blue crab Callinectes sapidus

PubMed Central

Arnaldo, Francis B.; Villar, Van Anthony M.; Konkalmatt, Prasad R.; Owens, Shaun A.; Asico, Laureano D.; Jones, John E.; Yang, Jian; Lovett, Donald L.; Armando, Ines; Concepcion, Gisela P.

2014-01-01

Dopamine-mediated regulation of Na+-K+-ATPase activity in the posterior gills of some crustaceans has been reported to be involved in osmoregulation. The dopamine receptors of invertebrates are classified into three groups based on their structure and pharmacology: D1- and D2-like receptors and a distinct invertebrate receptor subtype (INDR). We tested the hypothesis that a D1-like receptor is expressed in the blue crab Callinectes sapidus and regulates Na+-K+-ATPase activity. RT-PCR, using degenerate primers, showed the presence of D1βR mRNA in the posterior gill. The blue crab posterior gills showed positive immunostaining for a dopamine D5 receptor (D5R or D1βR) antibody in the basolateral membrane and cytoplasm. Confocal microscopy showed colocalization of Na+-K+-ATPase and D1βR in the basolateral membrane. To determine the effect of D1-like receptor stimulation on Na+-K+-ATPase activity, intact crabs acclimated to low salinity for 6 days were given an intracardiac infusion of the D1-like receptor agonist fenoldopam, with or without the D1-like receptor antagonist SCH23390. Fenoldopam increased cAMP production twofold and decreased Na+-K+-ATPase activity by 50% in the posterior gills. This effect was blocked by coinfusion with SCH23390, which had no effect on Na+-K+-ATPase activity by itself. Fenoldopam minimally decreased D1βR protein expression (10%) but did not affect Na+-K+-ATPase α-subunit protein expression. This study shows the presence of functional D1βR in the posterior gills of euryhaline crabs chronically exposed to low salinity and highlights the evolutionarily conserved function of the dopamine receptors on sodium homeostasis. PMID:25080496

D1-like dopamine receptors downregulate Na+-K+-ATPase activity and increase cAMP production in the posterior gills of the blue crab Callinectes sapidus.

PubMed

Arnaldo, Francis B; Villar, Van Anthony M; Konkalmatt, Prasad R; Owens, Shaun A; Asico, Laureano D; Jones, John E; Yang, Jian; Lovett, Donald L; Armando, Ines; Jose, Pedro A; Concepcion, Gisela P

2014-09-15

Dopamine-mediated regulation of Na(+)-K(+)-ATPase activity in the posterior gills of some crustaceans has been reported to be involved in osmoregulation. The dopamine receptors of invertebrates are classified into three groups based on their structure and pharmacology: D1- and D2-like receptors and a distinct invertebrate receptor subtype (INDR). We tested the hypothesis that a D1-like receptor is expressed in the blue crab Callinectes sapidus and regulates Na(+)-K(+)-ATPase activity. RT-PCR, using degenerate primers, showed the presence of D1βR mRNA in the posterior gill. The blue crab posterior gills showed positive immunostaining for a dopamine D5 receptor (D5R or D1βR) antibody in the basolateral membrane and cytoplasm. Confocal microscopy showed colocalization of Na(+)-K(+)-ATPase and D1βR in the basolateral membrane. To determine the effect of D1-like receptor stimulation on Na(+)-K(+)-ATPase activity, intact crabs acclimated to low salinity for 6 days were given an intracardiac infusion of the D1-like receptor agonist fenoldopam, with or without the D1-like receptor antagonist SCH23390. Fenoldopam increased cAMP production twofold and decreased Na(+)-K(+)-ATPase activity by 50% in the posterior gills. This effect was blocked by coinfusion with SCH23390, which had no effect on Na(+)-K(+)-ATPase activity by itself. Fenoldopam minimally decreased D1βR protein expression (10%) but did not affect Na(+)-K(+)-ATPase α-subunit protein expression. This study shows the presence of functional D1βR in the posterior gills of euryhaline crabs chronically exposed to low salinity and highlights the evolutionarily conserved function of the dopamine receptors on sodium homeostasis. Copyright © 2014 the American Physiological Society.

Adaptation of H+-pumping and plasma membrane H+ ATPase activity in proteoid roots of white lupin under phosphate deficiency.

PubMed

Yan, Feng; Zhu, Yiyong; Müller, Caroline; Zörb, Christian; Schubert, Sven

2002-05-01

White lupin (Lupinus albus) is able to adapt to phosphorus deficiency by producing proteoid roots that release a huge amount of organic acids, resulting in mobilization of sparingly soluble soil phosphate in rhizosphere. The mechanisms responsible for the release of organic acids by proteoid root cells, especially the trans-membrane transport processes, have not been elucidated. Because of high cytosolic pH, the release of undissociated organic acids is not probable. In the present study, we focused on H+ export by plasma membrane H+ ATPase in active proteoid roots. In vivo, rhizosphere acidification of active proteoid roots was vanadate sensitive. Plasma membranes were isolated from proteoid roots and lateral roots from P-deficient and -sufficient plants. In vitro, in comparison with two types of lateral roots and proteoid roots of P-sufficient plants, the following increase of the various parameters was induced in active proteoid roots of P-deficient plants: (a) hydrolytic ATPase activity, (b) Vmax and Km, (c) H+ ATPase enzyme concentration of plasma membrane, (d) H+-pumping activity, (e) pH gradient across the membrane of plasmalemma vesicles, and (f) passive H+ permeability of plasma membrane. In addition, lower vanadate sensitivity and more acidic pH optimum were determined for plasma membrane ATPase of active proteoid roots. Our data support the hypothesis that in active proteoid root cells, H+ and organic anions are exported separately, and that modification of plasma membrane H+ ATPase is essential for enhanced rhizosphere acidification by active proteoid roots.

Some assembly required: Contributions of Tom Stevens' lab to the V-ATPase field.

PubMed

Graham, Laurie A; Finnigan, Gregory C; Kane, Patricia M

2018-06-01

Tom Stevens' lab has explored the subunit composition and assembly of the yeast V-ATPase for more than 30 years. Early studies helped establish yeast as the predominant model system for study of V-ATPase proton pumps and led to the discovery of protein splicing of the V-ATPase catalytic subunit. The Vma - phenotype, characteristic of loss-of-V-ATPase activity in yeast was key in determining the enzyme's subunit composition via yeast genetics. V-ATPase subunit composition proved to be highly conserved among eukaryotes. Genetic screens for new vma mutants led to identification of a set of dedicated V-ATPase assembly factors and helped unravel the complex pathways for V-ATPase assembly. In later years, exploration of the evolutionary history of several V-ATPase subunits provided new information about the enzyme's structure and function. This review highlights V-ATPase work in the Stevens' lab between 1987 and 2017. © 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Caffeine prevents high-intensity exercise-induced increase in enzymatic antioxidant and Na+-K+-ATPase activities and reduction of anxiolytic like-behaviour in rats.

PubMed

Vieira, Juliano M; Carvalho, Fabiano B; Gutierres, Jessié M; Soares, Mayara S P; Oliveira, Pathise S; Rubin, Maribel A; Morsch, Vera M; Schetinger, Maria Rosa; Spanevello, Roselia M

2017-11-01

Here we investigated the impact of chronic high-intensity interval training (HIIT) and caffeine consumption on the activities of Na + -K + -ATPase and enzymes of the antioxidant system, as well as anxiolytic-like behaviour in the rat brain. Animals were divided into groups: control, caffeine (4 mg/kg), caffeine (8 mg/kg), HIIT, HIIT plus caffeine (4 mg/kg) and HIIT plus caffeine (8 mg/kg). Rats were trained three times per week for 6 weeks, and caffeine was administered 30 minutes before training. We assessed the anxiolytic-like behaviour, Na + -K + -ATPase, superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) activities, levels of reduced glutathione (GSH) and thiobarbituric acid reactive substances (TBARS) in the brain. HIIT-induced anxiolytic-like behaviour increased Na + -K + -ATPase and GPx activities and TBARS levels, altered the activities of SOD and CAT in different brain regions, and decreased GSH levels. Caffeine, however, elicited anxiogenic-like behaviour and blocked HIIT effects. The combination of caffeine and HIIT prevented the increase in SOD activity in the cerebral cortex and GPx activity in three brain regions. Our results show that caffeine promoted anxiogenic behaviour and prevented HIIT-induced changes in the antioxidant system and Na + -K + -ATPase activities.

An ATPase-deficient variant of the SNF2 family member HELLS shows altered dynamics at pericentromeric heterochromatin.

PubMed

Lungu, Cristiana; Muegge, Kathrin; Jeltsch, Albert; Jurkowska, Renata Z

2015-05-22

The HELLS (helicase, lymphoid specific, also known as lymphoid-specific helicase) protein is related to the SNF2 (sucrose non-fermentable 2) family of chromatin remodeling ATPases. It is required for efficient DNA methylation in mammals, particularly at heterochromatin-located repetitive sequences. In this study, we investigated the interaction of HELLS with chromatin and used an ATPase-deficient HELLS variant to address the role of ATP hydrolysis in this process. Chromatin fractionation experiments demonstrated that, in the absence of the ATPase activity, HELLS is retained at the nuclear matrix compartment, defined in part by lamin B1. Microscopy studies revealed a stronger association of the ATPase-deficient mutant with heterochromatin. These results were further supported by fluorescence recovery after photobleaching measurements, which showed that, at heterochromatic sites, wild-type HELLS is very dynamic, with a recovery half-time of 0.8s and a mobile protein fraction of 61%. In contrast, the ATPase-deficient mutant displayed 4.5-s recovery half-time and a reduction in the mobile fraction to 30%. We also present evidence suggesting that, in addition to the ATPase activity, a functional H3K9me3 signaling pathway contributes to an efficient release of HELLS from pericentromeric chromatin. Overall, our results show that a functional ATPase activity is not required for the recruitment of HELLS to heterochromatin, but it is important for the release of the enzyme from these sites. Copyright © 2015 Elsevier Ltd. All rights reserved.

Mutations in the Putative Zinc-Binding Motif of UL52 Demonstrate a Complex Interdependence between the UL5 and UL52 Subunits of the Human Herpes Simplex Virus Type 1 Helicase/Primase Complex

PubMed Central

Chen, Yan; Carrington-Lawrence, Stacy D.; Bai, Ping; Weller, Sandra K.

2005-01-01

Herpes simplex virus type 1 (HSV-1) encodes a heterotrimeric helicase-primase (UL5/8/52) complex. UL5 contains seven motifs found in helicase superfamily 1, and UL52 contains conserved motifs found in primases. The contributions of each subunit to the biochemical activities of the complex, however, remain unclear. We have previously demonstrated that a mutation in the putative zinc finger at UL52 C terminus abrogates not only primase but also ATPase, helicase, and DNA-binding activities of a UL5/UL52 subcomplex, indicating a complex interdependence between the two subunits. To test this hypothesis and to further investigate the role of the zinc finger in the enzymatic activities of the helicase-primase, a series of mutations were constructed in this motif. They differed in their ability to complement a UL52 null virus: totally defective, partial complementation, and potentiating. In this study, four of these mutants were studied biochemically after expression and purification from insect cells infected with recombinant baculoviruses. All mutants show greatly reduced primase activity. Complementation-defective mutants exhibited severe defects in ATPase, helicase, and DNA-binding activities. Partially complementing mutants displayed intermediate levels of these activities, except that one showed a wild-type level of helicase activity. These data suggest that the UL52 zinc finger motif plays an important role in the activities of the helicase-primase complex. The observation that mutations in UL52 affected helicase, ATPase, and DNA-binding activities indicates that UL52 binding to DNA via the zinc finger may be necessary for loading UL5. Alternatively, UL5 and UL52 may share a DNA-binding interface. PMID:15994803

Mutations in the putative zinc-binding motif of UL52 demonstrate a complex interdependence between the UL5 and UL52 subunits of the human herpes simplex virus type 1 helicase/primase complex.

PubMed

Chen, Yan; Carrington-Lawrence, Stacy D; Bai, Ping; Weller, Sandra K

2005-07-01

Herpes simplex virus type 1 (HSV-1) encodes a heterotrimeric helicase-primase (UL5/8/52) complex. UL5 contains seven motifs found in helicase superfamily 1, and UL52 contains conserved motifs found in primases. The contributions of each subunit to the biochemical activities of the complex, however, remain unclear. We have previously demonstrated that a mutation in the putative zinc finger at UL52 C terminus abrogates not only primase but also ATPase, helicase, and DNA-binding activities of a UL5/UL52 subcomplex, indicating a complex interdependence between the two subunits. To test this hypothesis and to further investigate the role of the zinc finger in the enzymatic activities of the helicase-primase, a series of mutations were constructed in this motif. They differed in their ability to complement a UL52 null virus: totally defective, partial complementation, and potentiating. In this study, four of these mutants were studied biochemically after expression and purification from insect cells infected with recombinant baculoviruses. All mutants show greatly reduced primase activity. Complementation-defective mutants exhibited severe defects in ATPase, helicase, and DNA-binding activities. Partially complementing mutants displayed intermediate levels of these activities, except that one showed a wild-type level of helicase activity. These data suggest that the UL52 zinc finger motif plays an important role in the activities of the helicase-primase complex. The observation that mutations in UL52 affected helicase, ATPase, and DNA-binding activities indicates that UL52 binding to DNA via the zinc finger may be necessary for loading UL5. Alternatively, UL5 and UL52 may share a DNA-binding interface.

Intracellular pH regulation in unstimulated Calliphora salivary glands is Na+ dependent and requires V-ATPase activity.

PubMed

Schewe, Bettina; Blenau, Wolfgang; Walz, Bernd

2012-04-15

Salivary gland cells of the blowfly Calliphora vicina have a vacuolar-type H(+)-ATPase (V-ATPase) that lies in their apical membrane and energizes the secretion of a KCl-rich primary saliva upon stimulation with serotonin (5-hydroxytryptamine). Whether and to what extent V-ATPase contributes to intracellular pH (pH(i)) regulation in unstimulated gland cells is unknown. We used the fluorescent dye BCECF to study intracellular pH(i) regulation microfluorometrically and show that: (1) under resting conditions, the application of Na(+)-free physiological saline induces an intracellular alkalinization attributable to the inhibition of the activity of a Na(+)-dependent glutamate transporter; (2) the maintenance of resting pH(i) is Na(+), Cl(-), concanamycin A and DIDS sensitive; (3) recovery from an intracellular acid load is Na(+) sensitive and requires V-ATPase activity; (4) the Na(+)/H(+) antiporter is not involved in pH(i) recovery after a NH(4)Cl prepulse; and (5) at least one Na(+)-dependent transporter and the V-ATPase maintain recovery from an intracellular acid load. Thus, under resting conditions, the V-ATPase and at least one Na(+)-dependent transporter maintain normal pH(i) values of pH 7.5. We have also detected the presence of a Na(+)-dependent glutamate transporter, which seems to act as an acid loader. Despite this not being a common pH(i)-regulating transporter, its activity affects steady-state pH(i) in C. vicina salivary gland cells.

Pumping ions: rapid parallel evolution of ionic regulation following habitat invasions.

PubMed

Lee, Carol Eunmi; Kiergaard, Michael; Gelembiuk, Gregory William; Eads, Brian Donovan; Posavi, Marijan

2011-08-01

Marine to freshwater colonizations constitute among the most dramatic evolutionary transitions in the history of life. This study examined evolution of ionic regulation following saline-to-freshwater transitions in an invasive species. In recent years, the copepod Eurytemora affinis has invaded freshwater habitats multiple times independently. We found parallel evolutionary shifts in ion-motive enzyme activity (V-type H(+) ATPase, Na(+) /K(+) -ATPase) across independent invasions and in replicate laboratory selection experiments. Freshwater populations exhibited increased V-type H(+) ATPase activity in fresh water (0 PSU) and declines at higher salinity (15 PSU) relative to saline populations. This shift represented marked evolutionary increases in plasticity. In contrast, freshwater populations displayed reduced Na(+) /K(+) -ATPase activity across all salinities. Most notably, modifying salinity alone during laboratory selection experiments recapitulated the evolutionary shifts in V-type H(+) ATPase activity observed in nature. Maternal and embryonic acclimation could not account for the observed shifts in enzyme activity. V-type H(+) ATPase function has been hypothesized to be critical for freshwater and terrestrial adaptations, but evolution of this enzyme function had not been previously demonstrated in the context of habitat transitions. Moreover, the speed of these evolutionary shifts was remarkable, within a few generations in the laboratory and a few decades in the wild. © 2011 The Author(s). Evolution© 2011 The Society for the Study of Evolution.

Induction of Phosphoenolpyruvate Carboxykinase (PEPCK) during Acute Acidosis and Its Role in Acid Secretion by V-ATPase-Expressing Ionocytes.

PubMed

Furukawa, Fumiya; Tseng, Yung-Che; Liu, Sian-Tai; Chou, Yi-Ling; Lin, Ching-Chun; Sung, Po-Hsuan; Uchida, Katsuhisa; Lin, Li-Yih; Hwang, Pung-Pung

2015-01-01

Vacuolar-Type H(+)-ATPase (V-ATPase) takes the central role in pumping H(+) through cell membranes of diverse organisms, which is essential for surviving acid-base fluctuating lifestyles or environments. In mammals, although glucose is believed to be an important energy source to drive V-ATPase, and phosphoenolpyruvate carboxykinase (PEPCK), a key enzyme for gluconeogenesis, is known to be activated in response to acidosis, the link between acid secretion and PEPCK activation remains unclear. In the present study, we used zebrafish larva as an in vivo model to show the role of acid-inducible PEPCK activity in glucose production to support higher rate of H(+) secretion via V-ATPase, by utilizing gene knockdown, glucose supplementation, and non-invasive scanning ion-selective electrode technique (SIET). Zebrafish larvae increased V-ATPase-mediated acid secretion and transiently expression of Pck1, a zebrafish homolog of PEPCK, in response to acid stress. When pck1 gene was knocked down by specific morpholino, the H(+) secretion via V-ATPase decreased, but this effect was rescued by supplementation of glucose into the yolk. By assessing changes in amino acid content and gene expression of respective enzymes, glutamine and glutamate appeared to be the major source for replenishment of Krebs cycle intermediates, which are subtracted by Pck1 activity. Unexpectedly, pck1 knockdown did not affect glutamine/glutamate catalysis, which implies that Pck1 does not necessarily drive this process. The present study provides the first in vivo evidence that acid-induced PEPCK provides glucose for acid-base homeostasis at an individual level, which is supported by rapid pumping of H(+) via V-ATPase at the cellular level.

Effect of frozen storage on the structure and enzymatic activities of myofibrillar proteins of rabbit skeletal muscle.

PubMed

Kang, J O; Ito, T; Fukazawa, T

1983-01-01

The effect of frozen storage on the biochemical properties of myofibrils, and of their major constituents, actin and myosin, was investigated. Extractability of myofibrillar proteins increased slightly for 3 weeks during frozen storage of muscle, decreasing thereafter. The change in myofibrillar ATPase activity during frozen storage was consistent with that of a reconstituted acto-heavy meromyosin (HMM) complex prepared from frozen stored muscle at the same weight ratio of actin to myosin as in situ. However, myosin ATPase activity showed a different pattern of change when compared with myofibrillar ATPase activity. The maximum velocity of acto-HMM ATPase activity and the apparent dissociation constant of the acto-HMM complex decreased for 1 week during frozen storage, increasing thereafter, indicating that the affinity of actin for myosin was greatest in muscle which had been frozen for 1 week. Copyright © 1983. Published by Elsevier Ltd.

Conformational changes in human Hsp70 induced by high hydrostatic pressure produce oligomers with ATPase activity but without chaperone activity.

PubMed

Araujo, Thaís L S; Borges, Julio Cesar; Ramos, Carlos H; Meyer-Fernandes, José Roberto; Oliveira Júnior, Reinaldo S; Pascutti, Pedro G; Foguel, Debora; Palhano, Fernando L

2014-05-13

We investigated the folding of the 70 kDa human cytosolic inducible protein (Hsp70) in vitro using high hydrostatic pressure as a denaturing agent. We followed the structural changes in Hsp70 induced by high hydrostatic pressure using tryptophan fluorescence, molecular dynamics, circular dichroism, high-performance liquid chromatography gel filtration, dynamic light scattering, ATPase activity, and chaperone activity. Although monomeric, Hsp70 is very sensitive to hydrostatic pressure; after pressure had been removed, the protein did not return to its native sate but instead formed oligomeric species that lost chaperone activity but retained ATPase activity.

Phenylarsine Oxide Inhibits the Fusicoccin-Induced Activation of Plasma Membrane H+-ATPase1

PubMed Central

Olivari, Claudio; Albumi, Cristina; Pugliarello, Maria Chiara; De Michelis, Maria Ida

2000-01-01

To investigate the mechanism by which fusicoccin (FC) induces the activation of the plasma membrane (PM) H+-ATPase, we used phenylarsine oxide (PAO), a known inhibitor of protein tyrosine-phosphatases. PAO was supplied in vivo in the absence or presence of FC to radish (Raphanus sativus L.) seedlings and cultured Arabidopsis cells prior to PM extraction. Treatment with PAO alone caused a slight decrease of PM H+-ATPase activity and, in radish, a decrease of PM-associated 14-3-3 proteins. When supplied prior to FC, PAO drastically inhibited FC-induced activation of PM H+-ATPase, FC binding to the PM, and the FC-induced increase of the amount of 14-3-3 associated with the PM. On the contrary, PAO was completely ineffective on all of the above-mentioned parameters when supplied after FC. The H+-ATPase isolated from PAO-treated Arabidopsis cells maintained the ability to respond to FC if supplied with exogenous, nonphosphorylated 14-3-3 proteins. Altogether, these results are consistent with a model in which the dephosphorylated state of tyrosine residues of a protein(s), such as 14-3-3 protein, is required to permit FC-induced association between the 14-3-3 protein and the PM H+-ATPase. PMID:10677439

Effect of bacoside A on membrane-bound ATPases in the brain of rats exposed to cigarette smoke.

PubMed

Anbarasi, K; Vani, G; Balakrishna, K; Devi, C S Shyamala

2005-01-01

Membrane-bound enzymes play a vital role in neuronal function through maintenance of membrane potential and impulse propagation. We have evaluated the harmful effects of chronic cigarette smoking on membrane-bound ATPases and the protective effect of Bacoside A in rat brain. Adult male albino rats were exposed to cigarette smoke for a period of 12 weeks and simultaneously administered with Bacoside A (the active principle isolated from Bacopa monniera) at a dosage of 10 mg/kg b.w/day, p.o. The levels of lipid peroxides as marker for evaluating the extent of membrane damage, the activities of Na+/K+-ATPase, Ca2+-ATPase and Mg2+-ATPase, and associated cations sodium (Na+), potassium (K+), calcium (Ca2+), and magnesium (Mg2+) were investigated in the brain. Neuronal membrane damage was evident from the elevated levels of lipid peroxides and decreased activities of membrane-bound enzymes. Disturbances in the electrolyte balance with accumulation of Na+ and Ca2+ and depletion of K+ and Mg2+ were also observed. Administration of Bacoside A inhibited lipid peroxidation, improved the activities of ATPases, and maintained the ionic equilibrium. The results of our study indicate that Bacoside A protects the brain from cigarette smoking induced membrane damage. Copyright 2005 Wiley Periodicals, Inc.

Prolonged Exposure of Primary Human Muscle Cells to Plasma Fatty Acids Associated with Obese Phenotype Induces Persistent Suppression of Muscle Mitochondrial ATP Synthase β Subunit

PubMed Central

Tran, Lee; Hanavan, Paul D.; Campbell, Latoya E.; De Filippis, Elena; Lake, Douglas F.; Coletta, Dawn K.; Roust, Lori R.; Mandarino, Lawrence J.; Carroll, Chad C.; Katsanos, Christos S.

2016-01-01

Our previous studies show reduced abundance of the β-subunit of mitochondrial H+-ATP synthase (β-F1-ATPase) in skeletal muscle of obese individuals. The β-F1-ATPase forms the catalytic core of the ATP synthase, and it is critical for ATP production in muscle. The mechanism(s) impairing β-F1-ATPase metabolism in obesity, however, are not completely understood. First, we studied total muscle protein synthesis and the translation efficiency of β-F1-ATPase in obese (BMI, 36±1 kg/m2) and lean (BMI, 22±1 kg/m2) subjects. Both total protein synthesis (0.044±0.006 vs 0.066±0.006%·h-1) and translation efficiency of β-F1-ATPase (0.0031±0.0007 vs 0.0073±0.0004) were lower in muscle from the obese subjects when compared to the lean controls (P<0.05). We then evaluated these same responses in a primary cell culture model, and tested the specific hypothesis that circulating non-esterified fatty acids (NEFA) in obesity play a role in the responses observed in humans. The findings on total protein synthesis and translation efficiency of β-F1-ATPase in primary myotubes cultured from a lean subject, and after exposure to NEFA extracted from serum of an obese subject, were similar to those obtained in humans. Among candidate microRNAs (i.e., non-coding RNAs regulating gene expression), we identified miR-127-5p in preventing the production of β-F1-ATPase. Muscle expression of miR-127-5p negatively correlated with β-F1-ATPase protein translation efficiency in humans (r = – 0.6744; P<0.01), and could be modeled in vitro by prolonged exposure of primary myotubes derived from the lean subject to NEFA extracted from the obese subject. On the other hand, locked nucleic acid inhibitor synthesized to target miR-127-5p significantly increased β-F1-ATPase translation efficiency in myotubes (0.6±0.1 vs 1.3±0.3, in control vs exposure to 50 nM inhibitor; P<0.05). Our experiments implicate circulating NEFA in obesity in suppressing muscle protein metabolism, and establish impaired β-F1-ATPase translation as an important consequence of obesity. PMID:27532680

Oryza sativa H+-ATPase (OSA) is Involved in the Regulation of Dumbbell-Shaped Guard Cells of Rice.

PubMed

Toda, Yosuke; Wang, Yin; Takahashi, Akira; Kawai, Yuya; Tada, Yasuomi; Yamaji, Naoki; Feng Ma, Jian; Ashikari, Motoyuki; Kinoshita, Toshinori

2016-06-01

The stomatal apparatus consists of a pair of guard cells and regulates gas exchange between the leaf and atmosphere. In guard cells, blue light (BL) activates H(+)-ATPase in the plasma membrane through the phosphorylation of its penultimate threonine, mediating stomatal opening. Although this regulation is thought to be widely adopted among kidney-shaped guard cells in dicots, the molecular basis underlying that of dumbbell-shaped guard cells in monocots remains unclear. Here, we show that H(+)-ATPases are involved in the regulation of dumbbell-shaped guard cells. Stomatal opening of rice was promoted by the H(+)-ATPase activator fusicoccin and by BL, and the latter was suppressed by the H(+)-ATPase inhibitor vanadate. Using H(+)-ATPase antibodies, we showed the presence of phosphoregulation of the penultimate threonine in Oryza sativa H(+)-ATPases (OSAs) and localization of OSAs in the plasma membrane of guard cells. Interestingly, we identified one H(+)-ATPase isoform, OSA7, that is preferentially expressed among the OSA genes in guard cells, and found that loss of function of OSA7 resulted in partial insensitivity to BL. We conclude that H(+)-ATPase is involved in BL-induced stomatal opening of dumbbell-shaped guard cells in monocotyledon species. © The Author 2016. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists.

Selenium Administration Alleviates Toxicity of Chromium(VI) in the Chicken Brain.

PubMed

Hao, Pan; Zhu, Yiran; Wang, Shenghua; Wan, Huiyu; Chen, Peng; Wang, Yang; Cheng, Ziqiang; Liu, Yongxia; Liu, Jianzhu

2017-07-01

Selenium (Se) can play a protective role against heavy metal toxicity. This experiment aims to evaluate the effect of Se supplementation at different doses on the chicken brains. Oxidative stress was induced in the chicken brains by chromium(VI). A total of 105 Hyland brown male chickens were randomly divided into seven groups, including the control group, poisoned group [6%LD 50 K 2 Cr 2 O 7 body weight (B.W.)], and detoxification groups K 2 Cr 2 O 7 (6%LD 50 ) + Se (0.31, 0.63, 1.25, 2.50, and 5.00 Na 2 SeO 3 mg/kg B.W.) orally in water for 42 days. The chickens were detected by the activities of mitochondrial membrane potential, 2'-benzoyloxycinnamaldehyde, superoxide dismutase (SOD), malondialdehyde (MDA), glutathione (GSH), and Ca 2+ -ATPase. Cr(VI) administration caused histopathological damage. In addition, changes in oxidative stress indicators were observed in the chicken's brains. Se supplement increased the levels of GSH, mitochondrial membrane potential (MMP), and Ca 2+ -ATPase and reduced MDA activity in the detoxification groups. However, the high-dose Se supplementation groups of 2.50 and 5.00 mg/kg reduced the activities of GSH, MMP, and Ca 2+ -ATPase; increased the brain-body ratio; and increased SOD activity. In conclusion, Cr(VI) exposure caused oxidative stress. Se exerted a remission effect on toxic responses in the chicken brains. However, a high Se concentration was synergistic to the toxic effect of Cr(VI).

Effects of anti-inflammatory and anti-rheumatic drugs on the activities of purified and membrane-bound Na+/K+ adenosine triphosphatase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chan, M.K.; Minta, J.O.

1985-08-01

The authors have examined the effects of anti-inflammatory and anti-rheumatic drugs on membrane-bound and purified Na /K -ATPase activity in vitro. Only the gold-containing compounds (gold sodium thiomalate and auranofin) were found to inhibit the enzyme activity in a dose-dependent manner. Sodium thiomalate and triethylphosphine, the ligand compounds for gold sodium thiomalate and auranofin, respectively, had no effect on ATPase activity. The antagonistic properties was abolished by preincubation of the gold compounds with dithiothreitol. Lineweaver-Burke analysis of the inhibitions of purified ATPase by the gold compounds was found to follow uncompetitive kinetics. Inhibition of ATPase by gold may cause disruptionmore » of transmembrane cation transport and thus result in impairment of several metabolic processes and cellular functions.« less

Active Detergent-solubilized H+,K+-ATPase Is a Monomer*

PubMed Central

Dach, Ingrid; Olesen, Claus; Signor, Luca; Nissen, Poul; le Maire, Marc; Møller, Jesper V.; Ebel, Christine

2012-01-01

The H+,K+-ATPase pumps protons or hydronium ions and is responsible for the acidification of the gastric fluid. It is made up of an α-catalytic and a β-glycosylated subunit. The relation between cation translocation and the organization of the protein in the membrane are not well understood. We describe here how pure and functionally active pig gastric H+,K+-ATPase with an apparent Stokes radius of 6.3 nm can be obtained after solubilization with the non-ionic detergent C12E8, followed by exchange of C12E8 with Tween 20 on a Superose 6 column. Mass spectroscopy indicates that the β-subunit bears an excess mass of 9 kDa attributable to glycosylation. From chemical analysis, there are 0.25 g of phospholipids and around 0.024 g of cholesterol bound per g of protein. Analytical ultracentrifugation shows one main complex, sedimenting at s20,w = 7.2 ± 0.1 S, together with minor amounts of irreversibly aggregated material. From these data, a buoyant molecular mass is calculated, corresponding to an H+,K+-ATPase α,β-protomer of 147.3 kDa. Complementary sedimentation velocity with deuterated water gives a picture of an α,β-protomer with 0.9–1.4 g/g of bound detergent and lipids and a reasonable frictional ratio of 1.5, corresponding to a Stokes radius of 7.1 nm. An α2,β2 dimer is rejected by the data. Light scattering coupled to gel filtration confirms the monomeric state of solubilized H+,K+-ATPase. Thus, α,β H+,K+-ATPase is active at least in detergent and may plausibly function as a monomer, as has been established for other P-type ATPases, Ca2+-ATPase and Na+,K+-ATPase. PMID:23055529

The mechanism of Cu+ transport ATPases: interaction with CU+ chaperones and the role of transient metal-binding sites.

PubMed

Padilla-Benavides, Teresita; McCann, Courtney J; Argüello, José M

2013-01-04

Cu(+)-ATPases are membrane proteins that couple the hydrolysis of ATP to the efflux of cytoplasmic Cu(+). In cells, soluble chaperone proteins bind and distribute cytoplasmic Cu(+), delivering the ion to the transmembrane metal-binding sites in the ATPase. The structure of Legionella pneumophila Cu(+)-ATPase (Gourdon, P., Liu, X. Y., Skjørringe, T., Morth, J. P., Møller, L. B., Pedersen, B. P., and Nissen, P. (2011) Nature 475, 59-64) shows that a kinked transmembrane segment forms a "platform" exposed to the cytoplasm. In addition, neighboring invariant Met, Asp, and Glu are located at the "entrance" of the ion path. Mutations of amino acids in these regions of the Archaeoglobus fulgidus Cu(+)-ATPase CopA do not affect ATPase activity in the presence of Cu(+) free in solution. However, Cu(+) bound to the corresponding chaperone (CopZ) could not activate the mutated ATPases, and in parallel experiments, CopZ was unable to transfer Cu(+) to CopA. Furthermore, mutation of a specific electronegative patch on the CopZ surface abolishes the ATPase activation and Cu(+) transference, indicating that the region is required for the CopZ-CopA interaction. Moreover, the data suggest that the interaction is driven by the complementation of the electropositive platform in the ATPase and the electronegative Cu(+) chaperone. This docking likely places the Cu(+) proximal to the conserved carboxyl and thiol groups in the entrance site that induce metal release from the chaperone via ligand exchange. The initial interaction of Cu(+) with the pump is transient because Cu(+) is transferred from the entrance site to transmembrane metal-binding sites involved in transmembrane translocation.

NO levels in diabetes mellitus: Effects of l-NAME and insulin on LCAT, Na(+)/K(+) ATPase activity and lipid profile.

PubMed

Tekin, Neslihan; Akyüz, Fahrettin; Temel, Halide Edip

2011-01-01

Diabetes mellitus (DM) is a chronic disease and one of the most important health problems. Several factors may be responsible for the complications of diabetes mellitus including alterations in the activities of sodium-potassium adenosine triphosphatase (Na(+)/K(+) ATPase) and lecithin:cholesterol acyltransferase (LCAT) and also levels of nitric oxide (NO). We have investigated the effects of alterations in serum NO levels on activities of erythrocyte membran Na/K ATPase and serum LCAT enzymes. The experiments were performed on male rats divided into four groups: group 1, control (standart diet); group 2, diabetic control (single dose of 65mg/kg of streptozotocin (STZ), i.p); group 3, STZ+insulin (8IU/kg/day s.c.); group 4 (STZ+l-NAME 5mg/kg/day orally). Streptozotocin-induced diabetic rats, showed a significant increase in blood glucose and serum cholesterol (C) and triglyceride (TG). Compared to the control group with diabetic group plasma LCAT concentrations and erythrocyte membrane Na(+)/K(+) ATPase were found to be decreased. Activities of Na(+)/K(+) ATPase and serum NO level were decreased with the administration of l-NAME. We observed that insulin was ameliorated in all parameters. Serum NO levels is related to erythrocyte membrane Na(+)/K(+) ATPase activity. But serum NO levels did not affect the plasma LCAT activity and serum lipid profiles. Copyright © 2010 Diabetes India. Published by Elsevier Ltd. All rights reserved.

Structural Insights into the Unusually Strong ATPase Activity of the AAA Domain of the Caenorhabditis elegans Fidgetin-like 1 (FIGL-1) Protein*

PubMed Central

Peng, Wentao; Lin, Zhijie; Li, Weirong; Lu, Jing; Shen, Yuequan; Wang, Chunguang

2013-01-01

The FIGL-1 (fidgetin like-1) protein is a homolog of fidgetin, a protein whose mutation leads to multiple developmental defects. The FIGL-1 protein contains an AAA (ATPase associated with various activities) domain and belongs to the AAA superfamily. However, the biological functions and developmental implications of this protein remain unknown. Here, we show that the AAA domain of the Caenorhabditis elegans FIGL-1 protein (CeFIGL-1-AAA), in clear contrast to homologous AAA domains, has an unusually high ATPase activity and forms a hexamer in solution. By determining the crystal structure of CeFIGL-1-AAA, we found that the loop linking helices α9 and α10 folds into the short helix α9a, which has an acidic surface and interacts with a positively charged surface of the neighboring subunit. Disruption of this charge interaction by mutagenesis diminishes both the ATPase activity and oligomerization capacity of the protein. Interestingly, the acidic residues in helix α9a of CeFIGL-1-AAA are not conserved in other homologous AAA domains that have relatively low ATPase activities. These results demonstrate that the sequence of CeFIGL-1-AAA has adapted to establish an intersubunit charge interaction, which contributes to its strong oligomerization and ATPase activity. These unique properties of CeFIGL-1-AAA distinguish it from other homologous proteins, suggesting that CeFIGL-1 may have a distinct biological function. PMID:23979136

Structural insights into the unusually strong ATPase activity of the AAA domain of the Caenorhabditis elegans fidgetin-like 1 (FIGL-1) protein.

PubMed

Peng, Wentao; Lin, Zhijie; Li, Weirong; Lu, Jing; Shen, Yuequan; Wang, Chunguang

2013-10-11

The FIGL-1 (fidgetin like-1) protein is a homolog of fidgetin, a protein whose mutation leads to multiple developmental defects. The FIGL-1 protein contains an AAA (ATPase associated with various activities) domain and belongs to the AAA superfamily. However, the biological functions and developmental implications of this protein remain unknown. Here, we show that the AAA domain of the Caenorhabditis elegans FIGL-1 protein (CeFIGL-1-AAA), in clear contrast to homologous AAA domains, has an unusually high ATPase activity and forms a hexamer in solution. By determining the crystal structure of CeFIGL-1-AAA, we found that the loop linking helices α9 and α10 folds into the short helix α9a, which has an acidic surface and interacts with a positively charged surface of the neighboring subunit. Disruption of this charge interaction by mutagenesis diminishes both the ATPase activity and oligomerization capacity of the protein. Interestingly, the acidic residues in helix α9a of CeFIGL-1-AAA are not conserved in other homologous AAA domains that have relatively low ATPase activities. These results demonstrate that the sequence of CeFIGL-1-AAA has adapted to establish an intersubunit charge interaction, which contributes to its strong oligomerization and ATPase activity. These unique properties of CeFIGL-1-AAA distinguish it from other homologous proteins, suggesting that CeFIGL-1 may have a distinct biological function.

ATP binding at noncatalytic sites of soluble chloroplast F1-ATPase is required for expression of the enzyme activity.

PubMed

Milgrom, Y M; Ehler, L L; Boyer, P D

1990-11-05

The F1-ATPase from chloroplasts (CF1) lacks catalytic capacity for ATP hydrolysis if ATP is not bound at noncatalytic sites. CF1 heat activated in the presence of ADP, with less than one ADP and no ATP at non-catalytic sites, shows a pronounced lag in the onset of ATP hydrolysis after exposure to 5-20 microM ATP. The onset of activity correlates well with the binding of ATP at the last two of the three noncatalytic sites. The dependence of activity on the presence of ATP at non-catalytic sites is shown at relatively low or high free Mg2+ concentrations, with or without bicarbonate as an activating anion, and when the binding of ATP at noncatalytic sites is slowed 3-4-fold by sulfate. The latent CF1 activated by dithiothreitol also requires ATP at noncatalytic sites for ATPase activity. A similar requirement by other F1-ATPases and by ATP synthases seems plausible.

Biochemical changes in the skin of rats exposed to radiation against the background of thermal stress. [X rays; ATPase and creatine kinase activities

DOE Office of Scientific and Technical Information (OSTI.GOV)

Matyushichev, V.B.; Taratukhin, V.R.; Shamratova, V.G.

1978-01-01

The effectiveness of exposing rats to different doses of x radiation after submitting them to a heat load, according to the tests of ATPase and creatine kinase activity of aqueous extracts of skin at the relatively late observation period was compared. The effects of the combined factors were monitored by means of a heat load (one group) and exposure to radiation alone in doses of 25, 50, 100, 250, and 400 R (5 groups). The obtained data are indicative of marked specificity of ATPase and creatine kinase reactions to the combined factors. Creatine kinase activity undergoes a 157% change, whereasmore » the mean relative deviation of ATPase activity constitutes only 71% of the normal level. The most effect loads are 36/sup 0/C + 25 R and 36/sup 0/C + 400 R. With all tested doses the extent of the effect of radiation on creatine kinase activity is only negligibly lower than the effectiveness of combined loads, whereas according to the ATPase test, radiation alone induces virtually the same changes in activity as combined factors. ATPase undergoes maximum change after irradiation in doses of 250 and 400 R; delivery of 25 to 100 R is associated with much less marked changes in activity. In contrast, creatine kinase demonstrates maximum sensitivity to radiation in a dosage of 25 R and minimum sensitivity, with a dosage of 100 R. Thermal stress (according to ATPase and creatine kinase activity) has a profound and quite substantial effect on processes of development of radiation lesion. It can be manifested by complete or partial summation of effects of each of the factors, mutual attenuation of effects, or absence of interaction between factors in the combination. All this is indicative of the complexity and differences in mechanisms of expression of effects of the factors used. (ERB)« less

V-ATPase proton pumping activity is required for adult zebrafish appendage regeneration.

PubMed

Monteiro, Joana; Aires, Rita; Becker, Jörg D; Jacinto, António; Certal, Ana C; Rodríguez-León, Joaquín

2014-01-01

The activity of ion channels and transporters generates ion-specific fluxes that encode electrical and/or chemical signals with biological significance. Even though it is long known that some of those signals are crucial for regeneration, only in recent years the corresponding molecular sources started to be identified using mainly invertebrate or larval vertebrate models. We used adult zebrafish caudal fin as a model to investigate which and how ion transporters affect regeneration in an adult vertebrate model. Through the combined use of biophysical and molecular approaches, we show that V-ATPase activity contributes to a regeneration-specific H+ ef`flux. The onset and intensity of both V-ATPase expression and H+ efflux correlate with the different regeneration rate along the proximal-distal axis. Moreover, we show that V-ATPase inhibition impairs regeneration in adult vertebrate. Notably, the activity of this H+ pump is necessary for aldh1a2 and mkp3 expression, blastema cell proliferation and fin innervation. To the best of our knowledge, this is the first report on the role of V-ATPase during adult vertebrate regeneration.

V-ATPase Proton Pumping Activity Is Required for Adult Zebrafish Appendage Regeneration

PubMed Central

Monteiro, Joana; Aires, Rita; Becker, Jörg D.; Jacinto, António; Certal, Ana C.; Rodríguez-León, Joaquín

2014-01-01

The activity of ion channels and transporters generates ion-specific fluxes that encode electrical and/or chemical signals with biological significance. Even though it is long known that some of those signals are crucial for regeneration, only in recent years the corresponding molecular sources started to be identified using mainly invertebrate or larval vertebrate models. We used adult zebrafish caudal fin as a model to investigate which and how ion transporters affect regeneration in an adult vertebrate model. Through the combined use of biophysical and molecular approaches, we show that V-ATPase activity contributes to a regeneration-specific H+ ef`flux. The onset and intensity of both V-ATPase expression and H+ efflux correlate with the different regeneration rate along the proximal-distal axis. Moreover, we show that V-ATPase inhibition impairs regeneration in adult vertebrate. Notably, the activity of this H+ pump is necessary for aldh1a2 and mkp3 expression, blastema cell proliferation and fin innervation. To the best of our knowledge, this is the first report on the role of V-ATPase during adult vertebrate regeneration. PMID:24671205

Glutamate transporter activity promotes enhanced Na+/K+‐ATPase‐mediated extracellular K+ management during neuronal activity

PubMed Central

Larsen, Brian Roland; Holm, Rikke; Vilsen, Bente

2016-01-01

Key points Management of glutamate and K+ in brain extracellular space is of critical importance to neuronal function.The astrocytic α2β2 Na+/K+‐ATPase isoform combination is activated by the K+ transients occurring during neuronal activity.In the present study, we report that glutamate transporter‐mediated astrocytic Na+ transients stimulate the Na+/K+‐ATPase and thus the clearance of extracellular K+.Specifically, the astrocytic α2β1 Na+/K+‐ATPase subunit combination displays an apparent Na+ affinity primed to react to physiological changes in intracellular Na+.Accordingly, we demonstrate a distinct physiological role in K+ management for each of the two astrocytic Na+/K+‐ATPase β‐subunits. Abstract Neuronal activity is associated with transient [K+]o increases. The excess K+ is cleared by surrounding astrocytes, partly by the Na+/K+‐ATPase of which several subunit isoform combinations exist. The astrocytic Na+/K+‐ATPase α2β2 isoform constellation responds directly to increased [K+]o but, in addition, Na+/K+‐ATPase‐mediated K+ clearance could be governed by astrocytic [Na+]i. During most neuronal activity, glutamate is released in the synaptic cleft and is re‐absorbed by astrocytic Na+‐coupled glutamate transporters, thereby elevating [Na+]i. It thus remains unresolved whether the different Na+/K+‐ATPase isoforms are controlled by [K+]o or [Na+]i during neuronal activity. Hippocampal slice recordings of stimulus‐induced [K+]o transients with ion‐sensitive microelectrodes revealed reduced Na+/K+‐ATPase‐mediated K+ management upon parallel inhibition of the glutamate transporter. The apparent intracellular Na+ affinity of isoform constellations involving the astrocytic β2 has remained elusive as a result of inherent expression of β1 in most cell systems, as well as technical challenges involved in measuring intracellular affinity in intact cells. We therefore expressed the different astrocytic isoform constellations in Xenopus oocytes and determined their apparent Na+ affinity in intact oocytes and isolated membranes. The Na+/K+‐ATPase was not fully saturated at basal astrocytic [Na+]i, irrespective of isoform constellation, although the β1 subunit conferred lower apparent Na+ affinity to the α1 and α2 isoforms than the β2 isoform. In summary, enhanced astrocytic Na+/K+‐ATPase‐dependent K+ clearance was obtained with parallel glutamate transport activity. The astrocytic Na+/K+‐ATPase isoform constellation α2β1 appeared to be specifically geared to respond to the [Na+]i transients associated with activity‐induced glutamate transporter activity. PMID:27231201

Antagonistic actions of renal dopamine and 5-hydroxytryptamine: increase in Na+, K(+)-ATPase activity in renal proximal tubules via activation of 5-HT1A receptors.

PubMed Central

Soares-da-Silva, P.; Pinto-do-O, P. C.; Bertorello, A. M.

1996-01-01

1. 5-Hydroxytryptamine (5-HT) is antinatriuretic. Since this effect of 5-HT is not accomplished by changes in glomerular haemodynamics, we have examined in this study whether 5-HT may influence sodium excretion by affecting the Na+, K(+)-ATPase activity in renal cortical tubules. 2. Na+, K(+)-ATPase activity was determined as the rate of [32P]-ATP hydrolysis in renal cortical tubules in suspension. Basal Na+, K(+)-ATPase activity in renal tubules was 4.8 +/- 0.4 mumol Pi mg-1 protein h-1 (n = 8). The 5-HT1A receptor agonist, (+/-)-8-hydroxy-2-(di-n-propylamino) tetraline (8-OH-DPAT) (10 to 3000 nM) induced a concentration-dependent increase (P < 0.05) in Na+, K(+)-ATPase activity with an EC50 value of 355 nM (95% confidence limits: 178, 708). Maximal stimulation elicited by 3000 nM of 8-OH-DPAT was antagonized by the selective 5-HT1A receptor antagonist, (+)-WAY 100135 10 to 1000 nM) with an IC50 value of 20 nM (14, 29); 0.3 microM (+)-WAY 100135 completely abolished (P < 0.01) the stimulatory effect of 8-OH-DPAT. The stimulatory effect of 8-OH-DPAT was found to be time-dependent (15 +/- 2% and 66 +/- 7% increase at 2.5 and 5.0 min, respectively). The 5-HT2 receptor agonist alpha-methyl-5-HT (100 to 3000 nM) did not induce any significant changes in Na+, K(+)-ATPase activity (5.0 +/- 1.5 mumol Pi mg-1 protein h-1; n = 4). 3. The stimulatory effect 8-OH-DPAT was absent when homogenates were used. Stimulation occurred at a Vmax concentration (70 mM) of sodium supporting the notion that stimulation occurs independently of increasing sodium permeability. 4. The inhibitory effect of dopamine (P < 0.05) on Na+, K(+)-ATPase activity was blunted by co-incubation with 8-OH-DPAT (0.5 microM). 5. It is concluded that activation of 5-HT1A receptors increases Na+, K(+)-ATPase activity in renal cortical tubules; this effect may represent an important cellular mechanism, at the tubule level, responsible for the antinatriuretic effect of 5-HT. Images Figure 4 PMID:8882616

The MLH1 ATPase domain is needed for suppressing aberrant formation of interstitial telomeric sequences.

PubMed

Jia, Pingping; Chai, Weihang

2018-05-01

Genome instability gives rise to cancer. MLH1, commonly known for its important role in mismatch repair (MMR), DNA damage signaling and double-strand break (DSB) repair, safeguards genome stability. Recently we have reported a novel role of MLH1 in preventing aberrant formation of interstitial telomeric sequences (ITSs) at intra-chromosomal regions. Deficiency in MLH1, in particular its N-terminus, leads to an increase of ITSs. Here, we identify that the ATPase activity in the MLH1 N-terminal domain is important for suppressing the formation of ITSs. The ATPase activity is also needed for recruiting MLH1 to DSBs. Moreover, defective ATPase activity of MLH1 causes an increase in micronuclei formation. Our results highlight the crucial role of MLH1's ATPase domain in preventing the aberrant formation of telomeric sequences at the intra-chromosomal regions and preserving genome stability. Copyright © 2018 Elsevier B.V. All rights reserved.

Na+/K+ ATPase regulates the expression and localization of acetylcholine receptors in a pump activity-independent manner

PubMed Central

Doi, Motomichi; Iwasaki, Kouichi

2008-01-01

Na+/K+ ATPase is a plasma membrane-localized sodium pump that maintains the ion gradients between the extracellular and intracellular environments, which in turn controls the cellular resting membrane potential. Recent evidence suggests that the pump is also localized at synapses and regulates synaptic efficacy. However, its precise function at the synapse is unknown. Here we show that two mutations in the α subunit of the eat-6 Na+/K+ ATPase in Caenorhabditis elegans dramatically increase the sensitivity to acetylcholine (Ach) agonists and alter the localization of nicotinic Ach receptors at the neuromuscular junction (NMJ). These defects can be rescued by mutated EAT-6 proteins which lack its pump activity, suggesting the presence of a novel function for Ach signaling. The Na+/K+ ATPase accumulates at postsynaptic sites and appears to surround Ach receptors to maintain rigid clusters at the NMJ. Our findings suggest a critical pump activity-independent, allele –specific role for Na+/K+ ATPase on postsynaptic organization and synaptic efficacy. PMID:18599311

Characterization of the plasma membrane H+-ATPase in the liverwort Marchantia polymorpha.

PubMed

Okumura, Masaki; Inoue, Shin-ichiro; Takahashi, Koji; Ishizaki, Kimitsune; Kohchi, Takayuki; Kinoshita, Toshinori

2012-06-01

The plasma membrane H(+)-ATPase generates an electrochemical gradient of H(+) across the plasma membrane that provides the driving force for solute transport and regulates pH homeostasis and membrane potential in plant cells. Recent studies have demonstrated that phosphorylation of the penultimate threonine in H(+)-ATPase and subsequent binding of a 14-3-3 protein is the major common activation mechanism for H(+)-ATPase in vascular plants. However, there is very little information on the plasma membrane H(+)-ATPase in nonvascular plant bryophytes. Here, we show that the liverwort Marchantia polymorpha, which is the most basal lineage of extant land plants, expresses both the penultimate threonine-containing H(+)-ATPase (pT H(+)-ATPase) and non-penultimate threonine-containing H(+)-ATPase (non-pT H(+)-ATPase) as in the green algae and that pT H(+)-ATPase is regulated by phosphorylation of its penultimate threonine. A search in the expressed sequence tag database of M. polymorpha revealed eight H(+)-ATPase genes, designated MpHA (for M. polymorpha H(+)-ATPase). Four isoforms are the pT H(+)-ATPase; the remaining isoforms are non-pT H(+)-ATPase. An apparent 95-kD protein was recognized by anti-H(+)-ATPase antibodies against an Arabidopsis (Arabidopsis thaliana) isoform and was phosphorylated on the penultimate threonine in response to the fungal toxin fusicoccin in thalli, indicating that the 95-kD protein contains pT H(+)-ATPase. Furthermore, we found that the pT H(+)-ATPase in thalli is phosphorylated in response to light, sucrose, and osmotic shock and that light-induced phosphorylation depends on photosynthesis. Our results define physiological signals for the regulation of pT H(+)-ATPase in the liverwort M. polymorpha, which is one of the earliest plants to acquire pT H(+)-ATPase.

The effect of chlorpyrifos upon ATPase activity of sarcoplasmic reticulum and biomechanics of skeleta l muscle contraction.

PubMed

Nozdrenko, D M; Miroshnychenko, M S; Soroca, V M; Korchins ka, L V; Zavodovskiy, D O

2016-01-01

We investigated the effect of chlorpyrifos, an organophosphate insecticide, on Ca2+,Mg2+-ATPase activity of sarcoplasmic reticulum and on contraction dynamics (force and length changes) of Rana temporaria m. tibialis anterior muscle fiber bundles. All of the used concentrations of chlorpyrifos (10-6 to 10-5 M) caused decrease of Ca2+,Mg2+-ATPase activity. The inhibition of Ca2+,Mg2+-ATPase activity by chlorpyriphos in concentrations of 10-6 M to 7.5·10-6 M is due to permeation of sarcoplasmic reticulum rather than due to direct enzyme inhibition by organophosphate insecticides. The inhibitory properties of the compound were higher at increased concentration and exposure timeframes. Chlorpyrifos in concentration range of 10-6 to 7.5·10-6 M causes changes in muscle fiber response force that were more pronounced than changes in contractile length. We demonstrated inhibition of Ca2+,Mg2+-ATPase activity caused by noncholinergic effects of chlorpyriphos. It is possible to conclude that influence of organophosphate insecticides happens not only in the neuromuscular transmission but also on the level of subcellular structures.

Comparative chemical genomics reveal that the spiroindolone antimalarial KAE609 (Cipargamin) is a P-type ATPase inhibitor

PubMed Central

Goldgof, Gregory M.; Durrant, Jacob D.; Ottilie, Sabine; Vigil, Edgar; Allen, Kenneth E.; Gunawan, Felicia; Kostylev, Maxim; Henderson, Kiersten A.; Yang, Jennifer; Schenken, Jake; LaMonte, Gregory M.; Manary, Micah J.; Murao, Ayako; Nachon, Marie; Stanhope, Rebecca; Prescott, Maximo; McNamara, Case W.; Slayman, Carolyn W.; Amaro, Rommie E.; Suzuki, Yo; Winzeler, Elizabeth A.

2016-01-01

The spiroindolones, a new class of antimalarial medicines discovered in a cellular screen, are rendered less active by mutations in a parasite P-type ATPase, PfATP4. We show here that S. cerevisiae also acquires mutations in a gene encoding a P-type ATPase (ScPMA1) after exposure to spiroindolones and that these mutations are sufficient for resistance. KAE609 resistance mutations in ScPMA1 do not confer resistance to unrelated antimicrobials, but do confer cross sensitivity to the alkyl-lysophospholipid edelfosine, which is known to displace ScPma1p from the plasma membrane. Using an in vitro cell-free assay, we demonstrate that KAE609 directly inhibits ScPma1p ATPase activity. KAE609 also increases cytoplasmic hydrogen ion concentrations in yeast cells. Computer docking into a ScPma1p homology model identifies a binding mode that supports genetic resistance determinants and in vitro experimental structure-activity relationships in both P. falciparum and S. cerevisiae. This model also suggests a shared binding site with the dihydroisoquinolones antimalarials. Our data support a model in which KAE609 exerts its antimalarial activity by directly interfering with P-type ATPase activity. PMID:27291296

Role of estrogen and progesterone in the modulation of CNG-A1 and Na/K+-ATPase expression in the renal cortex.

PubMed

Gracelli, Jones B; Souza-Menezes, Jackson; Barbosa, Carolina M L; Ornellas, Felipe S; Takiya, Christina M; Alves, Leandro M; Wengert, Mira; Feltran, Georgia da Silva; Caruso-Neves, Celso; Moyses, Margareth R; Prota, Luiz F M; Morales, Marcelo M

2012-01-01

The steroid hormones, estrogen and progesterone, are involved mainly in the control of female reproductive functions. Among other effects, estrogen and progesterone can modulate Na(+) reabsorption along the nephron altering the body's hydroelectrolyte balance. In this work, we analyzed the expression of cyclic nucleotide-gated channel A1 (CNG-A1) and α1 Na(+)/K(+)-ATPase subunit in the renal cortex and medulla of female ovariectomized rats and female ovariectomized rats subjected to 10 days of 17β-estradiol benzoate (2.0 µg/kg body weight) and progesterone (1.7 mg/kg body weight) replacement. Na(+)/K(+) ATPase activity was also measured. Immunofluorescence localization of CNG-A1 in the cortex and medulla was performed in control animals. We observed that CNG-A1 is localized at the basolateral membrane of proximal and distal tubules. Female ovariectomized rats showed low expression of CNG-A1 and low expression and activity of Na(+)/K(+) ATPase in the renal cortex. When female ovariectomized rats were subjected to 17β-estradiol benzoate replacement, normalization of CNG-A1 expression and Na(+)/K(+) ATPase expression and activity was observed. The replacement of progesterone was not able to recover CNG-A1 expression and Na(+)/K(+) ATPase expression at the control level. Only the activity of Na(+)/K(+) ATPase was able to be recovered at control levels in animals subjected to progesterone replacement. No changes in expression and activity were observed in the renal medulla. The expression of CNG-A1 is higher in cortex compared to medulla. In this work, we observed that estrogen and progesterone act in renal tissues modulating CNG-A1 and Na(+)/K(+) ATPase and these effects could be important in Na(+) and water balance. Copyright © 2012 S. Karger AG, Basel.

Impacts of Usher syndrome type IB mutations on human myosin VIIa motor function.

PubMed

Watanabe, Shinya; Umeki, Nobuhisa; Ikebe, Reiko; Ikebe, Mitsuo

2008-09-09

Usher syndrome (USH) is a human hereditary disorder characterized by profound congenital deafness, retinitis pigmentosa, and vestibular dysfunction. Myosin VIIa has been identified as the responsible gene for USH type 1B, and a number of missense mutations have been identified in the affected families. However, the molecular basis of the dysfunction of USH gene, myosin VIIa, in the affected families is unknown to date. Here we clarified the effects of USH1B mutations on human myosin VIIa motor function for the first time. The missense mutations of USH1B significantly inhibited the actin activation of ATPase activity of myosin VIIa. G25R, R212C, A397D, and E450Q mutations abolished the actin-activated ATPase activity completely. P503L mutation increased the basal ATPase activity for 2-3-fold but reduced the actin-activated ATPase activity to 50% of the wild type. While all of the mutations examined, except for R302H, reduced the affinity for actin and the ATP hydrolysis cycling rate, they did not largely decrease the rate of ADP release from actomyosin, suggesting that the mutations reduce the duty ratio of myosin VIIa. Taken together, the results suggest that the mutations responsible for USH1B cause the complete loss of the actin-activated ATPase activity or the reduction of duty ratio of myosin VIIa.

Impacts of Usher Syndrome Type IB Mutations on Human Myosin VIIa Motor Function†

PubMed Central

Watanabe, Shinya; Umeki, Nobuhisa; Ikebe, Reiko; Ikebe, Mitsuo

2010-01-01

Usher syndrome (USH) is a human hereditary disorder characterized by profound congenital deafness, retinitis pigmentosa and vestibular dysfunction. Myosin VIIa has been identified as the responsible gene for USH type 1B, and a number of missense mutations have been identified in the affected families. However, the molecular basis of the dysfunction of USH gene, myosin VIIa, in the affected families is unknown to date. Here we clarified the effects of USH1B mutations on human myosin VIIa motor function for the first time. The missense mutations of USH1B significantly inhibited the actin activation of ATPase activity of myosin VIIa. G25R, R212C, A397D and E450Q mutations abolished the actin-activated ATPase activity completely. P503L mutation increased the basal ATPase activity for 2-3 fold, but reduced the actin-activated ATPase activity to 50% of the wild type. While all the mutations examined, except for R302H, reduced the affinity for actin and the ATP hydrolysis cycling rate, they did not largely decrease the rate of ADP release from acto-myosin, suggesting that the mutations reduce the duty ratio of myosin VIIa. Taken together, the results suggest that the mutations responsible for USH1B cause the complete loss of the actin-activated ATPase activity or the reduction of duty ratio of myosin VIIa. PMID:18700726

Tandem phosphorylation of Ser-911 and Thr-912 at the C terminus of yeast plasma membrane H+-ATPase leads to glucose-dependent activation.

PubMed

Lecchi, Silvia; Nelson, Clark J; Allen, Kenneth E; Swaney, Danielle L; Thompson, Katie L; Coon, Joshua J; Sussman, Michael R; Slayman, Carolyn W

2007-12-07

In recent years there has been growing interest in the post-translational regulation of P-type ATPases by protein kinase-mediated phosphorylation. Pma1 H(+)-ATPase, which is responsible for H(+)-dependent nutrient uptake in yeast (Saccharomyces cerevisiae), is one such example, displaying a rapid 5-10-fold increase in activity when carbon-starved cells are exposed to glucose. Activation has been linked to Ser/Thr phosphorylation in the C-terminal tail of the ATPase, but the specific phosphorylation sites have not previously been mapped. The present study has used nanoflow high pressure liquid chromatography coupled with electrospray electron transfer dissociation tandem mass spectrometry to identify Ser-911 and Thr-912 as two major phosphorylation sites that are clearly related to glucose activation. In carbon-starved cells with low Pma1 activity, peptide 896-918, which was derived from the C terminus upon Lys-C proteolysis, was found to be singly phosphorylated at Thr-912, whereas in glucose-metabolizing cells with high ATPase activity, the same peptide was doubly phosphorylated at Ser-911 and Thr-912. Reciprocal (14)N/(15)N metabolic labeling of cells was used to measure the relative phosphorylation levels at the two sites. The addition of glucose to carbon-starved cells led to a 3-fold reduction in the singly phosphorylated form and an 11-fold increase in the doubly phosphorylated form. These results point to a mechanism in which the stepwise phosphorylation of two tandemly positioned residues near the C terminus mediates glucose-dependent activation of the H(+)-ATPase.

Intracellular sodium modulates the state of protein kinase C phosphorylation of rat proximal tubule Na+,K+-ATPase.

PubMed

Ibarra, F R; Cheng, S X Jun; Agrén, M; Svensson, L-B; Aizman, O; Aperia, A

2002-06-01

The natriuretic hormone dopamine and the antinatriuretic hormone noradrenaline, acting on alpha-adrenergic receptors, have been shown to bidirectionally modulate the activity of renal tubular Na+,K+-adenosine triphosphate (ATPase). Here we have examined whether intracellular sodium concentration influences the effects of these bidirectional forces on the state of phosphorylation of Na+,K+-ATPase. Proximal tubules dissected from rat kidney were incubated with dopamine or the alpha-adrenergic agonist, oxymetazoline, and transiently permeabilized in a medium where sodium concentration ranged between 5 and 70 mM. The variations of sodium concentration in the medium had a proportional effect on intracellular sodium. Dopamine and protein kinase C (PKC) phosphorylate the catalytic subunit of rat Na+,K+-ATPase on the Ser23 residue. The level of PKC induced Na+,K+-ATPase phosphorylation was determined using an antibody that only recognizes Na+,K+-ATPase, which is not phosphorylated on its PKC site. Under basal conditions Na+,K+-ATPase was predominantly in its phosphorylated state. When intracellular sodium was increased, Na+,K+-ATPase was predominantly in its dephosphorylated state. Phosphorylation of Na+,K+-ATPase by dopamine was most pronounced when intracellular sodium was high, and dephosphorylation by oxymetazoline was most pronounced when intracellular sodium was low. The oxymetazoline effect was mimicked by the calcium ionophore A23187. An inhibitor of the calcium-dependent protein phosphatase, calcineurin, increased the state of Na+,K+-ATPase phosphorylation. The results imply that phosphorylation of renal Na+,K+-ATPase activity is modulated by the level of intracellular sodium and that this effect involves PKC and calcium signalling pathways. The findings may have implication for the regulation of salt excretion and sodium homeostasis.

Glycolytic control of vacuolar-type ATPase activity: A mechanism to regulate influenza viral infection

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kohio, Hinissan P.; Adamson, Amy L., E-mail: aladamso@uncg.edu

As new influenza virus strains emerge, finding new mechanisms to control infection is imperative. In this study, we found that we could control influenza infection of mammalian cells by altering the level of glucose given to cells. Higher glucose concentrations induced a dose-specific increase in influenza infection. Linking influenza virus infection with glycolysis, we found that viral replication was significantly reduced after cells were treated with glycolytic inhibitors. Addition of extracellular ATP after glycolytic inhibition restored influenza infection. We also determined that higher levels of glucose promoted the assembly of the vacuolar-type ATPase within cells, and increased vacuolar-type ATPase proton-transportmore » activity. The increase of viral infection via high glucose levels could be reversed by inhibition of the proton pump, linking glucose metabolism, vacuolar-type ATPase activity, and influenza viral infection. Taken together, we propose that altering glucose metabolism may be a potential new approach to inhibit influenza viral infection. - Highlights: • Increased glucose levels increase Influenza A viral infection of MDCK cells. • Inhibition of the glycolytic enzyme hexokinase inhibited Influenza A viral infection. • Inhibition of hexokinase induced disassembly the V-ATPase. • Disassembly of the V-ATPase and Influenza A infection was bypassed with ATP. • The state of V-ATPase assembly correlated with Influenza A infection of cells.« less

Biochemical characterization of P-type copper ATPases

PubMed Central

Inesi, Giuseppe; Pilankatta, Rajendra; Tadini-Buoninsegni, Francesco

2014-01-01

Copper ATPases, in analogy with other members of the P-ATPase superfamily, contain a catalytic headpiece including an aspartate residue reacting with ATP to form a phosphoenzyme intermediate, and transmembrane helices containing cation-binding sites [TMBS (transmembrane metal-binding sites)] for catalytic activation and cation translocation. Following phosphoenzyme formation by utilization of ATP, bound copper undergoes displacement from the TMBS to the lumenal membrane surface, with no H+ exchange. Although PII-type ATPases sustain active transport of alkali/alkali-earth ions (i.e. Na+, Ca2+) against electrochemical gradients across defined membranes, PIB-type ATPases transfer transition metal ions (i.e. Cu+) from delivery to acceptor proteins and, prominently in mammalian cells, undergo trafficking from/to various membrane compartments. A specific component of copper ATPases is the NMBD (N-terminal metal-binding domain), containing up to six copper-binding sites in mammalian (ATP7A and ATP7B) enzymes. Copper occupancy of NMBD sites and interaction with the ATPase headpiece are required for catalytic activation. Furthermore, in the presence of copper, the NMBD allows interaction with protein kinase D, yielding phosphorylation of serine residues, ATP7B trafficking and protection from proteasome degradation. A specific feature of ATP7A is glycosylation and stabilization on plasma membranes. Cisplatin, a platinum-containing anti-cancer drug, binds to copper sites of ATP7A and ATP7B, and undergoes vectorial displacement in analogy with copper. PMID:25242165

Low erythrocyte Na/K-pump activity and number in northeast Thailand adults: evidence suggesting an acquired disorder.

PubMed

Tosukhowong, P; Tungsanga, K; Kittinantavorakoon, C; Chaitachawong, C; Pansin, P; Sriboonlue, P; Sitprija, V

1996-07-01

Healthy northeastern Thais have a higher erythrocyte sodium concentration and a lower erythrocyte membrane Na,K-adenosine triphosphatase (ATPase) activity than central Thais. To elucidate whether the defect is hereditary or acquired, we studied plasma sodium and potassium and erythrocyte sodium, potassium, Na,K-ATPase activity, and ouabain-binding sites (OBS) in the following groups: healthy newborns of ethnic central Thais (group 1), healthy newborns of ethnic northeast Thais (group 2), healthy adults of central Thailand ethnicity who lived in the rural central region (group 3) or in Bangkok (group 4), healthy adults of northeast Thailand ethnicity who lived in the rural northeast region (group 5) or who migrated to work in Bangkok for at least 1 year (group 6). Erythrocyte Na was higher in group 2 than in group 1. Group 3 had lower erythrocyte Na,K-ATPase activity than group 4, and it was lower in group 5 than in group 6. Among all groups, group 5 had the highest erythrocyte Na (11.6 mmol/L,F < 0.0001) and the lowest Na,K-ATPase activity (63 mmol Pi/mg x h, F < 0.0001) and erythrocyte OBS (397 sites per cell, F < 0.05) than the other adult groups. There was a positive correlation between erythrocyte Na,K-ATPase and erythrocyte OBS (r = .416, P < .0001). Multiple regression analysis demonstrated a correlation between erythrocyte Na as a dependent variable and erythrocyte OBS, plasma potassium, erythrocyte potassium, and erythrocyte Na,K-ATPase (r = .517, P < .0001). The erythrocyte Na,K-ATPase/OBS ratio, an expression of Na,K-ATPase activity equalized for the number of Na,K-pump units, was lowest among rural adults of the central region (group 3) and the northeast region (group 5) (F < 0.0002). Our data suggest that rural dwellers in Thailand tend to have lower erythrocyte Na,K-ATPase activity than urban dwellers and that this is probably acquired after birth. It was more severe among those from the northeast versus the central region, and was less severe among those who migrated to an urban area. This defect in northeast rural dwellers was probably associated with low numbers of Na,K-pump units and a defect of the pump to express activity, whereas in central rural dwellers it was probably associated with the latter condition. We postulate that there might be circulating Na,K-pump inhibitors and metabolic disturbances that cause attenuation of Na,K-ATPase function and synthesis in the northeast Thailand rural population, and that such substances may have an environmental origin. There may be a relationship between these abnormalities and sudden unexpected deaths.

Silybum marianum oil attenuates oxidative stress and ameliorates mitochondrial dysfunction in mice treated with D-galactose

PubMed Central

Zhu, Shu Yun; Dong, Ying; Tu, Jie; Zhou, Yue; Zhou, Xing Hua; Xu, Bin

2014-01-01

Background: Silybum marianum has been used as herbal medicine for the treatment of liver disease, liver cirrhosis, and to prevent liver cancer in Europe and Asia since ancient times. Silybum marianum oil (SMO), a by-product of silymarin production, is rich in essential fatty acids, phospholipids, sterols, and vitamin E. However, it has not been very good development and use. Objective: In the present study, we used olive oil as a control to investigate the antioxidant and anti-aging effect of SMO in D-galactose (D-gal)-induced aging mice. Materials and Methods: D-gal was injected intraperitoneally (500 mg/kg body weight daily) for 7 weeks while SMO was simultaneously administered orally. The triglycerides (TRIG) and cholesterol (CHOL) levels were estimated in the serum. Superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), total antioxidant capacity (T-AOC), monoamine oxidase (MAO), malondialdehyde (MDA), caspase-3, and Bcl-2 were determined in the liver and brain. The activities of Na+-K+-adenosine triphosphatase (ATPase), Ca2+-Mg2+-ATPase, membrane potential (ΔΨm), and membrane fluidity of the liver mitochondrial were estimated. Results: SMO decreased levels of TRIG and CHOL in aging mice. SMO administration elevated the activities of SOD, GSH-Px, and T-AOC, which are suppressed by aging. The levels of MAO and MDA in the liver and brain were reduced by SMO administration in aging mice. Enzyme linked immunosorbent assay showed that SMO significantly decreased the concentration of caspase-3 and improved the activity of Bcl-2 in the liver and brain of aging mice. Furthermore, SMO significantly attenuated the D-gal induced liver mitochondrial dysfunction by improving the activities of Na+-K+-ATPase, Ca2+-Mg2+-ATPase, membrane potential (ΔΨm), and membrane fluidity. Conclusion: These results indicate that SMO effectively attenuated oxidative damage and improved apoptosis related factors as well as liver mitochondrial dysfunction in aging mice. PMID:24914315

Organochlorine insecticide, herbicide and polychlorinated biphenyl (PCB) inhibition of NaK-ATPase in rainbow trout

USGS Publications Warehouse

Davis, Paul W.; Friedhoff, Jacqueline M.; Wedemeyer, Gary A.

1972-01-01

The current widespread presence of chlorinated insecticides, polychlorinated biphenyls (PCB's) and herbicides in world waterways has elicited much interest in the mechanisms of their toxicity in fishes. Inhibition of Na+,K+-activated adenosinetriphosphatase (NaK-ATPase) and Mg++-dependent ATPase (Mg-ATPase) by DDT, endosulfan and dicofol has been demonstrated in gill, brain and kidney microsomes of rainbow trout (1,2). Intestinal and gill ATPases in marine teleosts were recently reported to be sensitive to organochlorines (3). CutkonTp et al (4) noted inhibition of NaK-ATPase and Mg-ATPase in bluegill brain, liver, muscle and kidney by DDT and related chlorinated hydrocarbons. Inhibition of ATPases by PCB's has been recently shown in bluegill kidney, brain and liver (5). In the present study, we have further examined the NaK-ATPase enzyme system in trout gill as a site for the possible toxicity of selected organopolychlors, i.e., chlorinated insecticides, herbicides and PCB's.

Polar localization of plasma membrane Ca2+/Mg2+ ATPase correlates with the pattern of steady ionic currents in eggs ofLymnaea stagnalis andBithynia tentaculata (Mollusca).

PubMed

Zivkovic, Danica; Créton, Robbert; Zwaan, Gideon; de Bruijn, Willem C; Dohmen, M René

1990-11-01

During extrusion of the first polar body in eggs ofLymnaea stagnalis andBithynia tentaculata a localized Ca 2+ /Mg 2+ ATPase activity was detected, using Ando's enzyme-cytochemical method for electron microscopy [Ando et al. (1981) Acta Histochem Cytochem 14:705-726]. The enzyme activity was distributed in a polar fashion, along the cytoplasmic face of the plasma membrane. In the eggs ofLymnaea it was found only in the vegetal hemisphere, whereas inBithynia eggs it was localized both in the vegetal hemisphere and at the animal pole. This pattern of enzyme activity corresponds to the polar pattern of transcellular ionic currents measured with the vibrating probe, which we showed to be partially carried or regulated by calcium [Zivkovic and Dohmen (1989) Biol Bull (Woods Hole) 176 (Suppl):103-109]. The characteristics of the ATPase were studied using a variety of approaches such as ion and substrate depletions and substitutions, addition of specific inhibitors of ATPase activity, treatment with EDTA/EGTA and electron energy-loss spectrometry. The results indicate that, inLymnaea, there are at least two enzymatic entities. The first one is a Ca 2+ /Mg 2+ ATPase localized along the membrane and in the cortex of the vegetal hemisphere. The second one is a Ca 2+ -stimulated ATPase (calcium pump of the plasma membrane) localized in a small region of the membrane at the vegetal pole. We speculate that in the eggs ofLymnaea andBithynia a functional relationship exists between the plasma-membrane-associated ATPase activity and the transcellular ionic currents measured in the same region.

Dietary omega 6 fatty acids and the effects of hyperthyroidism in mice.

PubMed

Deshpande, N; Hulbert, A J

1995-03-01

The influence of the type of dietary fat on the effects of thyroid hormones was investigated in mice. Hyperthyroidism was achieved by providing thyroid hormones (T3 and T4) in the drinking water. Both hyperthyroid and euthyroid mice (Mus musculus) were fed isoenergetic diets containing 18% (w/w) total lipid but differing in fatty acid composition. Diets were either low in the polyunsaturated linoleic acid (18:2, omega 6) and high in saturated fatty acids (SFAs) or low in saturated fats and high in the polyunsaturated fatty acid (PUFA), linoleic acid. Treatments were maintained for 21-22 days. Plasma thyroid hormone levels, standard metabolic rate (SMR), changes in body mass, specific activities of malic enzyme (ME), Na-K-ATPase and glycerolphosphate dehydrogenase (GPDH) of the liver were measured. Fatty acid composition of the liver phospholipids was also determined. Levels of T3 (15-17 nM) and T4 (250-255 nM) were significantly higher in the respective hyperthyroid groups. There was no significant influence of the diet on hormone levels. Hyperthyroidism increased the SMR 37-44% above the euthyroid levels. A significant body weight loss of 14-18% was observed in hyperthyroid mice on the PUFA diet but not in those on the SFA diet. PUFA diet significantly reduced the activity of ME but had no effect on Na-K-ATPase or GPDH activity. Activities of Na-K-ATPase and GPDH were significantly elevated in all hyperthyroid groups. Mice on T4 and PUFA diet showed a highly significant 399% increase in GPDH activity above the euthyroid level.(ABSTRACT TRUNCATED AT 250 WORDS)

In vitro effects of fermented papaya (Carica papaya, L.) on platelets obtained from patients with type 2 diabetes.

PubMed

Raffaelli, F; Nanetti, L; Montecchiani, G; Borroni, F; Salvolini, E; Faloia, E; Ferretti, G; Mazzanti, L; Vignini, A

2015-02-01

Oxidative stress is associated with insulin resistance pathogenesis, insulin secretion deficiency, and complication onset. Fermented papaya preparation (FPP), a dietary supplement obtained by fermentation of the papaya fruit, may be used as an antioxidant in the prevention of diabetic complications. Platelets from 30 patients with type 2 diabetes mellitus (DM 2) and 15 healthy subjects were analyzed to evaluate the in vitro effects of FPP incubation. Na(+)/K(+)-adenosine triphosphatase (ATPase) activity, membrane fluidity, total antioxidant capacity (TAC), superoxide dismutase (SOD) activity, and conjugated diene levels were determined. In vitro FPP incubation improved platelet function, by enhancing Na(+)/K(+)-ATPase activity and membrane fluidity, and ameliorated the antioxidant system functionality, through an increase in TAC and SOD activity and a parallel decrease in conjugated diene levels in patients with DM 2. Our data suggest that the incubation with FPP may have a protective effect on platelets from patients with DM 2, by preventing the progression of oxidative damage associated with diabetes and its complications. Copyright © 2014 Elsevier B.V. All rights reserved.

Tumor cell cholesterol depletion and V-ATPase inhibition as an inhibitory mechanism to prevent cell migration and invasiveness in melanoma.

PubMed

Costa, Gildeíde Aparecida; de Souza, Sávio Bastos; da Silva Teixeira, Layz Ribeiro; Okorokov, Lev A; Arnholdt, Andrea Cristina Vetö; Okorokova-Façanha, Anna L; Façanha, Arnoldo Rocha

2018-03-01

V-ATPase interactions with cholesterol enriched membrane microdomains have been related to metastasis in a variety of cancers, but the underlying mechanism remains at its beginnings. It has recently been reported that the inhibition of this H + pump affects cholesterol mobilization to the plasma membrane. Inhibition of melanoma cell migration and invasiveness was assessed by wound healing and Transwell assays in murine cell lines (B16F10 and Melan-A). V-ATPase activity was measured in vitro by ATP hydrolysis and H + transport in membrane vesicles, and intact cell H + fluxes were measured by using a non-invasive Scanning Ion-selective Electrode Technique (SIET). Cholesterol depletion by 5mM MβCD was found to be inhibitory to the hydrolytic and H + pumping activities of the V-ATPase of melanoma cell lines, as well as to the migration and invasiveness capacities of these cells. Nearly the same effects were obtained using concanamycin A, a specific inhibitor of V-ATPase, which also promoted a decrease of the H + efflux in live cells at the same extent of MβCD. We found that cholesterol depletion significantly affects the V-ATPase activity and the initial metastatic processes following a profile similar to those observed in the presence of the V-ATPase specific inhibitor, concanamycin. The results shed new light on the functional role of the interactions between V-ATPases and cholesterol-enriched microdomains of cell membranes that contribute with malignant phenotypes in melanoma. Copyright © 2017 Elsevier B.V. All rights reserved.

Proteasome Inhibition Contributed to the Cytotoxicity of Arenobufagin after Its Binding with Na, K-ATPase in Human Cervical Carcinoma HeLa Cells

PubMed Central

Zhen, Hong; Huang, Ming; Zheng, Xi; Feng, Lixing; Jiang, Baohong; Yang, Min; Wu, Wanying; Liu, Xuan; Guo, Dean

2016-01-01

Although the possibility of developing cardiac steroids/cardiac glycosides as novel cancer therapeutic agents has been recognized, the mechanism of their anticancer activity is still not clear enough. Toad venom extract containing bufadienolides, which belong to cardiac steroids, has actually long been used as traditional Chinese medicine in clinic for cancer therapy in China. The cytotoxicity of arenobufagin, a bufadienolide isolated from toad venom, on human cervical carcinoma HeLa cells was checked. And, the protein expression profile of control HeLa cells and HeLa cells treated with arenobufagin for 48 h was analyzed using two-dimensional electrophoresis, respectively. Differently expressed proteins in HeLa cells treated with arenobufagin were identified and the pathways related to these proteins were mapped from KEGG database. Computational molecular docking was performed to verify the binding of arenobufagin and Na, K-ATPase. The effects of arenobufagin on Na, K-ATPase activity and proteasome activity of HeLa cells were checked. The protein-protein interaction network between Na, K-ATPase and proteasome was constructed and the expression of possible intermediate proteins ataxin-1 and translationally-controlled tumor protein in HeLa cells treated with arenobufagin was then checked. Arenobufagin induced apoptosis and G2/M cell cycle arrest in HeLa cells. The cytotoxic effect of arenobufagin was associated with 25 differently expressed proteins including proteasome-related proteins, calcium ion binding-related proteins, oxidative stress-related proteins, metabolism-related enzymes and others. The results of computational molecular docking revealed that arenobufagin was bound in the cavity formed by the transmembrane alpha subunits of Na, K-ATPase, which blocked the pathway of extracellular Na+/K+ cation exchange and inhibited the function of ion exchange. Arenobufagin inhibited the activity of Na, K-ATPase and proteasome, decreased the expression of Na, K-ATPase α1 and α3 subunits and increased the expression of WEE1 in HeLa cells. Antibodies against Na, K-ATPase α1 and α3 subunits alone or combinated with arenobufagin also inhibited the activity of proteasome. Furthermore, the expression of the possible intermediate proteins ataxin-1 and translationally-controlled tumor protein was increased in HeLa cells treated with arenobufagin by flow cytometry analysis, respectively. These results indicated that arenobufagin might directly bind with Na, K-ATPase α1 and α3 subunits and the inhibitive effect of arenobufagin on proteasomal activity of HeLa cells might be related to its binding with Na, K-ATPase. PMID:27428326

Proteasome Inhibition Contributed to the Cytotoxicity of Arenobufagin after Its Binding with Na, K-ATPase in Human Cervical Carcinoma HeLa Cells.

PubMed

Yue, Qingxi; Zhen, Hong; Huang, Ming; Zheng, Xi; Feng, Lixing; Jiang, Baohong; Yang, Min; Wu, Wanying; Liu, Xuan; Guo, Dean

2016-01-01

Although the possibility of developing cardiac steroids/cardiac glycosides as novel cancer therapeutic agents has been recognized, the mechanism of their anticancer activity is still not clear enough. Toad venom extract containing bufadienolides, which belong to cardiac steroids, has actually long been used as traditional Chinese medicine in clinic for cancer therapy in China. The cytotoxicity of arenobufagin, a bufadienolide isolated from toad venom, on human cervical carcinoma HeLa cells was checked. And, the protein expression profile of control HeLa cells and HeLa cells treated with arenobufagin for 48 h was analyzed using two-dimensional electrophoresis, respectively. Differently expressed proteins in HeLa cells treated with arenobufagin were identified and the pathways related to these proteins were mapped from KEGG database. Computational molecular docking was performed to verify the binding of arenobufagin and Na, K-ATPase. The effects of arenobufagin on Na, K-ATPase activity and proteasome activity of HeLa cells were checked. The protein-protein interaction network between Na, K-ATPase and proteasome was constructed and the expression of possible intermediate proteins ataxin-1 and translationally-controlled tumor protein in HeLa cells treated with arenobufagin was then checked. Arenobufagin induced apoptosis and G2/M cell cycle arrest in HeLa cells. The cytotoxic effect of arenobufagin was associated with 25 differently expressed proteins including proteasome-related proteins, calcium ion binding-related proteins, oxidative stress-related proteins, metabolism-related enzymes and others. The results of computational molecular docking revealed that arenobufagin was bound in the cavity formed by the transmembrane alpha subunits of Na, K-ATPase, which blocked the pathway of extracellular Na+/K+ cation exchange and inhibited the function of ion exchange. Arenobufagin inhibited the activity of Na, K-ATPase and proteasome, decreased the expression of Na, K-ATPase α1 and α3 subunits and increased the expression of WEE1 in HeLa cells. Antibodies against Na, K-ATPase α1 and α3 subunits alone or combinated with arenobufagin also inhibited the activity of proteasome. Furthermore, the expression of the possible intermediate proteins ataxin-1 and translationally-controlled tumor protein was increased in HeLa cells treated with arenobufagin by flow cytometry analysis, respectively. These results indicated that arenobufagin might directly bind with Na, K-ATPase α1 and α3 subunits and the inhibitive effect of arenobufagin on proteasomal activity of HeLa cells might be related to its binding with Na, K-ATPase.

Alterations of Na,K-ATPase isoenzymes in the rat diabetic neuropathy: protective effect of dietary supplementation with n-3 fatty acids.

PubMed

Gerbi, A; Maixent, J M; Barbey, O; Jamme, I; Pierlovisi, M; Coste, T; Pieroni, G; Nouvelot, A; Vague, P; Raccah, D

1998-08-01

Diabetic neuropathy is a degenerative complication of diabetes accompanied by an alteration of nerve conduction velocity (NCV) and Na,K-ATPase activity. The present study in rats was designed first to measure diabetes-induced abnormalities in Na,K-ATPase activity, isoenzyme expression, fatty acid content in sciatic nerve membranes, and NCV and second to assess the preventive ability of a fish oil-rich diet (rich in n-3 fatty acids) on these abnormalities. Diabetes was induced by intravenous streptozotocin injection. Diabetic animals (D) and nondiabetic control animals (C) were fed the standard rat chow either without supplementation or supplemented with either fish oil (DM, CM) or olive oil (DO, CO) at a daily dose of 0.5 g/kg by gavage during 8 weeks. Analysis of the fatty acid composition of purified sciatic nerve membranes from diabetic animals showed a decreased incorporation of C16:1(n-7) fatty acids and arachidonic acids. Fish oil supplementation changed the fatty acid content of sciatic nerve membranes, decreasing C18:2(n-6) fatty acids and preventing the decreases of arachidonic acids and C18:1(n-9) fatty acids. Protein expression of Na,K-ATPase alpha subunits, Na,K-ATPase activity, and ouabain affinity were assayed in purified sciatic nerve membranes from CO, DO, and DM. Na,K-ATPase activity was significantly lower in sciatic nerve membranes of diabetic rats and significantly restored in diabetic animals that received fish oil supplementation. Diabetes induced a specific decrease of alpha1- and alpha3-isoform activity and protein expression in sciatic nerve membranes. Fish oil supplementation restored partial activity and expression to varying degrees depending on the isoenzyme. These effects were associated with a significant beneficial effect on NCV. This study indicates that fish oil has beneficial effects on diabetes-induced alterations in sciatic nerve Na,K-ATPase activity and function.

Novel ATPase activity of the polyprotein intermediate, Viral Protein genome-linked-Nuclear Inclusion-a protease, of Pepper vein banding potyvirus

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mathur, Chhavi; Savithri, Handanahal S., E-mail: bchss@biochem.iisc.ernet.in

2012-10-12

Highlights: Black-Right-Pointing-Pointer Pepper vein banding potyvirus VPg harbors Walker motifs. Black-Right-Pointing-Pointer VPg exhibits ATPase activity in the presence of NIa-Pro. Black-Right-Pointing-Pointer Plausible structural and functional interplay between VPg and NIa-Pro. Black-Right-Pointing-Pointer Functional relevance of prolonged presence of VPg-Pro during infection. -- Abstract: Potyviruses temporally regulate their protein function by polyprotein processing. Previous studies have shown that VPg (Viral Protein genome-linked) of Pepper vein banding virus interacts with the NIa-Pro (Nuclear Inclusion-a protease) domain, and modulates the kinetics of the protease. In the present study, we report for the first time that VPg harbors the Walker motifs A and B, andmore » the presence of NIa-Pro, especially in cis (cleavage site (E191A) VPg-Pro mutant), is essential for manifestation of the ATPase activity. Mutation of Lys47 (Walker motif A) and Asp88:Glu89 (Walker motif B) to alanine in E191A VPg-Pro lead to reduced ATPase activity, confirming that this activity was inherent to VPg. We propose that potyviral VPg, established as an intrinsically disordered domain, undergoes plausible structural alterations upon interaction with globular NIa-Pro which induces the ATPase activity.« less

Nonproteolytic Roles of 19S ATPases in Transcription of CIITApIV Genes

PubMed Central

Maganti, Nagini; Moody, Tomika D.; Truax, Agnieszka D.; Thakkar, Meghna; Spring, Alexander M.; Germann, Markus W.; Greer, Susanna F.

2014-01-01

Accumulating evidence shows the 26S proteasome is involved in the regulation of gene expression. We and others have demonstrated that proteasome components bind to sites of gene transcription, regulate covalent modifications to histones, and are involved in the assembly of activator complexes in mammalian cells. The mechanisms by which the proteasome influences transcription remain unclear, although prior observations suggest both proteolytic and non-proteolytic activities. Here, we define novel, non-proteolytic, roles for each of the three 19S heterodimers, represented by the 19S ATPases Sug1, S7, and S6a, in mammalian gene expression using the inflammatory gene CIITApIV. These 19S ATPases are recruited to induced CIITApIV promoters and also associate with CIITA coding regions. Additionally, these ATPases interact with elongation factor PTEFb complex members CDK9 and Hexim-1 and with Ser5 phosphorylated RNA Pol II. Both the generation of transcripts from CIITApIV and efficient recruitment of RNA Pol II to CIITApIV are negatively impacted by siRNA mediated knockdown of these 19S ATPases. Together, these results define novel roles for 19S ATPases in mammalian gene expression and indicate roles for these ATPases in promoting transcription processes. PMID:24625964

Temperature influence on the development and loss of seawater tolerance in two fast-growing strains of Atlantic salmon

USGS Publications Warehouse

Handeland, S.O.; Wilkinson, E.; Sveinsbo, B.; McCormick, S.D.; Stefansson, S.O.

2004-01-01

Development of hypo-osmoregulatory ability, gill Na+,K +-ATPase activity, condition factor and growth in Atlantic salmon during parr-smolt transformation was studied in a 2??3 factorial design with three temperatures (12.0, 8.9??C and ambient, 2.4-11.9??C, mean: 6.0??C) and two farmed strains of smolts (Mowi and AquaGen). The development of hypo-osmoregulatory ability and gill Na+,K+-ATPase activity were significantly influenced by freshwater temperature. In smolts raised at 12.0??C, maximum gill Na+,K+-ATPase activity was reached in late April, compared with late May and mid-June in the 8.9??C and ambient groups, respectively. In all groups, peak gill Na+,K +-ATPase activity was seen 350 degree days (d??C) after the onset of the smolt-related increase in enzyme activity (30 March) The period of high enzyme activity (>90% of maximum) lasted approximately 250 d??C. No distinct peak level in gill Na+,K+-ATPase activity was seen in the AquaGen strain at ambient temperature. Elevated temperatures also accelerated the loss of hypo-osmoregulatory capacity. In all groups, gill Na+,K+-ATPase activity reached pre-smolt levels approximately 500 d??C after the calculated peak level. Growth rate in freshwater was influenced by strain, temperature and their interaction, with the Mowi strain showing a higher growth rate than the AquaGen strain at 8.9??C and ambient temperatures. Following transfer to seawater, a higher growth rate was recorded in smolts from the Mowi strain than the AquaGen strain from the ambient temperature regime. Temperature influences the development and loss of smolt characteristics in both strains, and has long-term effects on post-smolt performance in seawater. ?? 2004 Elsevier B.V. All rights reserved.

Expression of a constitutively activated plasma membrane H+-ATPase in Nicotiana tabacum BY-2 cells results in cell expansion.

PubMed

Niczyj, Marta; Champagne, Antoine; Alam, Iftekhar; Nader, Joseph; Boutry, Marc

2016-11-01

Increased acidification of the external medium by an activated H + -ATPase results in cell expansion, in the absence of upstream activating signaling. The plasma membrane H + -ATPase couples ATP hydrolysis with proton transport outside the cell, and thus creates an electrochemical gradient, which energizes secondary transporters. According to the acid growth theory, this enzyme is also proposed to play a major role in cell expansion, by acidifying the external medium and so activating enzymes that are involved in cell wall-loosening. However, this theory is still debated. To challenge it, we made use of a plasma membrane H + -ATPase isoform from Nicotiana plumbaginifolia truncated from its C-terminal auto-inhibitory domain (ΔCPMA4), and thus constitutively activated. This protein was expressed in Nicotiana tabacum BY-2 suspension cells using a heat shock inducible promoter. The characterization of several independent transgenic lines showed that the expression of activated ΔCPMA4 resulted in a reduced external pH by 0.3-1.2 units, as well as in an increased H + -ATPase activity by 77-155 % (ATP hydrolysis), or 70-306 % (proton pumping) of isolated plasma membranes. In addition, ΔCPMA4-expressing cells were 17-57 % larger than the wild-type cells and displayed abnormal shapes. A proteomic comparison of plasma membranes isolated from ΔCPMA4-expressing and wild-type cells revealed the altered abundance of several proteins involved in cell wall synthesis, transport, and signal transduction. In conclusion, the data obtained in this work showed that H + -ATPase activation is sufficient to induce cell expansion and identified possible actors which intervene in this process.

Developmental and hormone-induced changes of mitochondrial electron transport chain enzyme activities during the last instar larval development of maize stem borer, Chilo partellus (Lepidoptera: Crambidae).

PubMed

VenkatRao, V; Chaitanya, R K; Naresh Kumar, D; Bramhaiah, M; Dutta-Gupta, A

2016-12-01

The energy demand for structural remodelling in holometabolous insects is met by cellular mitochondria. Developmental and hormone-induced changes in the mitochondrial respiratory activity during insect metamorphosis are not well documented. The present study investigates activities of enzymes of mitochondrial electron transport chain (ETC) namely, NADH:ubiquinone oxidoreductase or complex I, Succinate: ubiquinone oxidoreductase or complex II, Ubiquinol:ferricytochrome c oxidoreductase or complex III, cytochrome c oxidase or complex IV and F 1 F 0 ATPase (ATPase), during Chilo partellus development. Further, the effect of juvenile hormone (JH) analog, methoprene, and brain and corpora-allata-corpora-cardiaca (CC-CA) homogenates that represent neurohormones, on the ETC enzyme activities was monitored. The enzymatic activities increased from penultimate to last larval stage and thereafter declined during pupal development with an exception of ATPase which showed high enzyme activity during last larval and pupal stages compared to the penultimate stage. JH analog, methoprene differentially modulated ETC enzyme activities. It stimulated complex I and IV enzyme activities, but did not alter the activities of complex II, III and ATPase. On the other hand, brain homogenate declined the ATPase activity while the injected CC-CA homogenate stimulated complex I and IV enzyme activities. Cumulatively, the present study is the first to show that mitochondrial ETC enzyme system is under hormone control, particularly of JH and neurohormones during insect development. Copyright © 2015 Elsevier Inc. All rights reserved.

Regulation of Vacuolar H+-ATPase (V-ATPase) Reassembly by Glycolysis Flow in 6-Phosphofructo-1-kinase (PFK-1)-deficient Yeast Cells*

PubMed Central

Chan, Chun-Yuan; Dominguez, Dennis; Parra, Karlett J.

2016-01-01

Yeast 6-phosphofructo-1-kinase (PFK-1) has two subunits, Pfk1p and Pfk2p. Deletion of Pfk2p alters glucose-dependent V-ATPase reassembly and vacuolar acidification (Chan, C. Y., and Parra, K. J. (2014) Yeast phosphofructokinase-1 subunit Pfk2p is necessary for pH homeostasis and glucose-dependent vacuolar ATPase reassembly. J. Biol. Chem. 289, 19448–19457). This study capitalized on the mechanisms suppressing vacuolar H+-ATPase (V-ATPase) in pfk2Δ to gain new knowledge of the mechanisms underlying glucose-dependent V-ATPase regulation. Because V-ATPase is fully assembled in pfk2Δ, and glycolysis partially suppressed at steady state, we manipulated glycolysis and assessed its direct involvement on V-ATPase function. At steady state, the ratio of proton transport to ATP hydrolysis increased 24% after increasing the glucose concentration from 2% to 4% to enhance the glycolysis flow in pfk2Δ. Tighter coupling restored vacuolar pH when glucose was abundant and glycolysis operated below capacity. After readdition of glucose to glucose-deprived cells, glucose-dependent V1Vo reassembly was proportional to the glycolysis flow. Readdition of 2% glucose to pfk2Δ cells, which restored 62% of ethanol concentration, led to equivalent 60% V1Vo reassembly levels. Steady-state level of assembly (100% reassembly) was reached at 4% glucose when glycolysis reached a threshold in pfk2Δ (≥40% the wild-type flow). At 4% glucose, the level of Pfk1p co-immunoprecipitated with V-ATPase decreased 58% in pfk2Δ, suggesting that Pfk1p binding to V-ATPase may be inhibitory in the mutant. We concluded that V-ATPase activity at steady state and V-ATPase reassembly after readdition of glucose to glucose-deprived cells are controlled by the glycolysis flow. We propose a new mechanism by which glucose regulates V-ATPase catalytic activity that occurs at steady state without changing V1Vo assembly. PMID:27226568

Effect of detergents on the H(+)-ATPase activity of inside-out and right-side-out plant plasma membrane vesicles.

PubMed

Palmgren, M G; Sommarin, M; Ulvskov, P; Larsson, C

1990-01-29

In search for a detergent to be used to assess the sidedness of plant plasma membrane vesicles by enzyme latency we tested the effect of 42 detergents on the ATPase activity of right-side-out and inside-out plasma membrane vesicles from sugar beet leaves. Most of the detergents seemed to inactivate the ATPase in addition to disrupting the permeability barrier to ATP. There were two main exceptions, namely long chain polyoxyethylene acyl ethers, such as detergents of the Brij series and Lubrol WX, and long chain lysophospholipids. These two types of detergents permeabilized the membranes at low concentrations and did not inhibit the ATPase at higher concentrations. Unmasking of latent active sites seemed to explain the activation of the plasma membrane H(+)-ATPase produced by long chain polyoxyethylene acyl ethers. These detergents should therefore be ideal for determination of vesicle orientation based on ATPase latency. By contrast, long chain lysophospholipids were found to be highly specific activators of the enzyme. In addition, long chain fatty acids were found to strongly inhibit ATP-dependent proton accumulation in the vesicles without inhibiting ATP hydrolysis. This uncoupling effect of the fatty acids could be abolished by the addition of fatty acid-free bovine serum albumin (BSA). Similarly, the proton transport capacity of ageing vesicles could be restored by addition of BSA. The latter findings may explain why isolated plasma membranes so often exhibit increased permeability to protons on ageing.

Ionoregulatory changes in different populations of maturing sockeye salmon Oncorhynchus nerka during ocean and river migration.

PubMed

Shrimpton, J M; Patterson, D A; Richards, J G; Cooke, S J; Schulte, P M; Hinch, S G; Farrell, A P

2005-11-01

We present the first data on changes in ionoregulatory physiology of maturing, migratory adult sockeye salmon Oncorhynchus nerka. Fraser River sockeye were intercepted in the ocean as far away as the Queen Charlotte Islands (approximately 850 km from the Fraser River) and during freshwater migration to the spawning grounds; for some populations this was a distance of over 700 km. Sockeye migrating in seawater toward the mouth of the Fraser River and upriver to spawning grounds showed a decline in gill Na+,K+-ATPase activity. As a result, gill Na+,K+-ATPase activity of fish arriving at the spawning grounds was significantly lower than values obtained from fish captured before entry into freshwater. Plasma osmolality and chloride levels also showed significant decreases from seawater values during the freshwater migration to spawning areas. Movement from seawater to freshwater increased mRNA expression of a freshwater-specific Na+,K+-ATPase isoform (alpha1a) while having no effect on the seawater-specific isoform (alpha1b). In addition, gill Na+,K+-ATPase activity generally increased in active spawners compared with unspawned fish on the spawning grounds and this was associated with a marked increase in Na+,K+-ATPase alpha1b mRNA. Increases in gill Na+,K+-ATPase activities observed in spawners suggests that the fish may be attempting to compensate for the osmotic perturbation associated with the decline in plasma chloride concentration and osmolality.

[Protective effects of luteolin on neurons against oxygen-glucose deprivation/reperfusion injury via improving Na+/K+ -ATPase activity].

PubMed

Fang, Lumei; Zhang, Mingming; Ding, Yuemin; Fang, Yuting; Yao, Chunlei; Zhang, Xiong

2010-04-01

Luteolin, a flavone, has considerable neuroprotective effects by its anti-oxidative mechanism. However, it is still unclear whether luteolin can protect neurons against oxygen-glucose deprivation/reperfusion (OGD/R) induced injury. After 2 hours oxygen-glucose deprivation and 24 hours reperfusion treatment in primary cultured hippocampal neurons, the neuron viability, survival rate and apoptosis rate were evaluated by MTT assay, lactate dehydrogenase (LDH) leakage assay and Hoechst staining, respectively. The activity of Na+/K+ -ATPase was examined in cultured neurons or in the hippocampus of SD rats treated by 10 minutes global cerebral ischemia and followed 24 hours reperfusion. Treatment by OGD/R markedly reduced neuronal viability, increased LDH leakage rate and increased apoptosis rate. Application of luteolin (10-100 micromol x L(-1)) during OGD inhibited OGD/R induced neuron injury and apoptosis in a dose-dependent manner. Compared to the control group or OGP/R-treated neurons, the activity of Na+/K+ -ATPase was significantly suppressed in global ischemia/reperfusion group or OGD/R-treated neurons. Application of luteolin during ischemia or OGD preserved the Na+/K+ -ATPase activity. Furthermore, inhibition of Na+/K+ -ATPase with ouabain attenuated the protective effect afforded by luteolin. The data provide the evidence that luteolin has neuroprotective effect against OGD/R induced injury and the protective effect may be associated with its ability to improve Na+/K+ -ATPase activity after OGD/R.

Phospholipid Requirements of Ca++-Stimulated, Mg++-Dependent ATP hydrolysis in Rat Brain Synaptic Membranes

DTIC Science & Technology

1989-01-01

ATPase is a negative charge around the enzyme based on the observation that Ca++/Mg++-ATPase reconstituted in phosphotidylcholine vesicles is...stimulated by calmodulin, but purified ATPase in phosphotidylserine vesicles is not because the enzyme is already maximally active. Stimulation of the

Purification and Characterization of a Glycerol-Resistant CF0-CF1 and CF1-ATPase from the Halotolerant Alga Dunaliella bardawil1

PubMed Central

Finel, Moshe; Pick, Uri; Selman-Reimer, Susanne; Selman, Bruce R.

1984-01-01

The isolation of the chloroplast ATP synthase complex (CF0-CF1) and of CF1 from Dunaliella bardawil is described. The subunit structure of the D. bardawil ATPase differs from that of the spinach in that the D. bardawil α subunit migrates ahead of the β subunit and ε-migrates ahead of subunit II of CF0 when separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The CF1 isolated from D. bardawil resembles the CF1 isolated from Chladmydomonas reinhardi in that a reversible, Mg2+-dependent ATPase is induced by selected organic solvents. Glycerol stimulates cyclic photophosphorylation catalyzed by D. bardawil thylakoid membranes but inhibits photophosphorylation catalyzed by spinach thylakoid membranes. Glycerol (20%) also stimulates the rate of ATP-Pi exchange catalyzed by D. bardawil CF0-CF1 proteoliposomes but inhibits the activity with the spinach enzyme. The ethanol-activated, Mg2+-ATPase of the D. bardawil CF1 is more resistant to glycerol inhibition than the octylglucoside-activated, Mg2+-ATPase of spinach CF1 or the ethanol-activated, Mg2+-dependent ATPase of the C. reinhardi CF1. Both cyclic photophosphorylation and ATP-Pi exchange catalyzed by D. bardawil CF0-CF1 are more sensitive to high concentrations of NaCl than is the spinach complex. Images Fig. 5 PMID:16663507

Ectopic adenine nucleotide translocase activity controls extracellular ADP levels and regulates the F1-ATPase-mediated HDL endocytosis pathway on hepatocytes.

PubMed

Cardouat, G; Duparc, T; Fried, S; Perret, B; Najib, S; Martinez, L O

2017-09-01

Ecto-F 1 -ATPase is a complex related to mitochondrial ATP synthase which has been identified as a plasma membrane receptor for apolipoprotein A-I (apoA-I), the major protein of high-density lipoprotein (HDL), and has been shown to contribute to HDL endocytosis in several cell types. On hepatocytes, apoA-I binding to ecto-F 1 -ATPase stimulates extracellular ATP hydrolysis into ADP, which subsequently activates a P2Y 13 -mediated HDL endocytosis pathway. Interestingly, other mitochondrial proteins have been found to be expressed at the plasma membrane of several cell types. Among these, adenine nucleotide translocase (ANT) is an ADP/ATP carrier but its role in controlling extracellular ADP levels and F 1 -ATPase-mediated HDL endocytosis has never been investigated. Here we confirmed the presence of ANT at the plasma membrane of human hepatocytes. We then showed that ecto-ANT activity increases or reduces extracellular ADP level, depending on the extracellular ADP/ATP ratio. Interestingly, ecto-ANT co-localized with ecto-F 1 -ATPase at the hepatocyte plasma membrane and pharmacological inhibition of ecto-ANT activity increased extracellular ADP level when ecto-F 1 -ATPase was activated by apoA-I. This increase in the bioavailability of extracellular ADP accordingly translated into an increase of HDL endocytosis on human hepatocytes. This study thus uncovered a new location and function of ANT for which activity at the cell surface of hepatocytes modulates the concentration of extracellular ADP and regulates HDL endocytosis. Copyright © 2017. Published by Elsevier B.V.

Preliminary Studies of Acute Cadmium Administration Effects on the Calcium-Activated Potassium (SKCa and BKCa) Channels and Na+/K+-ATPase Activity in Isolated Aortic Rings of Rats.

PubMed

Vassallo, Dalton V; Almenara, Camila C P; Broseghini-Filho, Gilson Brás; Teixeira, Ariane Calazans; da Silva, David Chaves F; Angeli, Jhuli K; Padilha, Alessandra S

2018-06-01

Cadmium is an environmental pollutant closely linked with cardiovascular diseases that seems to involve endothelium dysfunction and reduced nitric oxide (NO) bioavailability. Knowing that NO causes dilatation through the activation of potassium channels and Na + /K + -ATPase, we aimed to determine whether acute cadmium administration (10 μM) alters the participation of K + channels, voltage-activated calcium channel, and Na + /K + -ATPase activity in vascular function of isolated aortic rings of rats. Cadmium did not modify the acetylcholine-induced relaxation. After L-NAME addition, the relaxation induced by acetylcholine was abolished in presence or absence of cadmium, suggesting that acutely, this metal did not change NO release. However, tetraethylammonium (a nonselective K + channels blocker) reduced acetylcholine-induced relaxation but this effect was lower in the preparations with cadmium, suggesting a decrease of K + channels function in acetylcholine response after cadmium incubation. Apamin (a selective blocker of small Ca 2+ -activated K + channels-SK Ca ), iberiotoxin (a selective blocker of large-conductance Ca 2+ -activated K + channels-BK Ca ), and verapamil (a blocker of calcium channel) reduced the endothelium-dependent relaxation only in the absence of cadmium. Finally, cadmium decreases Na + /K + -ATPase activity. Our results provide evidence that the cadmium acute incubation unaffected the calcium-activated potassium channels (SK Ca and BK Ca ) and voltage-calcium channels on the acetylcholine vasodilatation. In addition, acute cadmium incubation seems to reduce the Na + /K + -ATPase activity.

BOT-4-one attenuates NLRP3 inflammasome activation: NLRP3 alkylation leading to the regulation of its ATPase activity and ubiquitination.

PubMed

Shim, Do-Wan; Shin, Woo-Young; Yu, Sang-Hyeun; Kim, Byung-Hak; Ye, Sang-Kyu; Koppula, Sushruta; Won, Hyung-Sik; Kang, Tae-Bong; Lee, Kwang-Ho

2017-11-08

The ATPase activity of NLRP3 has pivotal role in inflammasome activation and is recognized as a good target for the development of the NLRP3 inflammasome-specific inhibitor. However, signals in the vicinity of the ATPase activity of NLRP3 have not been fully elucidated. Here, we demonstrate NLRP3 inflammasome-specific action of a benzoxathiole derivative, BOT-4-one. BOT-4-one exhibited an inhibition of NLRP3 inflammasome activation, which was attributable to its alkylating capability to NLRP3. In particular, the NLRP3 alkylation by BOT-4-one led to an impaired ATPase activity of NLRP3, thereby obstructing the assembly of the NLRP3 inflammasome. Additionally, we found that NLRP3 alkylators, including BOT-4-one, enhance the ubiquitination level of NLRP3, which might also contribute to the inhibition of NLRP3 inflammasome activation. Finally, BOT-4-one appeared to be superior to other known NLRP3 alkylators in inhibiting the functionality of the NLRP3 inflammasome and its resulting anti-inflammatory activity was confirmed in vivo using a monosodium urate-induced peritonitis mouse model. Collectively, the results suggest that NLRP3 alkylators function by inhibiting ATPase activity and increasing the ubiquitination level of NLRP3, and BOT-4-one could be the type of NLRP3 inhibitor that may be potentially useful for the novel development of a therapeutic agent in controlling NLRP3 inflammasome-related diseases.

Arresting a Torsin ATPase Reshapes the Endoplasmic Reticulum*

PubMed Central

Rose, April E.; Zhao, Chenguang; Turner, Elizabeth M.; Steyer, Anna M.; Schlieker, Christian

2014-01-01

Torsins are membrane-tethered AAA+ ATPases residing in the nuclear envelope (NE) and endoplasmic reticulum (ER). Here, we show that the induction of a conditional, dominant-negative TorsinB variant provokes a profound reorganization of the endomembrane system into foci containing double membrane structures that are derived from the ER. These double-membrane sinusoidal structures are formed by compressing the ER lumen to a constant width of 15 nm, and are highly enriched in the ATPase activator LULL1. Further, we define an important role for a highly conserved aromatic motif at the C terminus of Torsins. Mutations in this motif perturb LULL1 binding, reduce ATPase activity, and profoundly limit the induction of sinusoidal structures. PMID:24275647

Organophosphate inhibition of avian salt gland Na, K-ATPase activity

USGS Publications Warehouse

Eastin, W.C.; Fleming, W.J.; Murray, H.C.

1982-01-01

1. Adult black ducks (Anas rubripes) were given freshwater or saltwater (1.5% NaCl) for 11 days and half of each group was also given an organophosphate (17 p.p.m. fenthion) in the diet on days 6–11.2. After 11 days, ducks drinking saltrwater had lost more weight and had higher plasma Na and uric acid concentration and osmolalities than birds drinking freshwater.3. Saltwater treatment stimulated the salt gland to increased weight and Na, K-ATPase activity.4. Fenthion generally reduced plasma and brain cholinesterase activity and depressed cholinesterase and Na, K-ATPase activities in salt glands of birds drinking saltwater.

Preparation of a highly concentrated, completely monomeric, active sarcoplasmic reticulum Ca2+-ATPase.

PubMed

Lüdi, H; Hasselbach, W

1985-11-21

Sarcoplasmic reticulum vesicles from fast skeletal muscle were partially delipidated with sodium cholate at high ionic strength and sedimented in a discontinuous sucrose gradient. Phospholipid content was reduced from 0.777 mumol/mg protein to 0.242 mumol/mg protein. As judged from gel electrophoresis and high pressure liquid gel chromatography, accessory proteins were removed during centrifugation and the Ca2+-ATPase was obtained in an almost pure form. Addition of myristoylglycerophosphocholine (1 mg/mg protein) reactivates ATPase and dinitrophenylphosphatase activity to the same degree obtained with native vesicles. Using the analytical ultracentrifuge it could be demonstrated that the reactivated Ca2+-ATPase was present exclusively in a monomeric state. These results were obtained at high and low ionic strength and up to a protein concentration of 10 mg/ml. Therefore this preparation should be very useful to investigate differences between oligomeric and monomeric Ca2+-ATPase.

MgATP-concentration dependence of protection of yeast vacuolar V-ATPase from inactivation by 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole supports a bi-site catalytic mechanism of ATP hydrolysis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Milgrom, Elena M.; Milgrom, Yakov M., E-mail: milgromy@upstate.edu

2012-06-29

Highlights: Black-Right-Pointing-Pointer MgATP protects V-ATPase from inactivation by 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole. Black-Right-Pointing-Pointer V-ATPase activity saturation with MgATP is not sufficient for complete protection. Black-Right-Pointing-Pointer The results support a bi-site catalytic mechanism for V-ATPase. -- Abstract: Catalytic site occupancy of the yeast vacuolar V-ATPase during ATP hydrolysis in the presence of an ATP-regenerating system was probed using sensitivity of the enzyme to inhibition by 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl). The results show that, regardless of the presence or absence of the proton-motive force across the vacuolar membrane, saturation of V-ATPase activity at increasing MgATP concentrations is accompanied by only partial protection of the enzyme from inhibitionmore » by NBD-Cl. Both in the presence and absence of an uncoupler, complete protection of V-ATPase from inhibition by NBD-Cl requires MgATP concentrations that are significantly higher than those expected from the K{sub m} values for MgATP. The results are inconsistent with a tri-site model and support a bi-site model for a mechanism of ATP hydrolysis by V-ATPase.« less

Regulation of vacuolar H{sup +}-ATPase in microglia by RANKL

DOE Office of Scientific and Technical Information (OSTI.GOV)

Serrano, Eric M.; Ricofort, Ryan D.; Zuo, Jian

2009-11-06

Vacuolar H{sup +}-ATPases (V-ATPases) are large electrogenic proton pumps composed of numerous subunits that play vital housekeeping roles in the acidification of compartments of the endocytic pathway. Additionally, V-ATPases play specialized roles in certain cell types, a capacity that is linked to cell type selective expression of isoforms of some of the subunits. We detected low levels of the a3 isoform of the a-subunit in mouse brain extracts. Examination of various brain-derived cell types by immunoblotting showed a3 was expressed in the N9 microglia cell line and in primary microglia, but not in other cell types. The expression of a3more » in osteoclasts requires stimulation by Receptor Activator of Nuclear Factor {kappa}B-ligand (RANKL). We found that Receptor Activator of Nuclear Factor {kappa}B (RANK) was expressed by microglia. Stimulation of microglia with RANKL triggered increased expression of a3. V-ATPases in microglia were shown to bind microfilaments, and stimulation with RANKL increased the proportion of V-ATPase associated with the detergent-insoluble cytoskeletal fraction and with actin. In summary, microglia express the a3-subunit of V-ATPase. The expression of a3 and the interaction between V-ATPases and microfilaments was modulated by RANKL. These data suggest a novel molecular pathway for regulating microglia.« less

Curcumin-induced histone acetylation inhibition improves stress-induced gastric ulcer disease in rats.

PubMed

He, Ping; Zhou, Renmin; Hu, Guorui; Liu, Zhifeng; Jin, Yu; Yang, Guang; Li, Mei; Lin, Qian

2015-03-01

Curcumin is known to possess anti‑inflammatory properties. Despite the fact that curcumin is known to be a strong inhibitor of H+, K+‑ATPase activity, the mechanism underlying the curcumin‑induced inhibition of the transcription of the H+, K+‑ATPase α subunit in gastric mucosal parietal cells remains unclear. The present study investigated the possible mechanism by which curcumin inhibits stomach H+, K+‑ATPase activity during the acute phase of gastric ulcer disease. A rat model of stress‑induced gastric ulcers was produced, in which the anti‑ulcer effects of curcumin were examined. Curcumin‑induced inhibition of the H+, K+‑ATPase promoter via histone acetylation, was verified using a chromatin immunoprecipitation assay. The results showed that curcumin improved stress‑induced gastric ulcer disease in rats, as demonstrated by increased pH values and reduced gastric mucosal hemorrhage and ulcer index. These effects were accompanied by a significant reduction in the level of histone H3 acetylation at the site of the H+, K+‑ATPase promoter and in the expression of the gastric H+,K+‑ATPase α subunit gene and protein. In conclusion, curcumin downregulated the acetylation of histone H3 at the site of the H+, K+‑ATPase promoter gene, thereby inhibiting the transcription and expression of the H+, K+‑ATPase gene. Curcumin was shown to have a preventive and therapeutic effect in gastric ulcer disease.

The yeast H+-ATPase Pma1 promotes Rag/Gtr-dependent TORC1 activation in response to H+-coupled nutrient uptake.

PubMed

Saliba, Elie; Evangelinos, Minoas; Gournas, Christos; Corrillon, Florent; Georis, Isabelle; André, Bruno

2018-03-23

The yeast Target of Rapamycin Complex 1 (TORC1) plays a central role in controlling growth. How amino acids and other nutrients stimulate its activity via the Rag/Gtr GTPases remains poorly understood. We here report that the signal triggering Rag/Gtr-dependent TORC1 activation upon amino-acid uptake is the coupled H + influx catalyzed by amino-acid/H + symporters. H + -dependent uptake of other nutrients, ionophore-mediated H + diffusion, and inhibition of the vacuolar V-ATPase also activate TORC1. As the increase in cytosolic H + elicited by these processes stimulates the compensating H + -export activity of the plasma membrane H + -ATPase (Pma1), we have examined whether this major ATP-consuming enzyme might be involved in TORC1 control. We find that when the endogenous Pma1 is replaced with a plant H + -ATPase, H + influx or increase fails to activate TORC1. Our results show that H + influx coupled to nutrient uptake stimulates TORC1 activity and that Pma1 is a key actor in this mechanism. © 2018, Saliba et al.

Mechanism of the asymmetric activation of the MinD ATPase by MinE

PubMed Central

Park, Kyung-Tae; Wu, Wei; Lovell, Scott; Lutkenhaus, Joe

2012-01-01

Summary MinD is a component of the Min system involved in the spatial regulation of cell division. It is an ATPase in the MinD/ParA/Mrp deviant Walker A motif family which is within the P loop GTPase superfamily. Its ATPase activity is stimulated by MinE, however, the mechanism of this activation is unclear. MinD forms a symmetric dimer with two binding sites for MinE, however, a recent model suggested that MinE occupying one site was sufficient for ATP hydrolysis. By generating heterodimers with one binding site for MinE we show that one binding site is sufficient for stimulation of the MinD ATPase. Furthermore, comparison of structures of MinD and related proteins led us to examine the role of N45 in the switch I region. An asparagine at this position is conserved in four of the deviant Walker A motif subfamilies (MinD, chromosomal ParAs, Get3 and FleN) and we find that N45 in MinD is essential for MinE stimulated ATPase activity and suggest that it is a key residue affected by MinE binding. PMID:22651575

Constitutive activation of a plasma membrane H(+)-ATPase prevents abscisic acid-mediated stomatal closure.

PubMed

Merlot, Sylvain; Leonhardt, Nathalie; Fenzi, Francesca; Valon, Christiane; Costa, Miguel; Piette, Laurie; Vavasseur, Alain; Genty, Bernard; Boivin, Karine; Müller, Axel; Giraudat, Jérôme; Leung, Jeffrey

2007-07-11

Light activates proton (H(+))-ATPases in guard cells, to drive hyperpolarization of the plasma membrane to initiate stomatal opening, allowing diffusion of ambient CO(2) to photosynthetic tissues. Light to darkness transition, high CO(2) levels and the stress hormone abscisic acid (ABA) promote stomatal closing. The overall H(+)-ATPase activity is diminished by ABA treatments, but the significance of this phenomenon in relationship to stomatal closure is still debated. We report two dominant mutations in the OPEN STOMATA2 (OST2) locus of Arabidopsis that completely abolish stomatal response to ABA, but importantly, to a much lesser extent the responses to CO(2) and darkness. The OST2 gene encodes the major plasma membrane H(+)-ATPase AHA1, and both mutations cause constitutive activity of this pump, leading to necrotic lesions. H(+)-ATPases have been traditionally assumed to be general endpoints of all signaling pathways affecting membrane polarization and transport. Our results provide evidence that AHA1 is a distinct component of an ABA-directed signaling pathway, and that dynamic downregulation of this pump during drought is an essential step in membrane depolarization to initiate stomatal closure.

Effects of rare earth and acid rain pollution on plant chloroplast ATP synthase and element contents at different growth stages.

PubMed

Zhang, Fan; Hu, Huiqing; Wang, Lihong; Zhou, Qing; Huang, Xiaohua

2018-03-01

Combined rare earth and acid rain pollution has become a new environmental problem, seriously affecting plant survival. The effects of these two kinds of pollutants on plant photosynthesis have been reported, but the micro mechanisms are not very clear. In this research, we studied the effects of lanthanum [La(III), 0.08, 1.20 and 2.40 mM] and acid rain (pH value = 2.5, 3.5 and 4.5) on the ATPase activity and gene transcription level and the functional element contents in rice leaf chloroplasts. The results showed that the combined 0.08 mM La(III) and pH 4.5 acid rain increased the ATPase activity and gene transcription level as well as contents of some functional elements. But other combined treatments of acid rain and La(III) reduced the ATPase activity and gene transcription level as well as functional element contents. The change magnitude of the above indexes at rice booting stage was greater than that in seedling stage or grain filling stage. These results reveal that effects of La(III) and acid rain on ATPase activity and functional element contents in rice leaf chloroplasts are related to the combination of La(III) dose and acid rain intensity and the plant growth stage. In addition, the changes in the ATPase activity were related to ATPase gene transcription level. This study would provide a reference for understanding the microcosmic mechanism of rare earth and acid rain pollution on plant photosynthesis and contribute to evaluate the possible environmental risks associated with combined La(III) and acid rain pollution. The effects of La(III) and acid rain on activity and gene transcription level of rice chloroplast ATPase and contents of functional elements were different at different growth stages. Copyright © 2017 Elsevier Ltd. All rights reserved.

Cellular localization of Na(+), K(+)-ATPase in the mammalian vestibular system

NASA Technical Reports Server (NTRS)

Kerr, T. P.

1984-01-01

Two different, but complementary, procedures for cellular localization of Na+, K+-ATPase in the guinea pig vestibular system were employed. One of these techniques, devised by Stirling, depends upon the well documented ability of the specific inhibitor ouabain to bind selectively to Na+,K+-ATPase, blocking catalytic activity. Microdisected vestibular tissues are incubated with tritium-labelled (3H-) ouabain, and regions with a high concentration of Na+,K+-ATPase are subsequently identified by light microscope autoradiography. A second method, originated by Ernst, detects inorganic phosphate released from an artificial substrate (nitrophenyl phosphate) by catalytic activity of the enzyme. In the presence of strontium ion, phosphate is precipitated near regions of high activity, then converted to a product which may finally be visualized in the electron microscope. This cytochemical enzymatic reaction is inhibited by ouabain.

Quercetin treatment regulates the Na+,K+-ATPase activity, peripheral cholinergic enzymes, and oxidative stress in a rat model of demyelination.

PubMed

Carvalho, Fabiano B; Gutierres, Jessié M; Beckmann, Diego; Santos, Rosmarini P; Thomé, Gustavo R; Baldissarelli, Jucimara; Stefanello, Naiara; Andrades, Amanda; Aiello, Graciane; Ripplinger, Angel; Lucio, Bruna M; Ineu, Rafael; Mazzanti, Alexandre; Morsch, Vera; Schetinger, Maria Rosa; Andrade, Cinthia M

2018-07-01

Quercetin is reported to exert a plethora of health benefits through many different mechanisms of action. This versatility and presence in the human diet has attracted the attention of the scientific community, resulting in a huge output of in vitro and in vivo (preclinical) studies. Therefore, we hypothesized that quercetin can protect Na + ,K + -ATPase activity in the central nervous system, reestablish the peripheral cholinesterases activities, and reduce oxidative stress during demyelination events in rats. In line with this expectation, our study aims to find out how quercetin acts on the Na + ,K + -ATPase activity in the central nervous system, peripheral cholinesterases, and stress oxidative markers in an experimental model of demyelinating disease. Wistar rats were divided into 4 groups: vehicle, quercetin, ethidium bromide (EB), and EB plus quercetin groups. The animals were treated once a day with vehicle (ethanol 20%) or quercetin 50 mg/kg for 7 (demyelination phase, by gavage) or 21 days (remyelination phase) after EB (0.1%, 10 μL) injection (intrapontine).The encephalon was removed, and the pons, hypothalamus, cerebral cortex, hippocampus, striatum, and cerebellum were dissected to verify the Na + ,K + -ATPase activity. Our results showed that quercetin protected against reduction in Na + ,K + -ATPase in the pons and cerebellum in the demyelination phase, and it increased the activity of this enzyme in the remyelination phase. During the demyelination, quercetin promoted the increase in acetylcholinesterase activity in whole blood and lymphocytes induced by EB, and it reduced the increase in acetylcholinesterase activity in lymphocytes in the remyelination phase. On day 7, EB increased the superoxide dismutase and decreased catalase activities, as well as increased the thiobarbituric acid-reactive substance levels. Taken together, these results indicated that quercetin regulates the Na + ,K + -ATPase activity, affects the alterations of redox state, and participates in the reestablishment of peripheral cholinergic activity during demyelinating and remyelination events. Copyright © 2018 Elsevier Inc. All rights reserved.

Plasma membrane H(+)-ATPase is involved in methyl jasmonate-induced root hair formation in lettuce (Lactuca sativa L.) seedlings.

PubMed

Zhu, Changhua; Yang, Na; Ma, Xiaoling; Li, Guijun; Qian, Meng; Ng, Denny; Xia, Kai; Gan, Lijun

2015-06-01

Our results show that methyl jasmonate induces plasma membrane H (+) -ATPase activity and subsequently influences the apoplastic pH of trichoblasts to maintain a cell wall pH environment appropriate for root hair development. Root hairs, which arise from root epidermal cells, are tubular structures that increase the efficiency of water absorption and nutrient uptake. Plant hormones are critical regulators of root hair development. In this study, we investigated the regulatory role of the plasma membrane (PM) H(+)-ATPase in methyl jasmonate (MeJA)-induced root hair formation. We found that MeJA had a pronounced effect on the promotion of root hair formation in lettuce seedlings, but that this effect was blocked by the PM H(+)-ATPase inhibitor vanadate. Furthermore, MeJA treatment increased PM H(+)-ATPase activity in parallel with H(+) efflux from the root tips of lettuce seedlings and rhizosphere acidification. Our results also showed that MeJA-induced root hair formation was accompanied by hydrogen peroxide accumulation. The apoplastic acidification acted in concert with reactive oxygen species to modulate root hair formation. Our results suggest that the effect of MeJA on root hair formation is mediated by modulation of PM H(+)-ATPase activity.

Quantitative Proteomics of the Tonoplast Reveals a Role for Glycolytic Enzymes in Salt Tolerance[C][W

PubMed Central

Barkla, Bronwyn J.; Vera-Estrella, Rosario; Hernández-Coronado, Marcela; Pantoja, Omar

2009-01-01

To examine the role of the tonoplast in plant salt tolerance and identify proteins involved in the regulation of transporters for vacuolar Na+ sequestration, we exploited a targeted quantitative proteomics approach. Two-dimensional differential in-gel electrophoresis analysis of free flow zonal electrophoresis separated tonoplast fractions from control, and salt-treated Mesembryanthemum crystallinum plants revealed the membrane association of glycolytic enzymes aldolase and enolase, along with subunits of the vacuolar H+-ATPase V-ATPase. Protein blot analysis confirmed coordinated salt regulation of these proteins, and chaotrope treatment indicated a strong tonoplast association. Reciprocal coimmunoprecipitation studies revealed that the glycolytic enzymes interacted with the V-ATPase subunit B VHA-B, and aldolase was shown to stimulate V-ATPase activity in vitro by increasing the affinity for ATP. To investigate a physiological role for this association, the Arabidopsis thaliana cytoplasmic enolase mutant, los2, was characterized. These plants were salt sensitive, and there was a specific reduction in enolase abundance in the tonoplast from salt-treated plants. Moreover, tonoplast isolated from mutant plants showed an impaired ability for aldolase stimulation of V-ATPase hydrolytic activity. The association of glycolytic proteins with the tonoplast may not only channel ATP to the V-ATPase, but also directly upregulate H+-pump activity. PMID:20028841

Quantitative proteomics of the tonoplast reveals a role for glycolytic enzymes in salt tolerance.

PubMed

Barkla, Bronwyn J; Vera-Estrella, Rosario; Hernández-Coronado, Marcela; Pantoja, Omar

2009-12-01

To examine the role of the tonoplast in plant salt tolerance and identify proteins involved in the regulation of transporters for vacuolar Na(+) sequestration, we exploited a targeted quantitative proteomics approach. Two-dimensional differential in-gel electrophoresis analysis of free flow zonal electrophoresis separated tonoplast fractions from control, and salt-treated Mesembryanthemum crystallinum plants revealed the membrane association of glycolytic enzymes aldolase and enolase, along with subunits of the vacuolar H(+)-ATPase V-ATPase. Protein blot analysis confirmed coordinated salt regulation of these proteins, and chaotrope treatment indicated a strong tonoplast association. Reciprocal coimmunoprecipitation studies revealed that the glycolytic enzymes interacted with the V-ATPase subunit B VHA-B, and aldolase was shown to stimulate V-ATPase activity in vitro by increasing the affinity for ATP. To investigate a physiological role for this association, the Arabidopsis thaliana cytoplasmic enolase mutant, los2, was characterized. These plants were salt sensitive, and there was a specific reduction in enolase abundance in the tonoplast from salt-treated plants. Moreover, tonoplast isolated from mutant plants showed an impaired ability for aldolase stimulation of V-ATPase hydrolytic activity. The association of glycolytic proteins with the tonoplast may not only channel ATP to the V-ATPase, but also directly upregulate H(+)-pump activity.

Cell cycle-related fluctuations in transcellular ionic currents and plasma membrane Ca2+/Mg2+ ATPase activity during early cleavages of Lymnaea stagnalis embryos.

PubMed

Zivkovic, Danica; Créton, Robbert; Dohmen, René

1991-08-01

During the first four mitotic division cycles of Lymnaea stagnalis embryos, we have detected cell cycle-dependent changes in the pattern of transcellular ionic currents and membrane-bound Ca 2+ -stimulated ATPase activity. Ionic currents ranging from 0.05 to 2.50 μA/cm 2 have been measured using the vibrating probe technique. Enzyme activity was detected using Ando's cytochemical method (Ando et al. 1981) which reveals Ca 2+ /Mg 2+ ATPase localization at the ultrastructural level, and under high-stringency conditions with respect to calcium availability, it reveals Ca 2+ -stimulated ATPase. The ionic currents and Ca 2+ -stimulated ATPase localization have in common that important changes occur during the M-phase of the cell cycles. Minimal outward current at the vegetal pole coincides with metaphase/anaphase. Maximal inward current at the animal pole coincides with the onset of cytokinesis at that pole. Ca 2+ -stimulated ATPase is absent from one half of the embryo at metaphase/anaphase of the two- and four-cell stage, whereas it is present in all cells during the remaining part of the cell cycle. Since fluctuations of cytosolic free calcium concentrations appear to correlate with both karyokinesis and cytokinesis, we speculate that part of the cyclic pattern of Ca 2+ -stimulated ATPase localization and of the transcellular ionic currents reflects the elevation of cytosolic free calcium concentration during the M-phase.

Transcriptional, translational and systemic alterations during the time course of osmoregulatory acclimation in two palaemonid shrimps from distinct osmotic niches.

PubMed

Faleiros, Rogério Oliveira; Furriel, Rosa P M; McNamara, John Campbell

2017-10-01

Palaemonid shrimps exhibit numerous adaptive strategies, both in their life cycles and in biochemical, physiological, morphological and behavioral characteristics that reflect the wide variety of habitats in which they occur, including species that are of particular interest when analyzing adaptive osmoregulatory strategies. The present investigation evaluates the short- (hours) and long-term (days) time courses of responses of two palaemonid shrimps from separate yet overlapping osmotic niches, Palaemon northropi (marine) and Macrobrachium acanthurus (diadromous, fresh water), to differential salinity challenges at distinct levels of structural organization: (i) transcriptional, analyzing quantitative expression of gill mRNAs that encode for subunits of the Na + /K + -ATPase and V(H + )-ATPase ion transporters; (ii) translational, examining the kinetic behavior of gill Na + /K + -ATPase specific activity; and (iii) systemic, accompanying consequent adjustment of hemolymph osmolality. Palaemon northropi is an excellent hyper-hypo-osmoregulator in dilute and concentrated seawater, respectively. Macrobrachium acanthurus is a strong hyper-regulator in fresh water and hypo-regulates hemolymph osmolality and particularly [Cl - ] in brackish water. Hemolymph hyper-regulation in fresh water (Macrobrachium acanthurus) and dilute seawater (Palaemon northropi) is underlain by augmented expression of both the gill Na + /K + -ATPase and V(H + )-ATPase. In contrast, in neither species is hypo-regulation sustained by changes in Na + /K + -ATPase mRNA expression levels, but rather by regulating enzyme specific activity. The integrated time course of Na + /K + - and V(H + )-ATPase expression and Na + /K + -ATPase activity in the gills of these palaemonid shrimps during acclimation to different salinities reveals versatility in their levels of regulation, and in the roles of these ion transporting pumps in sustaining processes of hyper- and hypo-osmotic and chloride regulation. Copyright © 2017 Elsevier Inc. All rights reserved.

SOS2 Promotes Salt Tolerance in Part by Interacting with the Vacuolar H+-ATPase and Upregulating Its Transport Activity▿

PubMed Central

Batelli, Giorgia; Verslues, Paul E.; Agius, Fernanda; Qiu, Quansheng; Fujii, Hiroaki; Pan, Songqin; Schumaker, Karen S.; Grillo, Stefania; Zhu, Jian-Kang

2007-01-01

The salt overly sensitive (SOS) pathway is critical for plant salt stress tolerance and has a key role in regulating ion transport under salt stress. To further investigate salt tolerance factors regulated by the SOS pathway, we expressed an N-terminal fusion of the improved tandem affinity purification tag to SOS2 (NTAP-SOS2) in sos2-2 mutant plants. Expression of NTAP-SOS2 rescued the salt tolerance defect of sos2-2 plants, indicating that the fusion protein was functional in vivo. Tandem affinity purification of NTAP-SOS2-containing protein complexes and subsequent liquid chromatography-tandem mass spectrometry analysis indicated that subunits A, B, C, E, and G of the peripheral cytoplasmic domain of the vacuolar H+-ATPase (V-ATPase) were present in a SOS2-containing protein complex. Parallel purification of samples from control and salt-stressed NTAP-SOS2/sos2-2 plants demonstrated that each of these V-ATPase subunits was more abundant in NTAP-SOS2 complexes isolated from salt-stressed plants, suggesting that the interaction may be enhanced by salt stress. Yeast two-hybrid analysis showed that SOS2 interacted directly with V-ATPase regulatory subunits B1 and B2. The importance of the SOS2 interaction with the V-ATPase was shown at the cellular level by reduced H+ transport activity of tonoplast vesicles isolated from sos2-2 cells relative to vesicles from wild-type cells. In addition, seedlings of the det3 mutant, which has reduced V-ATPase activity, were found to be severely salt sensitive. Our results suggest that regulation of V-ATPase activity is an additional key function of SOS2 in coordinating changes in ion transport during salt stress and in promoting salt tolerance. PMID:17875927

Meiotic Clade AAA ATPases: Protein Polymer Disassembly Machines.

PubMed

Monroe, Nicole; Hill, Christopher P

2016-05-08

Meiotic clade AAA ATPases (ATPases associated with diverse cellular activities), which were initially grouped on the basis of phylogenetic classification of their AAA ATPase cassette, include four relatively well characterized family members, Vps4, spastin, katanin and fidgetin. These enzymes all function to disassemble specific polymeric protein structures, with Vps4 disassembling the ESCRT-III polymers that are central to the many membrane-remodeling activities of the ESCRT (endosomal sorting complexes required for transport) pathway and spastin, katanin p60 and fidgetin affecting multiple aspects of cellular dynamics by severing microtubules. They share a common domain architecture that features an N-terminal MIT (microtubule interacting and trafficking) domain followed by a single AAA ATPase cassette. Meiotic clade AAA ATPases function as hexamers that can cycle between the active assembly and inactive monomers/dimers in a regulated process, and they appear to disassemble their polymeric substrates by translocating subunits through the central pore of their hexameric ring. Recent studies with Vps4 have shown that nucleotide-induced asymmetry is a requirement for substrate binding to the pore loops and that recruitment to the protein lattice via MIT domains also relieves autoinhibition and primes the AAA ATPase cassettes for substrate binding. The most striking, unifying feature of meiotic clade AAA ATPases may be their MIT domain, which is a module that is found in a wide variety of proteins that localize to ESCRT-III polymers. Spastin also displays an adjacent microtubule binding sequence, and the presence of both ESCRT-III and microtubule binding elements may underlie the recent findings that the ESCRT-III disassembly function of Vps4 and the microtubule-severing function of spastin, as well as potentially katanin and fidgetin, are highly coordinated. Copyright © 2015 Elsevier Ltd. All rights reserved.

Effect of Hindlimb Unweighting on Single Soleus Fiber Maximal Shortening Velocity and ATPase Activity

NASA Technical Reports Server (NTRS)

McDonald, K. S.; Fitts, R. H.

1993-01-01

This study characterizes the time course of change in single soleus muscle fiber size and function elicited by hindlimb un weighting (HU) and analyzes the extent to which varying durations of HU altered maximal velocity of shortening (V(sub o)), myofibrillar adenosinetriphosphatase (ATPase), and relative content of slow and fast myosin in individual soleus fibers. After 1, 2, or 3 weeks of HU, soleus muscle bundles were prepared and stored in skinning solution at -20 C. Single fibers were isolated and mounted between a motor arm and a transducer, and fiber force, V(sub o), and ATPase activity were measured. Fiber myosin content was determined by one-dimensional sodium dodecyl sulfate- (SDS) polyacrylamide gel electrophoresis. After 1, 2, and 3 weeks of HU, soleus fibers exhibited a progressive reduction in fiber diameter (16, 22, and 42%, respectively) and peak force (42, 48, and 7%, respectively). Peak specific tension was significantly reduced after 1 week of HU (18%) and showed no further change in 2-3 weeks of HU. During 1 and 3 wk of HU, fiber V(sub o) and ATPase showed a significant increase. By 3 week, V(sub o) had increased from 1.32 +/- 0.04 to 2.94 +/- 0.17 fiber lengths/s and fiber ATPase from 291 +/- 16 to 1064 +/- 128 micro-M min(sub -1) mm(sub -3). The percent fibers expressing fast myosin heavy chain increased from 4% to 29% by 3 week of HU, and V(sub o) and ATPase activity within a fiber were highly correlated. However, a large population of fibers after 1, 2, and 3 weeks of HU showed increases in V(sub o) and ATPase but displayed the same myosin protein profile on SDS gels as control fibers. The mechanism eliciting increased fiber V(sub o) and ATPase activity was not obvious but may have been due to increases in fast myosin that went undetected on SDS gels and/or other factors unrelated to the myosin filament.

Circulating aldosterone induces the apical accumulation of the proton pumping V-ATPase and increases proton secretion in clear cells in the caput epididymis.

PubMed

Roy, Jeremy W; Hill, Eric; Ruan, Ye Chun; Vedovelli, Luca; Păunescu, Teodor G; Brown, Dennis; Breton, Sylvie

2013-08-15

Clear cells express the vacuolar proton-pumping H(+)-ATPase (V-ATPase) and acidify the lumen of the epididymis, a process that is essential for male fertility. The renin-angiotensin-aldosterone system (RAAS) regulates fluid and electrolyte balance in the epididymis, and a previous study showed binding of aldosterone exclusively to epididymal clear cells (Hinton BT, Keefer DA. Steroid Biochem 23: 231-233, 1985). We examined here the role of aldosterone in the regulation of V-ATPase in the epididymis. RT-PCR showed expression of the mineralocorticoid receptor [MR; nuclear receptor subfamily 3, group C member 2 (NR3C2)] and 11-β-dehydrogenase isozyme 2 (HSD11β2) mRNAs specifically in clear cells, isolated by fluorescence-activated cell sorting from B1-enhanced green fluorescent protein (EGFP) mice. Tail vein injection of adult rats with aldosterone, 1,2-dioctanoyl-sn-glycerol (DOG), or 8-(4-chlorophenylthio)-cAMP (cpt-cAMP) induced V-ATPase apical membrane accumulation and extension of V-ATPase-labeled microvilli in clear cells in the caput epididymis but not in the cauda. V-ATPase activity was measured in EGFP-expressing clear cells using the intracellular pH (pHi)-sensing dye seminaphthorhodafluor-5F-5-(and 6)-carboxylic acid, acetoxymethyl ester acetate (SNARF-5F). Aldosterone induced a rapid increase in the rate of Na(+)- and bicarbonate-independent pHi recovery following an NH4Cl-induced acid load in clear cells isolated from the caput but not the cauda. This effect was abolished by concanamycin A, spironolactone, and chelerythrine but not myristoylated-protein kinase inhibitor (mPKI) or mifepristone. Thus aldosterone increases V-ATPase-dependent proton secretion in clear cells in the caput epididymis via MR/NR3C2 and PKC activation. This study, therefore, identifies aldosterone as an active member of the RAAS for the regulation of luminal acidification in the proximal epididymis.

Stimulation, Inhibition, or Stabilization of Na,K-ATPase Caused by Specific Lipid Interactions at Distinct Sites

PubMed Central

Habeck, Michael; Haviv, Haim; Katz, Adriana; Kapri-Pardes, Einat; Ayciriex, Sophie; Shevchenko, Andrej; Ogawa, Haruo; Toyoshima, Chikashi; Karlish, Steven J. D.

2015-01-01

The activity of membrane proteins such as Na,K-ATPase depends strongly on the surrounding lipid environment. Interactions can be annular, depending on the physical properties of the membrane, or specific with lipids bound in pockets between transmembrane domains. This paper describes three specific lipid-protein interactions using purified recombinant Na,K-ATPase. (a) Thermal stability of the Na,K-ATPase depends crucially on a specific interaction with 18:0/18:1 phosphatidylserine (1-stearoyl-2-oleoyl-sn-glycero-3-phospho-l-serine; SOPS) and cholesterol, which strongly amplifies stabilization. We show here that cholesterol associates with SOPS, FXYD1, and the α subunit between trans-membrane segments αTM8 and -10 to stabilize the protein. (b) Polyunsaturated neutral lipids stimulate Na,K-ATPase turnover by >60%. A screen of the lipid specificity showed that 18:0/20:4 and 18:0/22:6 phosphatidylethanolamine (PE) are the optimal phospholipids for this effect. (c) Saturated phosphatidylcholine and sphingomyelin, but not saturated phosphatidylserine or PE, inhibit Na,K-ATPase activity by 70–80%. This effect depends strongly on the presence of cholesterol. Analysis of the Na,K-ATPase activity and E1-E2 conformational transitions reveals the kinetic mechanisms of these effects. Both stimulatory and inhibitory lipids poise the conformational equilibrium toward E2, but their detailed mechanisms of action are different. PE accelerates the rate of E1 → E2P but does not affect E2(2K)ATP → E13NaATP, whereas sphingomyelin inhibits the rate of E2(2K)ATP → E13NaATP, with very little effect on E1 → E2P. We discuss these lipid effects in relation to recent crystal structures of Na,K-ATPase and propose that there are three separate sites for the specific lipid interactions, with potential physiological roles to regulate activity and stability of the pump. PMID:25533463

Intracellular pH homeostasis and serotonin-induced pH changes in Calliphora salivary glands: the contribution of V-ATPase and carbonic anhydrase.

PubMed

Schewe, Bettina; Schmälzlin, Elmar; Walz, Bernd

2008-03-01

Blowfly salivary gland cells have a vacuolar-type H(+)-ATPase (V-ATPase) in their apical membrane that energizes secretion of a KCl-rich saliva upon stimulation with serotonin (5-hydroxytryptamine, 5-HT). We have used BCECF to study microfluometrically whether V-ATPase and carbonic anhydrase (CA) are involved in intracellular pH (pH(i)) regulation, and we have localized CA activity by histochemistry. We show: (1) mean pH(i) in salivary gland cells is 7.5+/-0.3 pH units (N=96), higher than that expected from passive H(+) distribution; (2) low 5-HT concentrations (0.3-3 nmol l(-1)) induce a dose-dependent acidification of up to 0.2 pH units, with 5-HT concentrations >10 nmol l(-1), causing monophasic or multiphasic pH changes; (3) the acidifying effect of 5-HT is mimicked by bath application of cAMP, forskolin or IBMX; (4) salivary gland cells exhibit CA activity; (5) CA inhibition with acetazolamide and V-ATPase inhibition with concanamycin A lead to a slow acidification of steady-state pH(i); (6) 5-HT stimuli in the presence of acetazolamide induce an alkalinization that can be decreased by simultaneous application of the V-ATPase inhibitor concanamycin A; (7) concanamycin A removes alkali-going components from multiphasic 5-HT-induced pH changes; (8) NHE activity and a Cl(-)-dependent process are involved in generating 5-HT-induced pH changes; (9) the salivary glands probably contain a Na(+)-driven amino acid transporter. We conclude that V-ATPase and CA contribute to steady-state pH(i) regulation and 5-HT-induced outward H(+) pumping does not cause an alkalinization of pH(i) because of cytosolic H(+) accumulation attributable to stimulated cellular respiration and AE activity, masking the alkalizing effect of V-ATPase-mediated acid extrusion.

A physiological basis for variation in the contractile properties of isolated rat heart.

PubMed Central

Lin, L E; McClellan, G; Weisberg, A; Winegrad, S

1991-01-01

1. The maximum Ca(2+)-activated force, maximum velocity of unloaded shortening and both Ca(2+)- and actin-activated ATPase activities of myosin have been measured in detergent-skinned preparations of isolated bundles of rat right ventricle after exposure of the intact tissue to different conditions of superfusion, mechanical activity and temperature. 2. Maximum Ca(2+)-activated force per unit cross-sectional area decreases with increasing cross-sectional area, and, in the absence of electrical stimulation, with the duration of superfusion. Maximum velocity of unloaded shortening is not influenced by these differences. 3. Actin-activated ATPase activity of myosin decreases as cross-sectional area increases and duration of superfusion increases, but the extent of the decrease in enzymatic activity is less than that of developed force. Ca(2+)-activated ATPase activity is independent of these differences. 4. Actin-activated ATPase activity in cryostatic sections of quickly frozen tissue is not uniform across the transverse section. In thin bundles, it is highest in the centre and lowest at the edge of the section, which correspond, respectively, to the centre and the surface of the tissue bundle. Exposure of the tissue section to 1 microM-cyclic AMP increases the actin-activated ATPase activity of myosin with the largest increase in activity occurring at or near the surface of the bundle. 5. Ca(2+)-activated ATPase activity of myosin is uniform across the transverse section and is not changed by cyclic AMP. 6. Electrical stimulation, elevated Ca2+ concentration in the superfusion medium, or isoprenaline partially or completely reverse the decline in maximum Ca(2+)-activated force produced by prolonged superfusion of the bundle before its skinning. 7. These observations are similar in many ways to those made on frog skeletal muscles by Elzinga, Howarth, Rull, Wilson & Woledge (1989a). An explanation based on the existence of a physiological mechanism for regulating the properties of force generators is proposed. Regulation of the attachment of the cross-bridge to an actin filament may be the basis for the regulatory mechanism. Images Fig. 4 Fig. 7 PMID:1667804

Regulation of the ATPase activity of ABCE1 from Pyrococcus abyssi by Fe-S cluster status and Mg²⁺: implication for ribosomal function.

PubMed

Sims, Lynn M; Igarashi, Robert Y

2012-08-15

Ribosomal function is dependent on multiple proteins. The ABCE1 ATPase, a unique ABC superfamily member that bears two Fe₄S₄ clusters, is crucial for ribosomal biogenesis and recycling. Here, the ATPase activity of the Pyrococcus abyssi ABCE1 (PabABCE1) was studied using both apo- (without reconstituted Fe-S clusters) and holo- (with full complement of Fe-S clusters reconstituted post-purification) forms, and is shown to be jointly regulated by the status of Fe-S clusters and Mg²⁺. Typically ATPases require Mg²⁺, as is true for PabABCE1, but Mg²⁺ also acts as a negative allosteric effector that modulates ATP affinity of PabABCE1. Physiological [Mg²⁺] inhibits the PabABCE1 ATPase (K(i) of ∼1 μM) for both apo- and holo-PabABCE1. Comparative kinetic analysis of Mg²⁺ inhibition shows differences in degree of allosteric regulation between the apo- and holo-PabABCE1 where the apparent ATP K(m) of apo-PabABCE1 increases >30-fold from ∼30 μM to over 1 mM with M²⁺. This effect would significantly convert the ATPase activity of PabABCE1 from being independent of cellular energy charge (φ) to being dependent on φ with cellular [Mg²⁺]. These findings uncover intricate overlapping effects by both [Mg²⁺] and the status of Fe-S clusters that regulate ABCE1's ATPase activity with implications to ribosomal function. Copyright © 2012 Elsevier Inc. All rights reserved.

K+ and NH4(+) modulate gill (Na+, K+)-ATPase activity in the blue crab, Callinectes ornatus: fine tuning of ammonia excretion.

PubMed

Garçon, D P; Masui, D C; Mantelatto, F L M; McNamara, J C; Furriel, R P M; Leone, F A

2007-05-01

To better comprehend the mechanisms of ionic regulation, we investigate the modulation by Na+, K+, NH4(+) and ATP of the (Na+, K+)-ATPase in a microsomal fraction from Callinectes ornatus gills. ATP hydrolysis obeyed Michaelis-Menten kinetics with KM=0.61+/-0.03 mmol L(-1) and maximal rate of V=116.3+/-5.4 U mg(-1). Stimulation by Na+ (V=110.6+/-6.1 U mg(-1); K0.5=6.3+/-0.2 mmol L(-1)), Mg2+ (V=111.0+/-4.7 U mg(-1); K0.5=0.53+/-0.03 mmol L(-1)), NH4(+) (V=173.3+/-6.9 U mg(-1); K0.5=5.4+/-0.2 mmol L(-1)) and K+ (V=116.0+/-4.9 U mg(-1); K0.5=1.5+/-0.1 mmol L(-1)) followed a single saturation curve, although revealing site-site interactions. In the absence of NH4(+), ouabain (K(I)=74.5+/-1.2 micromol L(-1)) and orthovanadate inhibited ATPase activity by up to 87%; the inhibition patterns suggest the presence of F0F1 and K+-ATPases but not Na+-, V- or Ca2+-ATPase as contaminants. (Na+, K+)-ATPase activity was synergistically modulated by K+ and NH4(+). At 10 mmol L(-1) K+, increasing NH4(+) concentrations stimulated maximum activity to V=185.9+/-7.4 U mg(-1). However, at saturating NH4(+) (50 mmol L(-1)), increasing K+ concentrations did not stimulate activity further. Our findings provide evidence that the C. ornatus gill (Na+, K+)-ATPase may be particularly well suited for extremely efficient active NH4(+) excretion. At elevated NH4(+) concentrations, the enzyme is fully active, regardless of hemolymph K+ concentration, and K+ cannot displace NH4(+) from its exclusive binding sites. Further, the binding of NH4(+) to its specific sites induces an increase in enzyme apparent affinity for K+, which may contribute to maintaining K+ transport, assuring that exposure to elevated ammonia concentrations does not lead to a decrease in intracellular potassium levels. This is the first report of modulation by ammonium ions of C. ornatus gill (Na+, K+)-ATPase, and should further our understanding of NH4(+) excretion in benthic crabs.

Structure-Based Mutational Analysis of the Hepatitis C Virus NS3 Helicase

PubMed Central

Tai, Chun-Ling; Pan, Wen-Ching; Liaw, Shwu-Huey; Yang, Ueng-Cheng; Hwang, Lih-Hwa; Chen, Ding-Shinn

2001-01-01

The carboxyl terminus of the hepatitis C virus (HCV) nonstructural protein 3 (NS3) possesses ATP-dependent RNA helicase activity. Based on the conserved sequence motifs and the crystal structures of the helicase domain, 17 mutants of the HCV NS3 helicase were generated. The ATP hydrolysis, RNA binding, and RNA unwinding activities of the mutant proteins were examined in vitro to determine the functional role of the mutated residues. The data revealed that Lys-210 in the Walker A motif and Asp-290, Glu-291, and His-293 in the Walker B motif were crucial to ATPase activity and that Thr-322 and Thr-324 in motif III and Arg-461 in motif VI significantly influenced ATPase activity. When the pairing between His-293 and Gln-460, referred to as gatekeepers, was replaced with the Asp-293/His-460 pair, which makes the NS3 helicase more like the DEAD helicase subgroup, ATPase activity was not restored. It thus indicated that the whole microenvironment surrounding the gatekeepers, rather than the residues per se, was important to the enzymatic activities. Arg-461 and Trp-501 are important residues for RNA binding, while Val-432 may only play a coadjutant role. The data demonstrated that RNA helicase activity was possibly abolished by the loss of ATPase activity or by reduced RNA binding activity. Nevertheless, a low threshold level of ATPase activity was found sufficient for helicase activity. Results in this study provide a valuable reference for efforts under way to develop anti-HCV therapeutic drugs targeting NS3. PMID:11483774

Effect of Salinity and Alkalinity on Luciobarbus capito Gill Na+/K+-ATPase Enzyme Activity, Plasma Ion Concentration, and Osmotic Pressure

PubMed Central

2016-01-01

We evaluated the individual and combined effects of salinity and alkalinity on gill Na+/K+-ATPase enzyme activity, plasma ion concentration, and osmotic pressure in Luciobarbus capito. Increasing salinity concentrations (5, 8, 11, and 14 g/L) were associated with an initial increase and then decrease in L. capito gill Na+/K+-ATPase activity. Activity was affected by the difference between internal and external Na+ ion concentrations and osmotic pressure (P < 0.05). Both plasma ion (Na+, K+, and Cl−) concentration and osmotic pressure increased significantly (P < 0.05). An increase in alkalinity (15, 30, 45, and 60 mM) caused a significant increase in plasma K+ and urea nitrogen concentrations (P < 0.05) but had no effect on either plasma osmotic pressure or gill filament ATPase activity. In the two-factor experiment, the saline-alkaline interaction caused a significant increase in plasma ion (Na+, Cl−, and urea nitrogen) and osmotic pressure (P < 0.05). Variance analysis revealed that salinity, alkalinity, and their interaction significantly affected osmotic pressure, with salinity being most affected, followed by alkalinity, and their interaction. Gill filament ATPase activity increased at first and then decreased; peak values were observed in the orthogonal experiment group at a salinity of 8 g/L and alkalinity of 30 mM. PMID:27981049

Effects of gas periodic stimulation on key enzyme activity in gas double-dynamic solid state fermentation (GDD-SSF).

PubMed

Chen, Hongzhang; Shao, Meixue; Li, Hongqiang

2014-03-05

The heat and mass transfer have been proved to be the important factors in air pressure pulsation for cellulase production. However, as process of enzyme secretion, the cellulase formation has not been studied in the view of microorganism metabolism and metabolic key enzyme activity under air pressure pulsation condition. Two fermentation methods in ATPase activity, cellulase productivity, weight lose rate and membrane permeability were systematically compared. Results indicated that gas double-dynamic solid state fermentation had no obviously effect on cell membrane permeability. However, the relation between ATPase activity and weight loss rate was linearly dependent with r=0.9784. Meanwhile, the results also implied that gas periodic stimulation had apparently strengthened microbial metabolism through increasing ATPase activity during gas double-dynamic solid state fermentation, resulting in motivating the production of cellulase by Trichoderma reesei YG3. Therefore, the increase of ATPase activity would be another crucial factor to strengthen fermentation process for cellulase production under gas double-dynamic solid state fermentation. Copyright © 2013 Elsevier Inc. All rights reserved.

Effects of Iron Overload on the Activity of Na,K-ATPase and Lipid Profile of the Human Erythrocyte Membrane

PubMed Central

Sousa, Leilismara; Garcia, Israel J. P.; Costa, Tamara G. F.; Silva, Lilian N. D.; Renó, Cristiane O.; Oliveira, Eneida S.; Tilelli, Cristiane Q.; Santos, Luciana L.; Cortes, Vanessa F.; Santos, Herica L.; Barbosa, Leandro A.

2015-01-01

Iron is an essential chemical element for human life. However, in some pathological conditions, such as hereditary hemochromatosis type 1 (HH1), iron overload induces the production of reactive oxygen species that may lead to lipid peroxidation and a change in the plasma-membrane lipid profile. In this study, we investigated whether iron overload interferes with the Na,K-ATPase activity of the plasma membrane by studying erythrocytes that were obtained from the whole blood of patients suffering from iron overload. Additionally, we treated erythrocytes of normal subjects with 0.8 mM H2O2 and 1 μM FeCl3 for 24 h. We then analyzed the lipid profile, lipid peroxidation and Na,K-ATPase activity of plasma membranes derived from these cells. Iron overload was more frequent in men (87.5%) than in women and was associated with an increase (446%) in lipid peroxidation, as indicated by the amount of the thiobarbituric acid reactive substances (TBARS) and an increase (327%) in the Na,K-ATPase activity in the plasma membrane of erythrocytes. Erythrocytes treated with 1 μM FeCl3 for 24 h showed an increase (132%) in the Na,K-ATPase activity but no change in the TBARS levels. Iron treatment also decreased the cholesterol and phospholipid content of the erythrocyte membranes and similar decreases were observed in iron overload patients. In contrast, erythrocytes treated with 0.8 mM H2O2 for 24 h showed no change in the measured parameters. These results indicate that erythrocytes from patients with iron overload exhibit higher Na,K-ATPase activity compared with normal subjects and that this effect is specifically associated with altered iron levels. PMID:26197432

Inhibition of ATP Hydrolysis by Thermoalkaliphilic F1Fo-ATP Synthase Is Controlled by the C Terminus of the ɛ Subunit

PubMed Central

Keis, Stefanie; Stocker, Achim; Dimroth, Peter; Cook, Gregory M.

2006-01-01

The F1Fo-ATP synthases of alkaliphilic bacteria exhibit latent ATPase activity, and for the thermoalkaliphile Bacillus sp. strain TA2.A1, this activity is intrinsic to the F1 moiety. To study the mechanism of ATPase inhibition, we developed a heterologous expression system in Escherichia coli to produce TA2F1 complexes from this thermoalkaliphile. Like the native F1Fo-ATP synthase, the recombinant TA2F1 was blocked in ATP hydrolysis activity, and this activity was stimulated by the detergent lauryldimethylamine oxide. To determine if the C-terminal domain of the ɛ subunit acts as an inhibitor of ATPase activity and if an electrostatic interaction plays a role, a TA2F1 mutant with either a truncated ɛ subunit [i.e., TA2F1(ɛΔC)] or substitution of basic residues in the second α-helix of ɛ with nonpolar alanines [i.e., TA2F1(ɛ6A)] was constructed. Both mutants showed ATP hydrolysis activity at low and high concentrations of ATP. Treatment of the purified F1Fo-ATP synthase and TA2F1(ɛWT) complex with proteases revealed that the ɛ subunit was resistant to proteolytic digestion. In contrast, the ɛ subunit of TA2F1(ɛ6A) was completely degraded by trypsin, indicating that the C-terminal arm was in a conformation where it was no longer protected from proteolytic digestion. In addition, ATPase activity was not further activated by protease treatment when compared to the untreated control, supporting the observation that ɛ was responsible for inhibition of ATPase activity. To study the effect of the alanine substitutions in the ɛ subunit in the entire holoenzyme, we reconstituted recombinant TA2F1 complexes with F1-stripped native membranes of strain TA2.A1. The reconstituted TA2FoF1(ɛWT) was blocked in ATP hydrolysis and exhibited low levels of ATP-driven proton pumping consistent with the F1Fo-ATP synthase in native membranes. Reconstituted TA2FoF1(ɛ6A) exhibited ATPase activity that correlated with increased ATP-driven proton pumping, confirming that the ɛ subunit also inhibits ATPase activity of TA2FoF1. PMID:16707672

Mouse Myosin-19 Is a Plus-end-directed, High-duty Ratio Molecular Motor*

PubMed Central

Lu, Zekuan; Ma, Xiao-Nan; Zhang, Hai-Man; Ji, Huan-Hong; Ding, Hao; Zhang, Jie; Luo, Dan; Sun, Yujie; Li, Xiang-dong

2014-01-01

Class XIX myosin (Myo19) is a vertebrate-specific unconventional myosin, responsible for the transport of mitochondria. To characterize biochemical properties of Myo19, we prepared recombinant mouse Myo19-truncated constructs containing the motor domain and the IQ motifs using the baculovirus/Sf9 expression system. We identified regulatory light chain (RLC) of smooth muscle/non-muscle myosin-2 as the light chain of Myo19. The actin-activated ATPase activity and the actin-gliding velocity of Myo19-truncated constructs were about one-third and one-sixth as those of myosin-5a, respectively. The apparent affinity of Myo19 to actin was about the same as that of myosin-5a. The RLCs bound to Myo19 could be phosphorylated by myosin light chain kinase, but this phosphorylation had little effect on the actin-activated ATPase activity and the actin-gliding activity of Myo19-truncated constructs. Using dual fluorescence-labeled actin filaments, we determined that Myo19 is a plus-end-directed molecular motor. We found that, similar to that of the high-duty ratio myosin, such as myosin-5a, ADP release rate was comparable with the maximal actin-activated ATPase activity of Myo19, indicating that ADP release is a rate-limiting step for the ATPase cycle of acto-Myo19. ADP strongly inhibited the actin-activated ATPase activity and actin-gliding activity of Myo19-truncated constructs. Based on the above results, we concluded that Myo19 is a high-duty ratio molecular motor moving to the plus-end of the actin filament. PMID:24825904

The effect of cold acclimation on active ion transport in cricket ionoregulatory tissues.

PubMed

Des Marteaux, Lauren E; Khazraeenia, Soheila; Yerushalmi, Gil Y; Donini, Andrew; Li, Natalia G; Sinclair, Brent J

2018-02-01

Cold-acclimated insects defend ion and water transport function during cold exposure. We hypothesized that this is achieved via enhanced active transport. The Malpighian tubules and rectum are likely targets for such transport modifications, and recent transcriptomic studies indicate shifts in Na + -K + ATPase (NKA) and V-ATPase expression in these tissues following cold acclimation. Here we quantify the effect of cold acclimation (one week at 12°C) on active transport in the ionoregulatory organs of adult Gryllus pennsylvanicus field crickets. We compared primary urine production of warm- and cold-acclimated crickets in excised Malpighian tubules via Ramsay assay at a range of temperatures between 4 and 25°C. We then compared NKA and V-ATPase activities in Malpighian tubule and rectal homogenates from warm- and cold-acclimated crickets via NADH-linked photometric assays. Malpighian tubules of cold-acclimated crickets excreted fluid at lower rates at all temperatures compared to warm-acclimated crickets. This reduction in Malpighian tubule excretion rates may be attributed to increased NKA activity that we observed for cold-acclimated crickets, but V-ATPase activity was unchanged. Cold acclimation had no effect on rectal NKA activity at either 21°C or 6°C, and did not modify rectal V-ATPase activity. Our results suggest that an overall reduction, rather than enhancement of active transport in the Malpighian tubules allows crickets to maintain hemolymph water balance during cold exposure, and increased Malpighian tubule NKA activity may help to defend and/or re-establish ion homeostasis. Copyright © 2017 Elsevier Inc. All rights reserved.

Adrenaline and triiodothyronine modify the iron handling in the freshwater air-breathing fish Anabas testudineus Bloch: role of ferric reductase in iron acquisition.

PubMed

Rejitha, V; Peter, M C Subhash

2013-01-15

The effects of in vivo adrenaline and triiodothyronine (T(3)) on ferric reductase (FR) activity, a membrane-bound enzyme that reduces Fe(III) to Fe(II) iron, were studied in the organs of climbing perch (Anabas testudineus Bloch). Adrenaline injection (10 ng g(-1)) for 30 min produced significant inhibition of FR activity in the liver and kidney and that suggests a role for this stress hormone in iron acquisition in this fish. Short-term T(3) injection (40 ng g(-1)) reduced FR activity in the gills of fed fish but not in the unfed fish. Similar reduction of FR activity was also obtained in the intestine and kidney of fed fish after T(3) injection. Feeding produced pronounced decline in FR activity in the spleen but T(3) challenge in fed and unfed fish increased its activity in this iron storing organ and that point to the sensitivity of FR system to feeding activity. The in vitro effects of Fe on FR activity in the gill explants of freshwater fish showed correlations of FR with Na(+), K(+)-ATPase and H(+)-ATPase activities. Substantial increase in the FR activity was found in the gill explants incubated with all the tested doses of Fe(II) iron (1.80, 3.59 and 7.18 μM) and Fe(III) iron (1.25, 2.51 and 5.02 μM) and this indicate that FR and Na pump activity are positively correlated. On the contrary, substantial reduction of gill H(+)-ATPase activity was found in the gill explants incubated with Fe(II) iron and Fe(III) iron indicating that perch gills may not require a high acidic microenvironment for the reduction of Fe(III) iron. Accumulation of iron in the gill explants after Fe(III) iron incubation implies a direct relationship between Fe acquisition and FR activity in this tissue. The inverse correlation between FR activity and H(+)-ATPase activity in Fe(II) or Fe(III) loaded gills and the significant positive correlations of FR activity with total [Fe] content in the Fe(III) loaded gills substantiate that FR which shows sensitivity to sodium and proton pumps, has a vital role in Fe(II) and Fe(III) iron handling in this fish. Our data also provide evidence that adrenaline, T(3) and the feeding status are the vital factors that can regulate the storage and handling of iron in fish. Copyright © 2012 Elsevier Inc. All rights reserved.

Isolation, purification, and partial characterization of a membrane-bound Cl-/HCO3--activated ATPase complex from rat brain with sensitivity to GABAAergic ligands.

PubMed

Menzikov, Sergey A

2017-02-07

This study describes the isolation and purification of a protein complex with [Formula: see text]-ATPase activity and sensitivity to GABA A ergic ligands from rat brain plasma membranes. The ATPase complex was enriched using size-exclusion, affinity, and ion-exchange chromatography. The fractions obtained at each purification step were subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE), which revealed four subunits with molecular mass ∼48, 52, 56, and 59 kDa; these were retained at all stages of the purification process. Autoradiography revealed that the ∼52 and 56 kDa subunits could bind [ 3 H]muscimol. The [Formula: see text]-ATPase activity of this enriched protein complex was regulated by GABA A ergic ligands but was not sensitive to blockers of the NKCC or KCC cotransporters.

Studies with a reconstituted muscle glycolytic system. The anaerobic glycolytic response to simulated tetanic contraction

PubMed Central

Scopes, Robert K.

1974-01-01

By using a reconstituted glycolytic system and a highly active adenosine triphosphatase (ATPase), the metabolism during muscular tetanic contraction was simulated and observed. With an ATPase activity somewhat greater than can be maintained in muscle tissue, phosphocreatine was rapidly and completely utilized, lactate production commenced about 5s after the ATPase was added and after 15s adenine nucleotides were lost through deamination to IMP. By 40s, all metabolism ceased because of complete loss of adenine mononucleotides. With a lower ATPase activity, glycolytic regeneration of ATP was capable of maintaining the ATP concentration at its initial value and even by 80s, only one-half of the phosphocreatine had been utilized. No deamination occurred in this time. It is suggested that the metabolic events observed in the simulated system are basically the same as occur in muscle doing heavy work. PMID:4275706

Effect of Tributyltin, Cadmium, and Their Combination on Physiological Responses in Juvenile Grass Carp.

PubMed

Mu, Wei-Na; Li, Zhi-Hua; Zhong, Li-Qiao; Wu, Yan-Hua

2016-09-01

Tributyltin (TBT) and cadmium (Cd) are two common pollutants in aquatic environments. This study was designed to examine the physiological responses of juvenile Grass Carp Ctenopharyngodon idella to TBT, Cd, and their combination. Fish were apportioned into a control group, a TBT group (7.5 μg/L), a Cd group (2.97 mg/L), and a TBT-Cd group (7.5 μg/L TBT, 2.97 mg/L Cd(2+)) for 7 d. The following activities were measured: Na(+),K(+)-ATPase in gill tissues; nitric oxide synthase (NOS), acetylcholinesterase (AChE), and monoamine oxidase (MAO) in brain tissues; and lipid peroxidation (LPO), malondialdehyde (MDA), total antioxidative capacity (T-AOC), and glutathione (GSH) in liver tissues. Cadmium-induced stress was suggested by alterations in antioxidant responses (MDA, LPO, and T-AOC) and neurological parameters (AChE, MAO, and NOS). Cadmium also induced Na(+),K(+)-ATPase and GSH activity. Compared with the responses among the Cd group, the combination of TBT and Cd not only decreased the level of GSH and Na(+),K(+)-ATPase but also increased the levels of MDA, LPO, AChE, MAO, and NOS. These results suggest that a combination of TBT and Cd could reduce the adverse effects of Cd on Grass Carp. However, the exact mechanisms for the combined effects TBT and Cd on these biomarkers require further investigation. Received September 28, 2015; accepted April 17, 2016.

Adaptive responses of Bacillus cereus ATCC14579 cells upon exposure to acid conditions involve ATPase activity to maintain their internal pH

PubMed Central

Senouci-Rezkallah, Khadidja; Jobin, Michel P; Schmitt, Philippe

2015-01-01

This study examined the involvement of ATPase activity in the acid tolerance response (ATR) of Bacillus cereus ATCC14579 strain. In the current work, B. cereus cells were grown in anaerobic chemostat culture at external pH (pHe) 7.0 or 5.5 and at a growth rate of 0.2 h−1. Population reduction and internal pH (pHi) after acid shock at pH 4.0 was examined either with or without ATPase inhibitor N,N’-dicyclohexylcarbodiimide (DCCD) and ionophores valinomycin and nigericin. Population reduction after acid shock at pH 4.0 was strongly limited in cells grown at pH 5.5 (acid-adapted cells) compared with cells grown at pH 7.0 (unadapted cells), indicating that B. cereus cells grown at low pHe were able to induce a significant ATR and Exercise-induced increase in ATPase activity. However, DCCD and ionophores had a negative effect on the ability of B. cereus cells to survive and maintain their pHi during acid shock. When acid shock was achieved after DCCD treatment, pHi was markedly dropped in unadapted and acid-adapted cells. The ATPase activity was also significantly inhibited by DCCD and ionophores in acid-adapted cells. Furthermore, transcriptional analysis revealed that atpB (ATP beta chain) transcripts was increased in acid-adapted cells compared to unadapted cells before and after acid shock. Our data demonstrate that B. cereus is able to induce an ATR during growth at low pH. These adaptations depend on the ATPase activity induction and pHi homeostasis. Our data demonstrate that the ATPase enzyme can be implicated in the cytoplasmic pH regulation and in acid tolerance of B. cereus acid-adapted cells. PMID:25740257

Contributions of the Na+/K+-ATPase, NKCC1, and Kir4.1 to hippocampal K+ clearance and volume responses

PubMed Central

Larsen, Brian Roland; Assentoft, Mette; Cotrina, Maria L.; Hua, Susan Z.; Nedergaard, Maiken; Kaila, Kai; Voipio, Juha; MacAulay, Nanna

2015-01-01

Bursts of network activity in the brain are associated with a transient increase in extracellular K+ concentration. The excess K+ is removed from the extracellular space by mechanisms proposed to involve Kir4.1-mediated spatial buffering, the Na+/K+/2Cl− cotransporter (NKCC1), and/or Na+/K+-ATPase activity. Their individual contribution to [K+]o management has been of extended controversy. The present study aimed, by several complementary approaches, to delineate the transport characteristics of Kir4.1, NKCC1, and Na+/K+-ATPase and to resolve their involvement in clearance of extracellular K+ transients. Primary cultures of rat astrocytes displayed robust NKCC1 activity with [K+]o increases above basal levels. Increased [K+]o produced NKCC1-mediated swelling of cultured astrocytes and NKCC1 could thereby potentially act as a mechanism of K+ clearance while concomitantly mediate the associated shrinkage of the extracellular space. In rat hippocampal slices, inhibition of NKCC1 failed to affect the rate of K+ removal from the extracellular space while Kir4.1 enacted its spatial buffering only during a local [K+]o increase. In contrast, inhibition of the different isoforms of Na+/K+-ATPase reduced post-stimulusclearance of K+ transients. The glia-specific α2/β2 subunit composition of Na+/K+-ATPase, when expressed in Xenopus oocytes, displayed a K+ affinity and voltage-sensitivity that would render this astrocyte-specific subunit composition specifically geared for controlling [K+]o during neuronal activity. In rat hippocampal slices, simultaneous measurements of the extracellular space volume revealed that neither Kir4.1, NKCC1, nor Na+/K+-ATPase accounted for the stimulus-induced shrinkage of the extracellular space. Thus, NKCC1 plays no role in activity-induced extracellular K+ recovery in native hippocampal tissue while Kir4.1 and Na+/K+-ATPase serve temporally distinct roles. PMID:24482245

Crystal Structure and Biochemical Characterization of a Mycobacterium smegmatis AAA-Type Nucleoside Triphosphatase Phosphohydrolase (Msm0858).

PubMed

Unciuleac, Mihaela-Carmen; Smith, Paul C; Shuman, Stewart

2016-05-15

AAA proteins (ATPases associated with various cellular activities) use the energy of ATP hydrolysis to drive conformational changes in diverse macromolecular targets. Here, we report the biochemical characterization and 2.5-Å crystal structure of a Mycobacterium smegmatis AAA protein Msm0858, the ortholog of Mycobacterium tuberculosis Rv0435c. Msm0858 is a magnesium-dependent ATPase and is active with all nucleoside triphosphates (NTPs) and deoxynucleoside triphosphates (dNTPs) as substrates. The Msm0858 structure comprises (i) an N-terminal domain (amino acids [aa] 17 to 201) composed of two β-barrel modules and (ii) two AAA domains, D1 (aa 212 to 473) and D2 (aa 476 to 744), each of which has ADP in the active site. Msm0858-ADP is a monomer in solution and in crystallized form. Msm0858 domains are structurally homologous to the corresponding modules of mammalian p97. However, the position of the N-domain modules relative to the AAA domains in the Msm0858-ADP tertiary structure is different and would impede the formation of a p97-like hexameric quaternary structure. Mutational analysis of the A-box and B-box motifs indicated that the D1 and D2 AAA domains are both capable of ATP hydrolysis. Simultaneous mutations of the D1 and D2 active-site motifs were required to abolish ATPase activity. ATPase activity was effaced by mutation of the putative D2 arginine finger, suggesting that Msm0858 might oligomerize during the ATPase reaction cycle. A truncated variant Msm0858 (aa 212 to 745) that lacks the N domain was characterized as a catalytically active homodimer. Recent studies have underscored the importance of AAA proteins (ATPases associated with various cellular activities) in the physiology of mycobacteria. This study reports the ATPase activity and crystal structure of a previously uncharacterized mycobacterial AAA protein, Msm0858. Msm0858 consists of an N-terminal β-barrel domain and two AAA domains, each with ADP bound in the active site. Msm0858 is a structural homolog of mammalian p97, with respect to the linear order and tertiary structures of their domains. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

[The ability of cells to adjust to the low oxigen content associated with Na,K-ATPase glutationilation].

PubMed

Petrushanko, I Iu; Simonenko, O V; Burnysheva, K M; Klimanova, E A; Dergousova, E A; Mit'kevich, V A; Lopina, O D; Makarov, A A

2015-01-01

Decreasing the amount of oxygen in the tissues under hypoxic and ischemic conditions, observed at a number of pathologic processes, inevitably leads to their damage. One of the main causes of cell damage and death is a violation of the systems maintaining ionic balance. Na,K-ATPaseis a basic ion-transporting protein of animal cell plasma membrane and inhibition of the Na,K-ATPase activity at lower concentrations of oxygen is one of the earliest and most critical events for cell viability. Currently there is an active search for modulators of Na,K-ATPase activity. For this purpose traditionally used cardiac glycosides but the existence of serious adverse effects forced to look for alternative inhibitors of Na,K-ATPase. Previously we have found that the glutathionylation of Na,K-ATPase catalytic subunit leads to a complete-inhibition of the enzyme. In this paper it is shown that the agents which increase the level of Na,K-ATPase glutathionylation: ethyl glutathione (et-GSH), oxidized glutathione (GSSG) and N-acetyl cysteine (NAC), increase cell survival under oxygen deficiency conditions, prevent decline of ATP in the cells and normalize their redox status. Concentration range in which these substances have a maximum protective effect, and does not exhibit cytotoxic properties was defined: for et-GSH 0.2-0.5 mM, for GSSG 0.2-1 mM, for NAC 10 to 15 mM. The results show prospects for development of methods for tissues protection from damage caused by oxygen starvation by varying the degree of Na,K-ATPase glutathionylation.

ELECTRON MICROSCOPIC STUDY OF THE ATPASE ACTIVITY OF THE BAI STRAIN A (MYELOBLASTOSIS) AVIAN TUMOR VIRUS

PubMed Central

Novikoff, Alex B.; de Thé, Guy; Beard, D.; Beard, J. W.

1962-01-01

Thymus glands of chicks with leukemia induced by BAI strain A (myeloblastosis) virus were fixed in cold 4 per cent formaldehyde-sucrose. Frozen sections were incubated in the ATPase medium of Wachstein and Meisel and studied by light microscopy and electron microscopy. The ATPase activity of the virus is localized to the outermost membrane of the virus. The membrane of the blast-like cells of the thymus cortex from which the virus emerges, by budding, also possesses such activity. It appears likely that the outermost membrane of the virus is derived from the plasma membrane of these cells. PMID:13939125

ATPase activity measurement of DNA replicative helicase from Bacillus stearothermophilus by malachite green method.

PubMed

Yang, Mu; Wang, Ganggang

2016-09-15

The DnaB helicase from Bacillus stearothermophilus (DnaBBst) was a model protein for studying the bacterial DNA replication. In this work, a non-radioactive method for measuring ATPase activity of DnaBBst helicase was described. The working parameters and conditions were optimized. Furthermore, this method was applied to investigate effects of DnaG primase, ssDNA and helicase loader protein (DnaI) on ATPase activity of DnaBBst. Our results showed this method was sensitive and efficient. Moreover, it is suitable for the investigation of functional interaction between DnaB and related factors. Copyright © 2016 Elsevier Inc. All rights reserved.

Ultrastructure and ATPase activity of the rectal gland of the chondrichthyean fish Hydrolagus colliei (Holocephali).

PubMed

Lagios, M D; Stasko-Concannon, S

1979-05-18

The anatomy, histology, ultrastructure and ATPase activity of the intramural rectal gland of the chondrichthyean Hydrolagus colliei, are described. The cells of the rectal gland of Hydrolagus demonstrate the same well developed lateral and basal cisternae, elongate mitochondria and luminal border as those of their elasmobranch counterparts. ATPase activity within the rectal gland of Hydrolagus is as intense as that in a number of elasmobranchs examined in the course of the study. Despite its primitive intramural location the rectal gland of Hydrolagus represents a homolog of the more specialized and better known elasmobranch gland and appears as well suited for cation excretion.

Branchial ammonia excretion in the Asian weatherloach Misgurnus anguillicaudatus.

PubMed

Moreira-Silva, J; Tsui, T K N; Coimbra, J; Vijayan, M M; Ip, Y K; Wilson, J M

2010-01-01

The weatherloach, Misgurnus anguillicaudatus, is a freshwater, facultative air-breathing fish that lives in streams and rice paddy fields, where it may experience drought and/or high environmental ammonia (HEA) conditions. The aim of this study was to determine what roles branchial Na(+)/K(+)-ATPase, H(+)-ATPase, and Rhcg have in ammonia tolerance and how the weatherloach copes with ammonia loading conditions. The loach's high ammonia tolerance was confirmed as was evident from its high 96 h LC(50) value and high tissue tolerance to ammonia. The weatherloach does not appear to make use of Na(+)/NH(4)(+)-ATPase facilitated transport to excrete ammonia when exposed to HEA or to high environmental pH since no changes in activity were observed. Using immunofluorescence microscopy, distinct populations of vacuolar (V)-type H(+)-ATPase and Na(+)/K(+)-ATPase immunoreactive cells were identified in branchial epithelia, with apical and basolateral staining patterns, respectively. Rhesus C glycoprotein (Rhcg1), an ammonia transport protein, immunoreactivity was also found in a similar pattern as H(+)-ATPase. Rhcg1 (Slc42a3) mRNA expression also increased significantly during aerial exposure, although not significantly under ammonia loading conditions. The colocalization of H(+)-ATPase and Rhcg1 to the similar non-Na(+)/K(+)-ATPase immunoreactive cell type would support a role for H(+)-ATPase in ammonia excretion via Rhcg by NH(4)(+) trapping. The importance of gill boundary layer acidification in net ammonia excretion was confirmed in this fish; however, it was not associated with an increase in H(+)-ATPase expression, since tissue activity and protein levels did not increase with high environmental pH and/or HEA. However the V-ATPase inhibitor, bafilomycin, did decrease net ammonia flux whereas other ion transport inhibitors (amiloride, SITS) had no effect. H(+)-ATPase inhibition also resulted in a consequent elevation in plasma ammonia levels and a decrease in the net acid flux. In gill, aerial exposure was also associated with a significant increase in membrane fluidity (or increase in permeability) which would presumably enhance NH(3) permeation through the plasma membrane. Taken together, these results indicate the gill of the weatherloach is responsive to aerial conditions that would aid ammonia excretion.

Mutagenesis of the residues forming an ion binding pocket of the NtpK subunit of Enterococcus hirae V-ATPase.

PubMed

Kawano-Kawada, Miyuki; Iwaki, Tomoko; Hosaka, Toshiaki; Murata, Takeshi; Yamato, Ichiro; Homma, Michio; Kakinuma, Yoshimi

2012-09-01

The crystal structures of the Na(+)- and Li(+)-bound NtpK rings of Enterococcus hirae V-ATPase have been obtained. The coupling ion (Na(+) or Li(+)) was surrounded by five oxygen atoms contributed by residues T64, Q65, Q110, E139, and L61, and the hydrogen bonds of the side chains of Q110, Y68, and T64 stabilized the position of the E139 γ carboxylate essential for ion occlusion (PDB accession numbers 2BL2 and 2CYD). We previously indicated that an NtpK mutant strain (E139D) lost tolerance to sodium but not to lithium at alkaline pHs and suggested that the E139 residue is indispensable for the enzymatic activity of E. hirae V-ATPase linked with the sodium tolerance of this bacterium. In this study, we examined the activities of V-ATPase in which these four residues, except for E139, were substituted. The V-ATPase activities of the Q65A and Y68A mutants were slightly retained, but those of the T64A and Q110A mutants were negligible. Among the residues, T64 and Q110 are indispensable for the ion coupling of E. hirae V-ATPase, in addition to the essential residue E139.

Mutagenesis of the Residues Forming an Ion Binding Pocket of the NtpK Subunit of Enterococcus hirae V-ATPase

PubMed Central

Kawano-Kawada, Miyuki; Iwaki, Tomoko; Hosaka, Toshiaki; Murata, Takeshi; Yamato, Ichiro; Homma, Michio

2012-01-01

The crystal structures of the Na+- and Li+-bound NtpK rings of Enterococcus hirae V-ATPase have been obtained. The coupling ion (Na+ or Li+) was surrounded by five oxygen atoms contributed by residues T64, Q65, Q110, E139, and L61, and the hydrogen bonds of the side chains of Q110, Y68, and T64 stabilized the position of the E139 γ carboxylate essential for ion occlusion (PDB accession numbers 2BL2 and 2CYD). We previously indicated that an NtpK mutant strain (E139D) lost tolerance to sodium but not to lithium at alkaline pHs and suggested that the E139 residue is indispensable for the enzymatic activity of E. hirae V-ATPase linked with the sodium tolerance of this bacterium. In this study, we examined the activities of V-ATPase in which these four residues, except for E139, were substituted. The V-ATPase activities of the Q65A and Y68A mutants were slightly retained, but those of the T64A and Q110A mutants were negligible. Among the residues, T64 and Q110 are indispensable for the ion coupling of E. hirae V-ATPase, in addition to the essential residue E139. PMID:22730119

Omeprazole, a specific inhibitor of gastric (H/sup +/-K/sup +/)-ATPase, is a H/sup +/-activated oxidizing agent of sulfhydryl groups

DOE Office of Scientific and Technical Information (OSTI.GOV)

Im, W.B.; Sih, J.C.; Blakeman, D.P.

1985-04-25

Omeprazole (5-methoxy-2-(((4-methoxy-3,5- dimethylpyridinyl)methyl)sulfinyl)-1H-benzimidazole) appeared to inhibit gastric (H/sup +/-K/sup +/)-ATPase by oxidizing its essential sulfhydryl groups, since the gastric ATPase inactivated by the drug in vivo or in vitro recovered its K+-dependent ATP hydrolyzing activity upon incubation with mercaptoethanol. Biological reducing agents like cysteine or glutathione, however, were unable to reverse the inhibitory effect of omeprazole. Moreover, acidic environments enhanced the potency of omeprazole. The chemical reactivity of omeprazole with mercaptans is also consistent with the biological action of omeprazole. The N-sulfenylated compound reacted at neutral pH with another stoichiometric amount of ethyl mercaptan to produce omeprazole sulfide quantitatively. Themore » gastric polypeptides of 100 kilodaltons representing (H/sup +/-K/sup +/)-ATPase in the rat gastric mucosa or isolated hog gastric membranes were covalently labeled with (/sup 14/C)omeprazole. The radioactive label bound to the ATPase, however, could not be displaced by mercaptoethanol under the identical conditions where the ATPase activity was fully restored. These observations suggest that the essential sulfhydryl groups which reacted with omeprazole did not form a stable covalent bond with the drug, but rather that they further reacted with adjacent sulfhydryl groups to form disulfides which could be reduced by mercaptoethanol.« less

The role of brassinosteroids in the regulation of the plasma membrane H+-ATPase and NADPH oxidase under cadmium stress.

PubMed

Jakubowska, Dagmara; Janicka, Małgorzata

2017-11-01

The present research aim was to define the role of brassinosteroids (BRs) in plant adaptation to cadmium stress. We observed a stimulating effect of exogenous BR on the activity of two plasma membrane enzymes which play a key role in plants adaptation to cadmium stress, H + -ATPase (EC 3.6.3.14) and NADPH oxidase (EC 1.6.3.1). Using anti-phosphothreonine antibody we showed that modification of PM H + -ATPase activity under BR action could result from phosphorylation of the enzyme protein. Also the relative expression of genes encoding both PM H + -ATPase and NADPH oxidase was affected by BR. To confirm the role of BR in the cadmium stimulating effect on activity of both studied plasma membrane enzymes, an assay in the presence of a BR biosynthesis inhibitor (propiconazole) was performed. Moreover, as a tool in our work we used commercially available plant mutants unable to BR biosynthesis or with dysfunctional BR signaling pathway, to further confirm participation of BR in plant adaptation to heavy metal stress. Presented results demonstrate some elements of the brassinosteroid-induced pathway activated under cadmium stress, wherein H + -ATPase and NADPH oxidase are key factors. Copyright © 2017 Elsevier B.V. All rights reserved.

Thyroid thermogenesis. Relationships between Na+-dependent respiration and Na+ + K+-adenosine triphosphatase activity in rat skeletal muscle.

PubMed Central

Asano, Y; Liberman, U A; Edelman, I S

1976-01-01

The effect of thyroid status on QO2, QO2 (t) and NaK-ATPase activity was examined in rat skeletal muscle. QO2(t) (i.e. Na+-transport-dependent respiration) was estimated with ouabain or Na+-free media supplemented with K+. In contrast to the effects of ouabain on ion composition, intracellular K+ was maintained at about 125 meq/liter, and intracellular Na+ was almost nil in the Na+-free media. The estimates of QO2(t) were independent of the considerable differences in tissue ion concentrations. The increase in QO2(t) account for 47% of the increase in QO2 in the transition from the hypothyroid to the euthyroid state and 84% of the increase in the transition from the euthyroid to the hyperthyroid state. Surgical thyroidectomy lowered NaK-ATPase activity of the microsomal fraction (expressed per milligram protein) 32%; injections of triodothyronine (T3) increased this activity 75% in initially hypothyroid rats and 26% in initially euthyroid rats. Thyroidectomy was attended by significant falls in serum Ca and Pi concentrations. Administration of T3 resulted in further declines in serum Ca and marked increases in serum Ps concentrations. Similar effects were seen in 131I-treated rats, but the magnitude of the declines in serum Ca were less. The effects of T3 on QO2, QO2(t), and NaK-ATPase activity of skeletal muscle were indistinguishable in the 131I-ablated and surgically thyroidectomized rats. In thyroidectomized or euthyroid rats given repeated doses of T3, QO2(t) and NaA-ATPase activity increased proportionately. In thyroidectomized rats injected with single doses of T3, either 10, 50, or 250 mug/100 g body wt, QO2(t) increased linearly with NaK-ATPase activity. The kinetics of the NaK-ATPase activity was assessed with an ATP-generating system. T3 elicited a significant increase in Vmax with no change in Km for ATP. PMID:130385

Effects of stinging nettle root extracts and their steroidal components on the Na+,K(+)-ATPase of the benign prostatic hyperplasia.

PubMed

Hirano, T; Homma, M; Oka, K

1994-02-01

The effects of organic-solvent extracts of Urtica dioica (Urticaceae) on the Na+,K(+)-ATPase of the tissue of benign prostatic hyperplasia (BPH) were investigated. The membrane Na+,K(+)-ATPase fraction was prepared from a patient with BPH by a differential centrifugation of the tissue homogenate. The enzyme activity was inhibited by 10(-4)-10(-5) M of ouabain. The hexane extract, the ether extract, the ethyl acetate extract, and the butanol extract of the roots caused 27.6-81.5% inhibition of the enzyme activity at 0.1 mg/ml. In addition, a column extraction of stinging nettle roots using benzene as an eluent afforded efficient enzyme inhibiting activity. Steroidal components in stinging nettle roots, such as stigmast-4-en-3-one, stigmasterol, and campesterol inhibited the enzyme activity by 23.0-67.0% at concentrations ranging from 10(-3)-10(-6) M. These results suggest that some hydrophobic constituents such as steroids in the stinging nettle roots inhibited the membrane Na+,K(+)-ATPase activity of the prostate, which may subsequently suppress prostate-cell metabolism and growth.

Effects of intermittent fasting on age-related changes on Na,K-ATPase activity and oxidative status induced by lipopolysaccharide in rat hippocampus.

PubMed

Vasconcelos, Andrea Rodrigues; Kinoshita, Paula Fernanda; Yshii, Lidia Mitiko; Marques Orellana, Ana Maria; Böhmer, Ana Elisa; de Sá Lima, Larissa; Alves, Rosana; Andreotti, Diana Zukas; Marcourakis, Tania; Scavone, Cristoforo; Kawamoto, Elisa Mitiko

2015-05-01

Chronic neuroinflammation is a common characteristic of neurodegenerative diseases, and lipopolysaccharide (LPS) signaling is linked to glutamate-nitric oxide-Na,K-ATPase isoforms pathway in central nervous system (CNS) and also causes neuroinflammation. Intermittent fasting (IF) induces adaptive responses in the brain that can suppress inflammation, but the age-related effect of IF on LPS modulatory influence on nitric oxide-Na,K-ATPase isoforms is unknown. This work compared the effects of LPS on the activity of α1,α2,3 Na,K-ATPase, nitric oxide synthase gene expression and/or activity, cyclic guanosine monophosphate, 3-nitrotyrosine-containing proteins, and levels of thiobarbituric acid-reactive substances in CNS of young and older rats submitted to the IF protocol for 30 days. LPS induced an age-related effect in neuronal nitric oxide synthase activity, cyclic guanosine monophosphate, and levels of thiobarbituric acid-reactive substances in rat hippocampus that was linked to changes in α2,3-Na,K-ATPase activity, 3-nitrotyrosine proteins, and inducible nitric oxide synthase gene expression. IF induced adaptative cellular stress-response signaling pathways reverting LPS effects in rat hippocampus of young and older rats. The results suggest that IF in both ages would reduce the risk for deficits on brain function and neurodegenerative disorders linked to inflammatory response in the CNS. Copyright © 2015 Elsevier Inc. All rights reserved.

Muscular tissues of the squid Doryteuthis pealeii express identical myosin heavy chain isoforms: an alternative mechanism for tuning contractile speed

PubMed Central

Shaffer, Justin F.; Kier, William M.

2012-01-01

SUMMARY The speed of muscle contraction is largely controlled at the sarcomere level by the ATPase activity of the motor protein myosin. Differences in amino acid sequence in catalytically important regions of myosin yield different myosin isoforms with varying ATPase activities and resulting differences in cross-bridge cycling rates and interfilamentary sliding velocities. Modulation of whole-muscle performance by changes in myosin isoform ATPase activity is regarded as a universal mechanism to tune contractile properties, especially in vertebrate muscles. Invertebrates such as squid, however, may exhibit an alternative mechanism to tune contractile properties that is based on differences in muscle ultrastructure, including variable myofilament and sarcomere lengths. To determine definitively whether contractile properties of squid muscles are regulated via different myosin isoforms (i.e. different ATPase activities), the nucleotide and amino acid sequences of the myosin heavy chain from the squid Doryteuthis pealeii were determined from the mantle, arm, tentacle, fin and funnel retractor musculature. We identified three myosin heavy chain isoforms in squid muscular tissues, with differences arising at surface loop 1 and the carboxy terminus. All three isoforms were detected in all five tissues studied. These results suggest that the muscular tissues of D. pealeii express identical myosin isoforms, and it is likely that differences in muscle ultrastructure, not myosin ATPase activity, represent the most important mechanism for tuning contractile speeds. PMID:22189767

Clathrin coat controls synaptic vesicle acidification by blocking vacuolar ATPase activity

PubMed Central

Farsi, Zohreh; Rammner, Burkhard; Woehler, Andrew; Lafer, Eileen M; Mim, Carsten; Jahn, Reinhard

2018-01-01

Newly-formed synaptic vesicles (SVs) are rapidly acidified by vacuolar adenosine triphosphatases (vATPases), generating a proton electrochemical gradient that drives neurotransmitter loading. Clathrin-mediated endocytosis is needed for the formation of new SVs, yet it is unclear when endocytosed vesicles acidify and refill at the synapse. Here, we isolated clathrin-coated vesicles (CCVs) from mouse brain to measure their acidification directly at the single vesicle level. We observed that the ATP-induced acidification of CCVs was strikingly reduced in comparison to SVs. Remarkably, when the coat was removed from CCVs, uncoated vesicles regained ATP-dependent acidification, demonstrating that CCVs contain the functional vATPase, yet its function is inhibited by the clathrin coat. Considering the known structures of the vATPase and clathrin coat, we propose a model in which the formation of the coat surrounds the vATPase and blocks its activity. Such inhibition is likely fundamental for the proper timing of SV refilling. PMID:29652249

Conformational switching in the coiled-coil domains of a proteasomal ATPase regulates substrate processing.

PubMed

Snoberger, Aaron; Brettrager, Evan J; Smith, David M

2018-06-18

Protein degradation in all domains of life requires ATPases that unfold and inject proteins into compartmentalized proteolytic chambers. Proteasomal ATPases in eukaryotes and archaea contain poorly understood N-terminally conserved coiled-coil domains. In this study, we engineer disulfide crosslinks in the coiled-coils of the archaeal proteasomal ATPase (PAN) and report that its three identical coiled-coil domains can adopt three different conformations: (1) in-register and zipped, (2) in-register and partially unzipped, and (3) out-of-register. This conformational heterogeneity conflicts with PAN's symmetrical OB-coiled-coil crystal structure but resembles the conformational heterogeneity of the 26S proteasomal ATPases' coiled-coils. Furthermore, we find that one coiled-coil can be conformationally constrained even while unfolding substrates, and conformational changes in two of the coiled-coils regulate PAN switching between resting and active states. This switching functionally mimics similar states proposed for the 26S proteasome from cryo-EM. These findings thus build a mechanistic framework to understand regulation of proteasome activity.

Cation Transport Coupled to ATP Hydrolysis by the (Na, K)-ATPase: An Integrated, Animated Model

ERIC Educational Resources Information Center

Leone, Francisco A.; Furriel, Rosa P. M.; McNamara, John C.; Horisberger, Jean D.; Borin, Ivana A.

2010-01-01

An Adobe[R] animation is presented for use in undergraduate Biochemistry courses, illustrating the mechanism of Na[superscript +] and K[superscript +] translocation coupled to ATP hydrolysis by the (Na, K)-ATPase, a P[subscript 2c]-type ATPase, or ATP-powered ion pump that actively translocates cations across plasma membranes. The enzyme is also…

Bicarbonate-regulated adenylyl cyclase (sAC) is a sensor that regulates pH-dependent V-ATPase recycling.

PubMed

Pastor-Soler, Nuria; Beaulieu, Valerie; Litvin, Tatiana N; Da Silva, Nicolas; Chen, Yanqiu; Brown, Dennis; Buck, Jochen; Levin, Lonny R; Breton, Sylvie

2003-12-05

Modulation of environmental pH is critical for the function of many biological systems. However, the molecular identity of the pH sensor and its interaction with downstream effector proteins remain poorly understood. Using the male reproductive tract as a model system in which luminal acidification is critical for sperm maturation and storage, we now report a novel pathway for pH regulation linking the bicarbonate activated soluble adenylyl cyclase (sAC) to the vacuolar H+ATPase (V-ATPase). Clear cells of the epididymis and vas deferens contain abundant V-ATPase in their apical pole and are responsible for acidifying the lumen. Proton secretion is regulated via active recycling of V-ATPase. Here we demonstrate that this recycling is regulated by luminal pH and bicarbonate. sAC is highly expressed in clear cells, and apical membrane accumulation of V-ATPase is triggered by a sAC-dependent rise in cAMP in response to alkaline luminal pH. As sAC is expressed in other acid/base transporting epithelia, including kidney and choroid plexus, this cAMP-dependent signal transduction pathway may be a widespread mechanism that allows cells to sense and modulate extracellular pH.

Detection of endogenous lithium in neuropsychiatric disorders--a model for biological transmutation.

PubMed

Kurup, Ravi Kumar; Kurup, Parameswara Achutha

2002-01-01

The human hypothalamus produces an endogenous membrane Na(+)-K(+) ATPase inhibitor, digoxin. A digoxin induced model of cellular/neuronal quantal state and perception has been described by the authors. Biological transmutation has been described in microbial systems in the quantal state. The study focuses on the plasma levels of digoxin, RBC membrane Na(+)-K(+) ATPase activity, plasma levels of magnesium and lithium in neuropsychiatric and systemic disorders. Inhibition of RBC membrane Na(+)-K(+) ATPase activity was observed in most cases along with an increase in the levels of serum digoxin and lithium and a decrease in the level of serum Mg(++). The generation of endogenous lithium would obviously occur due to biological transmutation from magnesium. Digoxin and lithium together can produce added membrane Na(+)-K(+) ATPase inhibition. The role of membrane Na(+)-K(+) ATPase inhibition in the pathogenesis of neuropsychiatric and systemic disorders is discussed. The inhibition of membrane Na(+)-K(+) ATPase can contribute to an increase in intracellular calcium and a decrease in magnesium, which can result in a defective neurotransmitter transport mechanism, mitochondrial dysfunction and apoptosis, defective golgi body function and protein processing dysfunction, immune dysfunction and oncogenesis. Copyright 2002 John Wiley & Sons, Ltd.

Proton gradients produced by glucose oxidase microcapsules containing motor F0F1-ATPase for continuous ATP biosynthesis.

PubMed

Duan, Li; Qi, Wei; Yan, Xuehai; He, Qiang; Cui, Yue; Wang, Kewei; Li, Dongxiang; Li, Junbai

2009-01-15

Glucose oxidase (GOD) microcapsules held together by cross-linker, glutaraldehyde (GA), are fabricated by the layer-by-layer (LbL) assembly technique. The lipid bilayer containing CF(0)F(1)-ATPase was coated on the outer shell of GOD microcapsules. Driven under the proton gradients produced by catalysis of GOD microcapsules for glucose, ATP is synthesized from ADP and inorganic phosphate catalyzed by the ATPase rotary catalysis. The results show here that ATPase reconstituted on the GOD microcapsules retains its catalytic activity.

Disorders of lysosomal acidification - the emerging role of v-ATPase in aging and neurodegenerative disease

PubMed Central

Colacurcio, Daniel J.; Nixon, Ralph A.

2016-01-01

Autophagy and endocytosis deliver unneeded cellular materials to lysosomes for degradation. Beyond processing cellular waste, lysosomes release metabolites and ions that serve signaling and nutrient sensing roles, linking the functions of the lysosome to various pathways for intracellular metabolism and nutrient homeostasis. Each of these lysosomal behaviors is influenced by the intraluminal pH of the lysosome, which is maintained in the low acidic range by a proton pump, the vacuolar ATPase (v-ATPase). New reports implicate altered v-ATPase activity and lysosomal pH dysregulation in cellular aging, longevity, and adult-onset neurodegenerative diseases, including forms of Parkinson Disease and Alzheimer Disease. Genetic defects of subunits composing the v-ATPase or v-ATPase-related proteins occur in an increasingly recognized group of familial neurodegenerative diseases. Here, we review the expanding roles of the v-ATPase complex as a platform regulating lysosomal proteolysis and cellular homeostasis. We discuss the unique vulnerability of neurons to persistent low level lysosomal dysfunction and review recent clinical and experimental studies that link dysfunction of the v-ATPase complex to neurodegenerative diseases across the age spectrum. PMID:27197071

Specific phospholipid binding to Na,K-ATPase at two distinct sites.

PubMed

Habeck, Michael; Kapri-Pardes, Einat; Sharon, Michal; Karlish, Steven J D

2017-03-14

Membrane protein function can be affected by the physical state of the lipid bilayer and specific lipid-protein interactions. For Na,K-ATPase, bilayer properties can modulate pump activity, and, as observed in crystal structures, several lipids are bound within the transmembrane domain. Furthermore, Na,K-ATPase activity depends on phosphatidylserine (PS) and cholesterol, which stabilize the protein, and polyunsaturated phosphatidylcholine (PC) or phosphatidylethanolamine (PE), known to stimulate Na,K-ATPase activity. Based on lipid structural specificity and kinetic mechanisms, specific interactions of both PS and PC/PE have been inferred. Nevertheless, specific binding sites have not been identified definitively. We address this question with native mass spectrometry (MS) and site-directed mutagenesis. Native MS shows directly that one molecule each of 18:0/18:1 PS and 18:0/20:4 PC can bind specifically to purified human Na,K-ATPase (α 1 β 1 ). By replacing lysine residues at proposed phospholipid-binding sites with glutamines, the two sites have been identified. Mutations in the cytoplasmic αL8-9 loop destabilize the protein but do not affect Na,K-ATPase activity, whereas mutations in transmembrane helices (TM), αTM2 and αTM4, abolish the stimulation of activity by 18:0/20:4 PC but do not affect stability. When these data are linked to crystal structures, the underlying mechanism of PS and PC/PE effects emerges. PS (and cholesterol) bind between αTM 8, 9, 10, near the FXYD subunit, and maintain topological integrity of the labile C terminus of the α subunit (site A). PC/PE binds between αTM2, 4, 6, and 9 and accelerates the rate-limiting E 1 P-E 2 P conformational transition (site B). We discuss the potential physiological implications.

Managing Brain Extracellular K+ during Neuronal Activity: The Physiological Role of the Na+/K+-ATPase Subunit Isoforms

PubMed Central

Larsen, Brian Roland; Stoica, Anca; MacAulay, Nanna

2016-01-01

During neuronal activity in the brain, extracellular K+ rises and is subsequently removed to prevent a widespread depolarization. One of the key players in regulating extracellular K+ is the Na+/K+-ATPase, although the relative involvement and physiological impact of the different subunit isoform compositions of the Na+/K+-ATPase remain unresolved. The various cell types in the brain serve a certain temporal contribution in the face of network activity; astrocytes respond directly to the immediate release of K+ from neurons, whereas the neurons themselves become the primary K+ absorbers as activity ends. The kinetic characteristics of the catalytic α subunit isoforms of the Na+/K+-ATPase are, partly, determined by the accessory β subunit with which they combine. The isoform combinations expressed by astrocytes and neurons, respectively, appear to be in line with the kinetic characteristics required to fulfill their distinct physiological roles in clearance of K+ from the extracellular space in the face of neuronal activity. Understanding the nature, impact and effects of the various Na+/K+-ATPase isoform combinations in K+ management in the central nervous system might reveal insights into pathological conditions such as epilepsy, migraine, and spreading depolarization following cerebral ischemia. In addition, particular neurological diseases occur as a result of mutations in the α2- (familial hemiplegic migraine type 2) and α3 isoforms (rapid-onset dystonia parkinsonism/alternating hemiplegia of childhood). This review addresses aspects of the Na+/K+-ATPase in the regulation of extracellular K+ in the central nervous system as well as the related pathophysiology. Understanding the physiological setting in non-pathological tissue would provide a better understanding of the pathological events occurring during disease. PMID:27148079

Endogenous acetylcholine increases alveolar epithelial fluid transport via activation of alveolar epithelial Na,K-ATPase in mice.

PubMed

Li, Xia; Yan, Xi Xin; Li, Hong Lin; Li, Rong Qin

2015-10-01

The contribution of endogenous acetylcholine to alveolar fluid clearance (AFC) and related molecular mechanisms were explored. AFC was measured in Balb/c mice after vagotomy and vagus nerve stimulation. Effects of acetylcholine chloride on AFC in Kunming mice and Na,K-ATPase function in A549 alveolar epithelial cells also were determined. AFC significantly decreased in mice with left cervical vagus nerve transection compared with controls (48.69 ± 2.57 vs. 66.88 ± 2.64, P ≤ 0.01), which was reversed by stimulation of the peripheral (60.81 ± 1.96, P ≤ 0.01). Compared with control, acetylcholine chloride dose-dependently increased AFC and elevated Na,K-ATPase activity, and these increases were blocked or reversed by atropine. These effects were accompanied by recruitment of Na,K-ATPase α1 to the cell membrane. Thus, vagus nerves participate in alveolar epithelial fluid transport by releasing endogenous acetylcholine in the infusion-induced pulmonary edema mouse model. Effects of endogenous acetylcholine on AFC are likely mediated by Na,K-ATPase function through activation of muscarinic acetylcholine receptors on alveolar epithelia. Copyright © 2015 Elsevier B.V. All rights reserved.

Ethylene Mediates Alkaline-Induced Rice Growth Inhibition by Negatively Regulating Plasma Membrane H+-ATPase Activity in Roots

PubMed Central

Chen, Haifei; Zhang, Quan; Cai, Hongmei; Xu, Fangsen

2017-01-01

pH is an important factor regulating plant growth. Here, we found that rice was better adapted to low pH than alkaline conditions, as its growth was severely inhibited at high pH, with shorter root length and an extreme biomass reduction. Under alkaline stress, the expression of genes for ethylene biosynthesis enzymes in rice roots was strongly induced by high pH and exogenous ethylene precursor ACC and ethylene overproduction in etol1-1 mutant aggravated the alkaline stress-mediated inhibition of rice growth, especially for the root elongation with decreased cell length in root apical regions. Conversely, the ethylene perception antagonist silver (Ag+) and ein2-1 mutants could partly alleviate the alkaline-induced root elongation inhibition. The H+-ATPase activity was extremely inhibited by alkaline stress and exogenous ACC. However, the H+-ATPase-mediated rhizosphere acidification was enhanced by exogenous Ag+, while H+ efflux on the root surface was extremely inhibited by exogenous ACC, suggesting that ethylene negatively regulated H+-ATPase activity under high-pH stress. Our results demonstrate that H+-ATPase is involved in ethylene-mediated inhibition of rice growth under alkaline stress. PMID:29114258

A conserved inter-domain communication mechanism regulates the ATPase activity of the AAA-protein Drg1.

PubMed

Prattes, Michael; Loibl, Mathias; Zisser, Gertrude; Luschnig, Daniel; Kappel, Lisa; Rössler, Ingrid; Grassegger, Manuela; Hromic, Altijana; Krieger, Elmar; Gruber, Karl; Pertschy, Brigitte; Bergler, Helmut

2017-03-17

AAA-ATPases fulfil essential roles in different cellular pathways and often act in form of hexameric complexes. Interaction with pathway-specific substrate and adaptor proteins recruits them to their targets and modulates their catalytic activity. This substrate dependent regulation of ATP hydrolysis in the AAA-domains is mediated by a non-catalytic N-terminal domain. The exact mechanisms that transmit the signal from the N-domain and coordinate the individual AAA-domains in the hexameric complex are still the topic of intensive research. Here, we present the characterization of a novel mutant variant of the eukaryotic AAA-ATPase Drg1 that shows dysregulation of ATPase activity and altered interaction with Rlp24, its substrate in ribosome biogenesis. This defective regulation is the consequence of amino acid exchanges at the interface between the regulatory N-domain and the adjacent D1 AAA-domain. The effects caused by these mutations strongly resemble those of pathological mutations of the AAA-ATPase p97 which cause the hereditary proteinopathy IBMPFD (inclusion body myopathy associated with Paget's disease of the bone and frontotemporal dementia). Our results therefore suggest well conserved mechanisms of regulation between structurally, but not functionally related members of the AAA-family.

The F-ATPase operon from the oral streptococci S. mutans and S. sanguis: How structure relates to function

NASA Astrophysics Data System (ADS)

Kuhnert, Wendi Lee

1999-10-01

The oral microbe, Streptococcus mutans is known to be a primary contributor to the most common infection in humans, dental caries. In the plaque environment, resident bacteria metabolize dietary sucrose which results in the production of organic acids and a decrease in plaque pH. The proton-translocating ATPase (F-ATPase) protects the bacteria from acidification by extruding protons, at the expense of ATP, to maintain an internal pH which is more neutral than the external environment. Examination of this enzyme will help us to gain insight regarding its contribution to the aciduricity characteristics of oral bacteria. In this work, our goal was to begin the molecular dissection of the mechanism by which streptococcal ATPases are regulated and function enzymatically. Sequence analysis of the F-ATPase from the non-pathogenic S. sanguis revealed that the structural genes are homologous to S. mutans as well as other sequenced F-ATPases. Cloned subunits were functionally similar as shown by complementing E. coli ATPase mutants. S. sanguis/E. coli hybrid enzymes hydrolyzed ATP, but proton conduction was uncoupled as demonstrated with inhibition studies. Transcriptional regulation of the F-ATPase operon from S. mutans was examined using chloramphenicol acetyltransferase gene fusions. Fusions containing 136 bp of DNA upstream of the promoter showed higher levels of expression as compared to those with only 16 bp. Similar to ATPase enzymatic activity, CAT expression also increased during growth at low pH. Analysis of RNA demonstrated that ATPase mRNA levels were higher at low pH, which supported the CAT activity data. Therefore, the F-ATPase from S. mutans was regulated, at least partially, by both the DNA located upstream of the promoter as well as by pH. Examination of structural models of the F-ATPase from the pathogenic oral organisms S. mutans and Lactobacillus casei and the non- pathogenic S. sanguis showed that the differences noted in the sequence of the catalytic β subunit do not result in structural alterations. Therefore, the contribution that the F-ATPase makes towards the aciduricity of the oral streptococci is linked to its increased expression at low pH or perhaps to structural differences in the other, less-conserved, domains of the enzyme.

Functional Characterization of Ice Plant SKD1, an AAA-Type ATPase Associated with the Endoplasmic Reticulum-Golgi Network, and Its Role in Adaptation to Salt Stress1[W

PubMed Central

Jou, Yingtzy; Chiang, Chih-Pin; Jauh, Guang-Yuh; Yen, Hungchen Emilie

2006-01-01

A salt-induced gene mcSKD1 (suppressor of K+ transport growth defect) able to facilitate K+ uptake has previously been identified from the halophyte ice plant (Mesembryanthemum crystallinum). The sequence of mcSKD1 is homologous to vacuolar protein sorting 4, an ATPase associated with a variety of cellular activities-type ATPase that participates in the sorting of vacuolar proteins into multivesicular bodies in yeast (Saccharomyces cerevisiae). Recombinant mcSKD1 exhibited ATP hydrolytic activities in vitro with a half-maximal rate at an ATP concentration of 1.25 mm. Point mutations on active site residues abolished its ATPase activity. ADP is both a product and a strong inhibitor of the reaction. ADP-binding form of mcSDK1 greatly reduced its catalytic activity. The mcSKD1 protein accumulated ubiquitously in both vegetative and reproductive parts of plants. Highest accumulation was observed in cells actively engaging in the secretory processes, such as bladder cells of leaf epidermis. Membrane fractionation and double-labeling immunofluorescence showed the predominant localization of mcSKD1 in the endoplasmic reticulum-Golgi network. Immunoelectron microscopy identified the formation of mcSKD1 proteins into small aggregates in the cytosol and associated with membrane continuum within the endomembrane compartments. These results indicated that this ATPase participates in the endoplasmic reticulum-Golgi mediated protein sorting machinery for both housekeeping function and compartmentalization of excess Na+ under high salinity. PMID:16581876

Milrinone and thyroid hormone stimulate myocardial membrane Ca2+-ATPase activity and share structural homologies.

PubMed Central

Mylotte, K M; Cody, V; Davis, P J; Davis, F B; Blas, S D; Schoenl, M

1985-01-01

We have recently shown that thyroid hormone in physiological concentrations stimulates sarcolemma-enriched rabbit-myocardial-membrane Ca2+-ATPase in vitro. In this study, milrinone [2-methyl-5-cyano-(3,4'-bipyridin)-6(1H)-one], a cardiac inotropic agent, was thyromimetic in the same system. At clinically achievable concentrations (50-500 nM), milrinone significantly stimulated membrane Ca2+-ATPase in vitro. This action was antagonized by W-7 [N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide], an agent that also blocks thyroid hormone action on the Ca2+-ATPase, at concentrations as low as 5 microM. Progressive additions of milrinone to membranes incubated with a fixed concentration of thyroxine (0.10 nM) or triiodothyronine resulted in a progressive obliteration of the thyroid hormone effect on Ca2+-ATPase. Amrinone [5-amino-(3,4'-bipyridin)-6(1H)-one], the parent bipyridine of milrinone, had no effect on myocardial Ca2+-ATPase activity. X-ray crystallographic analysis of milrinone and amrinone revealed structural homologies between the phenolic ring of thyroxine and the substituted ring of milrinone, whereas amrinone did not share these homologies. The mechanism(s) of the inotropic actions of thyroxine and of milrinone is not clearly understood, but these observations implicate Ca2+-ATPase, a calcium pump-associated enzyme, as one mediator of the effects on the heart of these two compounds. PMID:2933747

Regulated Assembly of Vacuolar ATPase Is Increased during Cluster Disruption-induced Maturation of Dendritic Cells through a Phosphatidylinositol 3-Kinase/mTOR-dependent Pathway*

PubMed Central

Liberman, Rachel; Bond, Sarah; Shainheit, Mara G.; Stadecker, Miguel J.; Forgac, Michael

2014-01-01

The vacuolar (H+)-ATPases (V-ATPases) are ATP-driven proton pumps composed of a peripheral V1 domain and a membrane-embedded V0 domain. Regulated assembly of V1 and V0 represents an important regulatory mechanism for controlling V-ATPase activity in vivo. Previous work has shown that V-ATPase assembly increases during maturation of bone marrow-derived dendritic cells induced by activation of Toll-like receptors. This increased assembly is essential for antigen processing, which is dependent upon an acidic lysosomal pH. Cluster disruption of dendritic cells induces a semi-mature phenotype associated with immune tolerance. Thus, semi-mature dendritic cells are able to process and present self-peptides to suppress autoimmune responses. We have investigated V-ATPase assembly in bone marrow-derived, murine dendritic cells and observed an increase in assembly following cluster disruption. This increased assembly is not dependent upon new protein synthesis and is associated with an increase in concanamycin A-sensitive proton transport in FITC-loaded lysosomes. Inhibition of phosphatidylinositol 3-kinase with wortmannin or mTORC1 with rapamycin effectively inhibits the increased assembly observed upon cluster disruption. These results suggest that the phosphatidylinositol 3-kinase/mTOR pathway is involved in controlling V-ATPase assembly during dendritic cell maturation. PMID:24273170

The H,K-ATPase beta-subunit can act as a surrogate for the beta-subunit of Na,K-pumps.

PubMed

Horisberger, J D; Jaunin, P; Reuben, M A; Lasater, L S; Chow, D C; Forte, J G; Sachs, G; Rossier, B C; Geering, K

1991-10-15

Na,K-ATPase and H,K-ATPase are the only members of the P-type ATPases in which a glycosylated beta-subunit is part of the purified active enzyme. In this study, we have followed the synthesis and the posttranslational processing of the beta-subunit of H,K-ATPase (beta HK) in Xenopus oocytes injected with beta HK cRNA and have tested whether it can act as a surrogate for the beta-subunit of Na,K-ATPase (beta NaK) to support the functional expression of Na,K-pumps. In Xenopus oocytes, beta HK is processed from an Endo H-sensitive 51-kDa coreglycosylated form to an Endo H-resistant 71-kDa fully glycosylated form. Similar to beta NaK, beta HK can stabilize and increase the trypsin resistance of alpha-subunits of Na,K-ATPase (alpha NaK). Finally, expression of beta HK together with alpha NaK leads to an increased number of ouabain binding sites at the plasma membrane accompanied by an increased Rb+ uptake and Na,K-pump current. Our data suggest that beta HK, similar to beta NaK, can assemble to alpha NaK, support the structural maturation and the intracellular transport of catalytic alpha NaK, and ultimately form active alpha NaK-beta HK complexes with Na,K-pump transport properties.

A Non-Competitive Inhibitor of VCP/p97 and VPS4 Reveals Conserved Allosteric Circuits in Type I and II AAA ATPases.

PubMed

Pöhler, Robert; Krahn, Jan H; van den Boom, Johannes; Dobrynin, Grzegorz; Kaschani, Farnusch; Eggenweiler, Hans-Michael; Zenke, Frank T; Kaiser, Markus; Meyer, Hemmo

2018-02-05

AAA ATPases have pivotal functions in diverse cellular processes essential for survival and proliferation. Revealing strategies for chemical inhibition of this class of enzymes is therefore of great interest for the development of novel chemotherapies or chemical tools. Here, we characterize the compound MSC1094308 as a reversible, allosteric inhibitor of the type II AAA ATPase human ubiquitin-directed unfoldase (VCP)/p97 and the type I AAA ATPase VPS4B. Subsequent proteomic, genetic and biochemical studies indicate that MSC1094308 binds to a previously characterized drugable hotspot of p97, thereby inhibiting the D2 ATPase activity. Our results furthermore indicate that a similar allosteric site exists in VPS4B, suggesting conserved allosteric circuits and drugable sites in both type I and II AAA ATPases. Our results may thus guide future chemical tool and drug discovery efforts for the biomedically relevant AAA ATPases. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Vascular activation of K+ channels and Na+-K+ ATPase activity of estrogen-deficient female rats.

PubMed

Ribeiro Junior, Rogério Faustino; Fiorim, Jonaina; Marques, Vinicius Bermond; de Sousa Ronconi, Karoline; Botelho, Tatiani; Grando, Marcella D; Bendhack, Lusiane M; Vassallo, Dalton Valentim; Stefanon, Ivanita

2017-12-01

The goal of the present study was to evaluate vascular potassium channels and Na + -K + -ATPase activity in estrogen deficient female rats. Female rats that underwent ovariectomy were assigned to receive daily treatment with placebo (OVX) or estrogen replacement (OVX+E2, 1mg/kg, once a week, i.m.). Aortic rings were used to examine the involvement of K + channels and Na + -K + -ATPase in vascular reactivity. Acetylcholine (ACh)-induced relaxation was analyzed in the presence of L-NAME (100μM) and K + channels blockers: tetraethylammonium (TEA, 5mM), 4-aminopyridine (4-AP, 5mM), iberiotoxin (IbTX, 30nM), apamin (0.5mM), charybdotoxin (ChTX, 0.1mM) and iberiotoxin plus apamin. When aortic rings were pre-contracted with KCl (60mM) or pre-incubated with TEA (5mM), 4-aminopyridine (4-AP, 5mM) and iberiotoxin (IbTX, 30nM) plus apamin (0.5μM), the ACh-induced relaxation was less effective in the ovariectomized group. Additionally, 4-AP and IbTX decreased the relaxation by sodium nitroprusside in all groups but this reduction was greater in the ovariectomized group. Estrogen deficiency also increased aortic functional Na + -K + ATPase activity evaluated by K + -induced relaxation. L-NAME or endothelium removal were not able to block the increase in aortic functional Na + -K + ATPase activity, however, TEA (5mM) restored this increase to the control level. We also found that estrogen deficiency increased superoxide anion production and reduced nitric oxide release in aortic ring from ovariectomized animals. In summary, our results emphasize that the process underlying ACh-induced relaxation is preserved in ovariectomized animals due to the activation of K + channels and increased Na + -K + ATPase activity. Copyright © 2017 Elsevier Inc. All rights reserved.

Effect of gamma-hydroxybutyric acid on tissue Na+,K- ATPase levels after experimental head trauma.

PubMed

Yosunkaya, A; Ustün, M E; Bariskaner, H; Tavlan, A; Gürbilek, M

2004-05-01

A failure of the Na(+),K(+)-ATPase activity (which is essential for ion flux across the cell membranes) occurs in many pathological conditions and may lead to cell dysfunction or even cell death. By altering the concentration of specific opioid peptides, gamma-hydroxybutyric acid (GHB) may change ion flux across cell membranes and produce the 'channel arrest' which we assumed will inhibit the failure of Na+,K(+)-ATPase activity and therefore lead to energy conservation and cell protection. Therefore we planned this study to see the effects of GHB at two different doses on Na(+),K(+)-ATPase activity in an experimental head trauma model. Forty New Zealand rabbits were divided equally into four groups: group I was the sham-operated group, group II (untreated group), group III received head trauma and intravenous (i.v.) 500 mg/kg GHB and group IV received head trauma and i.v. 50 mg/kg GHB. Head trauma was delivered by performing a craniectomy over the right hemisphere and dropping a weight of 10 g from a height of 80 cm. The non-traumatized (left) side was named as 'a' and the traumatized (right) side as 'b'. One hour after the trauma in groups II and III and craniotomy in group I, brain cortices were resected from both sides and in group I only from the right side was the tissue Na-K-ATPase activity determined. The mean +/- SD of Na(+),K(+)-ATPase levels of each group are as follows: group I - 5.97 +/- 0.55; group IIa - 3.90 +/- 1.08; group IIb - 3.58 +/- 0.90; group IIIa - 5.53 +/- 0.60; group IIIb - 5.33 +/- 0.88; group IVa - 5.05 +/- 0.72; group IVb - 4.93 +/- 0.67. The Na(+),K(+)-ATPase levels of group IIa, IIb, IVa and IVb were significantly different from group S (P < 0.05). There were also significant differences between group IIa and groups IIIa and IVa; group IIb and groups IIIb and IVb (P < 0.05). We conclude that GHB is effective in suppressing the decrease in Na(+),K(+)-ATPase levels in brain tissue at two different dose schedules after head trauma.

The V-ATPase is expressed in the choroid plexus and mediates cAMP-induced intracellular pH alterations.

PubMed

Christensen, Henriette L; Păunescu, Teodor G; Matchkov, Vladimir; Barbuskaite, Dagne; Brown, Dennis; Damkier, Helle H; Praetorius, Jeppe

2017-01-01

The cerebrospinal fluid (CSF) pH influences brain interstitial pH and, therefore, brain function. We hypothesized that the choroid plexus epithelium (CPE) expresses the vacuolar H + -ATPase (V-ATPase) as an acid extrusion mechanism in the luminal membrane to counteract detrimental elevations in CSF pH. The expression of mRNA corresponding to several V-ATPase subunits was demonstrated by RT-PCR analysis of CPE cells (CPECs) isolated by fluorescence-activated cell sorting. Immunofluorescence and electron microscopy localized the V-ATPase primarily in intracellular vesicles with only a minor fraction in the luminal microvillus area. The vesicles did not translocate to the luminal membrane in two in vivo models of hypocapnia-induced alkalosis. The Na + -independent intracellular pH (pH i ) recovery from acidification was studied in freshly isolated clusters of CPECs. At extracellular pH (pH o ) 7.4, the cells failed to display significant concanamycin A-sensitive pH i recovery (i.e., V-ATPase activity). The recovery rate in the absence of Na + amounted to <10% of the pH i recovery rate observed in the presence of Na + Recovery of pH i was faster at pH o 7.8 and was abolished at pH o 7.0. The concanamycin A-sensitive pH i recovery was stimulated by cAMP at pH 7.4 in vitro, but intraventricular infusion of the membrane-permeant cAMP analog 8-CPT-cAMP did not result in trafficking of the V-ATPase. In conclusion, we find evidence for the expression of a minor fraction of V-ATPase in the luminal membrane of CPECs. This fraction does not contribute to enhanced acid extrusion at high extracellular pH, but seems to be activated by cAMP in a trafficking-independent manner. © 2017 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of The Physiological Society and the American Physiological Society.

AAA-ATPases in Protein Degradation

PubMed Central

Yedidi, Ravikiran S.; Wendler, Petra; Enenkel, Cordula

2017-01-01

Proteolytic machineries containing multisubunit protease complexes and AAA-ATPases play a key role in protein quality control and the regulation of protein homeostasis. In these protein degradation machineries, the proteolytically active sites are formed by either threonines or serines which are buried inside interior cavities of cylinder-shaped complexes. In eukaryotic cells, the proteasome is the most prominent protease complex harboring AAA-ATPases. To degrade protein substrates, the gates of the axial entry ports of the protease need to be open. Gate opening is accomplished by AAA-ATPases, which form a hexameric ring flanking the entry ports of the protease. Protein substrates with unstructured domains can loop into the entry ports without the assistance of AAA-ATPases. However, folded proteins require the action of AAA-ATPases to unveil an unstructured terminus or domain. Cycles of ATP binding/hydrolysis fuel the unfolding of protein substrates which are gripped by loops lining up the central pore of the AAA-ATPase ring. The AAA-ATPases pull on the unfolded polypeptide chain for translocation into the proteolytic cavity of the protease. Conformational changes within the AAA-ATPase ring and the adjacent protease chamber create a peristaltic movement for substrate degradation. The review focuses on new technologies toward the understanding of the function and structure of AAA-ATPases to achieve substrate recognition, unfolding and translocation into proteasomes in yeast and mammalian cells and into proteasome-equivalent proteases in bacteria and archaea. PMID:28676851

AAA-ATPases in Protein Degradation.

PubMed

Yedidi, Ravikiran S; Wendler, Petra; Enenkel, Cordula

2017-01-01

Proteolytic machineries containing multisubunit protease complexes and AAA-ATPases play a key role in protein quality control and the regulation of protein homeostasis. In these protein degradation machineries, the proteolytically active sites are formed by either threonines or serines which are buried inside interior cavities of cylinder-shaped complexes. In eukaryotic cells, the proteasome is the most prominent protease complex harboring AAA-ATPases. To degrade protein substrates, the gates of the axial entry ports of the protease need to be open. Gate opening is accomplished by AAA-ATPases, which form a hexameric ring flanking the entry ports of the protease. Protein substrates with unstructured domains can loop into the entry ports without the assistance of AAA-ATPases. However, folded proteins require the action of AAA-ATPases to unveil an unstructured terminus or domain. Cycles of ATP binding/hydrolysis fuel the unfolding of protein substrates which are gripped by loops lining up the central pore of the AAA-ATPase ring. The AAA-ATPases pull on the unfolded polypeptide chain for translocation into the proteolytic cavity of the protease. Conformational changes within the AAA-ATPase ring and the adjacent protease chamber create a peristaltic movement for substrate degradation. The review focuses on new technologies toward the understanding of the function and structure of AAA-ATPases to achieve substrate recognition, unfolding and translocation into proteasomes in yeast and mammalian cells and into proteasome-equivalent proteases in bacteria and archaea.

Modification of plasma membrane proton pumps in cucumber roots as an adaptation mechanism to salt stress.

PubMed

Janicka-Russak, Małgorzata; Kabała, Katarzyna; Wdowikowska, Anna; Kłobus, Grażyna

2013-07-01

The effect of salt stress (50mM NaCl) on modification of plasma membrane (PM) H(+)-ATPase (EC 3.6.3.14) activity in cucumber roots was studied. Plants were grown under salt stress for 1, 3 or 6 days. In salt-stressed plants, weak stimulation of ATP hydrolytic activity of PM H(+)-ATPase and significant stimulation of proton transport through the plasma membrane were observed. The H(+)/ATP coupling ratio in the plasma membrane of plants subjected to salt stress significantly increased. The greatest stimulation of PM H(+)-ATPase was in 6-day stressed plants. Increased H2O2 accumulation under salt stress conditions in cucumber roots was also observed, with the greatest accumulation observed in 6-day stressed plants. Additionally, during the sixth day of salinity, there appeared heat shock proteins (HSPs) 17.7 and 101, suggesting that repair processes and adaptation to stress occurred in plants. Under salt stress conditions, fast post-translational modifications took place. Protein blot analysis with antibody against phosphothreonine and 14-3-3 proteins showed that, under salinity, the level of those elements increased. Additionally, under salt stress, activity changes of PM H(+)-ATPase can partly result from changes in the pattern of expression of PM H(+)-ATPase genes. In cucumber seedlings, there was increased expression of CsHA10 under salt stress and the transcript of a new PM H(+)-ATPase gene isoform, CsHA1, also appeared. Accumulation of the CsHA1 transcript was induced by NaCl exposure, and was not expressed at detectable levels in roots of control plants. The appearance of a new PM H(+)-ATPase transcript, in addition to the increase in enzyme activity, indicates the important role of the enzyme in maintaining ion homeostasis in plants under salt stress. Copyright © 2013 Elsevier GmbH. All rights reserved.

Osmotic and ionic regulation in shore crabs Carcinus maenas inhabiting a tidal estuary

NASA Astrophysics Data System (ADS)

Winkler, A.; Siebers, D.; Becker, W.

1988-03-01

Shore crabs Carcinus maenas were exposed to salinities fluctuating according to the natural tidal rhythm. To this end they were maintained in net cages positioned in the estuarine waters of the river Elbe. The cages were lifted every hour, and between 8 12 specimens were analyzed for hemolymph concentrations of Na, K, Ca, Mg, and osmolality. The results obtained were compared with the respective data measured in external brackish water. In addition, the specific activity of Na-K-ATPase in a posterior gill was determined. Hemolymph Na and Mg as well as branchial Na-K-ATPase were also determined in crabs collected in the North Sea and the Baltic. The results show that in C. maenas living in salinities fluctuating with the tides by approx. 15‰ S, Na, K and Ca were hyperregulated, and Mg was effectively hyporegulated. The concentrations of all hemolymph ions and the activity of the Na-K-ATPase were kept constant over the whole tidal cycle. In Baltic crabs, Na was effectively hyperregulated and gill Na-K-ATPase was significantly elevated by a factor of ca 2 when compared with North Sea crabs. It is suggested that long-term hyperregulation of Na in constant salinities results from an increased number of Na-K-ATPase molecules which may change by synthesis or degradation following salinity stress. Constant hemolymph levels of hyperregulated Na in crabs inhabiting fluctuating brackish water are accomplished by activation of existing Na-K-ATPase by low Na and inhibition by higher ambient concentrations.

Exercise effects on the size and metabolic properties of soleus fibers in hindlimb-suspended rats

NASA Technical Reports Server (NTRS)

Graham, Scot C.; Roy, Roland R.; West, Steve P.; Thomason, Don; Baldwin, Kenneth M.

1989-01-01

The effects of four-week-long hind-limb suspension (HS) of rats on the size the soleus muscle fibers and the activity of succinate dehydrogenase (SDH) in dark and light ATPase fibers were investigated together with the efficacy of an endurance exercise (EX) program (daily treadmill exercise for 1.5 h/day at 20 m/min and a 30-percent grade) in ameliorating HS-induced changes. It was found that, in comparison to age-matched controls, the soleus wet weight decreased by 69 and 30 percent in HS and HS-EX rats, respectively, and the percent of dark ATPase fibers increased from 10 percent in controls to 19 and 17 percent, respectively. The values of the integrated fiber activity (activity/min times muscle area) showed a net loss of SDH in both the light and dark ATPase fibers of HS rats, but only in the light ATPase fibers of the HS-EX rats, indicating that exercise ameliorated but did not prevent the muscle fiber atrophy induced by HS.

Drug action of benzocaine on the sarcoplasmic reticulum Ca-ATPase from fast-twitch skeletal muscle.

PubMed

Di Croce, D; Trinks, P W; Grifo, M B; Takara, D; Sánchez, G A

2015-11-01

The effect of the local anesthetic benzocaine on sarcoplasmic reticulum membranes isolated from fast-twitch muscles was tested. The effects on Ca-ATPase activity, calcium binding and uptake, phosphoenzyme accumulation and decomposition were assessed using radioisotopic methods. The calcium binding to the Ca-ATPase was noncompetitively inhibited, and the enzymatic activity decreased in a concentration-dependent manner (IC50 47.1 mM). The inhibition of the activity depended on the presence of the calcium ionophore calcimycin and the membrane protein concentration. The pre-exposure of the membranes to benzocaine enhanced the enzymatic activity in the absence of calcimycin, supporting the benzocaine permeabilizing effect, which was prevented by calcium. Benzocaine also interfered with the calcium transport capability by decreasing the maximal uptake (IC50 40.3 mM) without modification of the calcium affinity for the ATPase. It inhibited the phosphorylation of the enzyme, and at high benzocaine concentration, the dephosphorylation step became rate-limiting as suggested by the biphasic profile of phosphoenzyme accumulation at different benzocaine concentrations. The data reported in this paper revealed a complex pattern of inhibition involving two sites for interaction with low and high benzocaine concentrations. It is concluded that benzocaine not only exerts an indirect action on the membrane permeability to calcium but also affects key steps of the Ca-ATPase enzymatic cycle.

Effect of LED photobiomodulation on fluorescent light induced changes in cellular ATPases and Cytochrome c oxidase activity in Wistar rat.

PubMed

A, Ahamed Basha; C, Mathangi D; R, Shyamala

2016-12-01

Fluorescent light exposure at night alters cellular enzyme activities resulting in health defects. Studies have demonstrated that light emitting diode photobiomodulation enhances cellular enzyme activities. The objectives of this study are to evaluate the effects of fluorescent light induced changes in cellular enzymes and to assess the protective role of pre exposure to 670 nm LED in rat model. Male Wistar albino rats were divided into 10 groups of 6 animals each based on duration of exposure (1, 15, and 30 days) and exposure regimen (cage control, exposure to fluorescent light [1800 lx], LED preexposure followed by fluorescent light exposure and only LED exposure). Na + -K + ATPase, Ca 2+ ATPase, and cytochrome c oxidase of the brain, heart, kidney, liver, and skeletal muscle were assayed. Animals of the fluorescent light exposure group showed a significant reduction in Na + -K + ATPase and Ca 2+ ATPase activities in 1 and 15 days and their increase in animals of 30-day group in most of the regions studied. Cytochrome c oxidase showed increase in their level at all the time points assessed in most of the tissues. LED light preexposure showed a significant enhancement in the degree of increase in the enzyme activities in almost all the tissues and at all the time points assessed. This study demonstrates the protective effect of 670 nm LED pre exposure on cellular enzymes against fluorescent light induced change.

N-Acetylcysteine-induced vasodilatation is modulated by KATP channels, Na+/K+-ATPase activity and intracellular calcium concentration: An in vitro study.

PubMed

Vezir, Özden; Çömelekoğlu, Ülkü; Sucu, Nehir; Yalın, Ali Erdinç; Yılmaz, Şakir Necat; Yalın, Serap; Söğüt, Fatma; Yaman, Selma; Kibar, Kezban; Akkapulu, Merih; Koç, Meryem İlkay; Seçer, Didem

2017-08-01

In this study, we aimed to investigate the role of ATP-sensitive potassium (K ATP ) channel, Na + /K + -ATPase activity, and intracellular calcium levels on the vasodilatory effect of N-acetylcysteine (NAC) in thoracic aorta by using electrophysiological and molecular techniques. Rat thoracic aorta ring preparations and cultured thoracic aorta cells were divided into four groups as control, 2mM NAC, 5mM NAC, and 10mM NAC. Thoracic aorta rings were isolated from rats for measurements of relaxation responses and Na + /K + -ATPase activity. In the cultured thoracic aorta cells, we measured the currents of K ATP channel, the concentration of intracellular calcium and mRNA expression level of K ATP channel subunits (KCNJ8, KCNJ11, ABCC8 and ABCC9). The relaxation rate significantly increased in all NAC groups compared to control. Similarly, Na + /K + - ATPase activity also significantly decreased in NAC groups. Outward K ATP channel current significantly increased in all NAC groups compared to the control group. Intracellular calcium concentration decreased significantly in all groups with compared control. mRNA expression level of ABCC8 subunit significantly increased in all NAC groups compared to the control group. Pearson correlation analysis showed that relaxation rate was significantly associated with K ATP current, intracellular calcium concentration, Na + /K + -ATPase activity and mRNA expression level of ABCC8 subunit. Our findings suggest that NAC relaxes vascular smooth muscle cells through a direct effect on K ATP channels, by increasing outward K+ flux, partly by increasing mRNA expression of K ATP subunit ABCC8, by decreasing in intracellular calcium and by decreasing in Na + /K + -ATPase activity. Copyright © 2017 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.

The H+/K+-ATPase inhibitory activities of Trametenolic acid B from Trametes lactinea (Berk.) Pat, and its effects on gastric cancer cells.

PubMed

Zhang, Qiaoyin; Huang, Nianyu; Wang, Junzhi; Luo, Huajun; He, Haibo; Ding, Mingruo; Deng, Wei-Qiao; Zou, Kun

2013-09-01

Trametenolic acid B (TAB), the bioactive component in the Trametes lactinea (Berk.) Pat, was reported to possess cytotoxic activities and thrombin inhibiting effects. This study was performed to investigate the effects of TAB on H(+)/K(+)-ATPase and gastric cancer. The H(+)/K(+)-ATPase inhibitory activity was determined by gastric parietal cells. Compared to the normal control group, TAB (10, 20, 40 and 80 μg/mL) inhibited the H(+)/K(+)-ATPase activity by 15.97, 16.96, 24.86 and 16.25%, respectively. In the study, 36 Kunming mice were randomly divided into six groups: control, model, TAB-L (TAB, 5 mg/kg/day, i.g.), TAB-M (TAB, 20 mg/kg/day, i.g.), TAB-H (TAB, 40 mg/kg/day, i.g.) and omeprazole (OL, 10 mg/kg/day, i.g.). All mice except the control group were administrated with anhydrous alcohol (5.0 mL/kg, i.g.) for induced gastric-ulcer 1h after the 5th day. At the same time, the control mice were given the same volume of physiological saline. After 4h, TAB was evaluated for H(+)/K(+)-ATPase inhibitory activities of ulcerative gaster, gastric ulcer index and ulcer inhibition. In vitro, the anti-proliferation effect of TAB to gastric cancer cell (HGC-27) in acid environment was detected by MTT, and the apoptosis morphological changes were also observed by Hoechst 33258 dye assay. The results indicated that TAB inhibited moderately H(+)/K(+)-ATPase activity in vitro. Compared to the model group, TAB showed anti-ulcer effects in gastric tissue with the dosages of 20 and 5 mg/kg in vivo. Apart from that, TAB could selectively inhibit gastric cancer cell viability and reduce cell apoptosis against HGC-27 cells at low doses in acid environment. Copyright © 2013 The Authors. Published by Elsevier B.V. All rights reserved.

LRRK2 phosphorylates pre-synaptic N-ethylmaleimide sensitive fusion (NSF) protein enhancing its ATPase activity and SNARE complex disassembling rate.

PubMed

Belluzzi, Elisa; Gonnelli, Adriano; Cirnaru, Maria-Daniela; Marte, Antonella; Plotegher, Nicoletta; Russo, Isabella; Civiero, Laura; Cogo, Susanna; Carrion, Maria Perèz; Franchin, Cinzia; Arrigoni, Giorgio; Beltramini, Mariano; Bubacco, Luigi; Onofri, Franco; Piccoli, Giovanni; Greggio, Elisa

2016-01-13

Lrrk2, a gene linked to Parkinson's disease, encodes a large scaffolding protein with kinase and GTPase activities implicated in vesicle and cytoskeletal-related processes. At the presynaptic site, LRRK2 associates with synaptic vesicles through interaction with a panel of presynaptic proteins. Here, we show that LRRK2 kinase activity influences the dynamics of synaptic vesicle fusion. We therefore investigated whether LRRK2 phosphorylates component(s) of the exo/endocytosis machinery. We have previously observed that LRRK2 interacts with NSF, a hexameric AAA+ ATPase that couples ATP hydrolysis to the disassembling of SNARE proteins allowing them to enter another fusion cycle during synaptic exocytosis. Here, we demonstrate that NSF is a substrate of LRRK2 kinase activity. LRRK2 phosphorylates full-length NSF at threonine 645 in the ATP binding pocket of D2 domain. Functionally, NSF phosphorylated by LRRK2 displays enhanced ATPase activity and increased rate of SNARE complex disassembling. Substitution of threonine 645 with alanine abrogates LRRK2-mediated increased ATPase activity. Given that the most common Parkinson's disease LRRK2 G2019S mutation displays increased kinase activity, our results suggest that mutant LRRK2 may impair synaptic vesicle dynamics via aberrant phosphorylation of NSF.

Specific Activation of the Plant P-type Plasma Membrane H+-ATPase by Lysophospholipids Depends on the Autoinhibitory N- and C-terminal Domains.

PubMed

Wielandt, Alex Green; Pedersen, Jesper Torbøl; Falhof, Janus; Kemmer, Gerdi Christine; Lund, Anette; Ekberg, Kira; Fuglsang, Anja Thoe; Pomorski, Thomas Günther; Buch-Pedersen, Morten Jeppe; Palmgren, Michael

2015-06-26

Eukaryotic P-type plasma membrane H(+)-ATPases are primary active transport systems that are regulated at the post-translation level by cis-acting autoinhibitory domains, which can be relieved by protein kinase-mediated phosphorylation or binding of specific lipid species. Here we show that lysophospholipids specifically activate a plant plasma membrane H(+)-ATPase (Arabidopsis thaliana AHA2) by a mechanism that involves both cytoplasmic terminal domains of AHA2, whereas they have no effect on the fungal counterpart (Saccharomyces cerevisiae Pma1p). The activation was dependent on the glycerol backbone of the lysophospholipid and increased with acyl chain length, whereas the headgroup had little effect on activation. Activation of the plant pump by lysophospholipids did not involve the penultimate residue, Thr-947, which is known to be phosphorylated as part of a binding site for activating 14-3-3 protein, but was critically dependent on a single autoinhibitory residue (Leu-919) upstream of the C-terminal cytoplasmic domain in AHA2. A corresponding residue is absent in the fungal counterpart. These data indicate that plant plasma membrane H(+)-ATPases evolved as specific receptors for lysophospholipids and support the hypothesis that lysophospholipids are important plant signaling molecules. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

A comparison of the effects of substance P and shorter analogues on the synaptosomal ATPases activities in the rat brain.

PubMed

Lachowicz, L; Janiszewska, G

1987-01-01

The influence in vitro of SP and C-terminal fragments of analogues SP(5-11) (pyroGlu5, Tyr8); SP(6-11) (pyroGlu6, Tyr8); SP(6-11) (pyroGlu6, D-Phe7); SP(6-11) (pyroGlu6, D-Phe8) on the (Ca, Mg) and (Na, K) ATPases activities from synaptosomal membranes of cerebral cortex and hippocampus of rat brain were compared. The data obtained in this study indicate the following: 1. Substance P stimulates the activities of (Na, K) and (Ca, Mg) ATPases more effectively in synaptosomal membranes from hippocampus than cerebral cortex. 2. Heptapeptide SP(5-11) (pyroGlu5, Tyr8) causes a more distinct increase of (Ca, Mg) ATPase activity in cortical synaptosomal membranes than SP does. 3. The change of L-Phe conformation to D in position 7 in hexapeptide induces reduction of enzymes activities in hippocampus. 4. Especially important for the maintenance of biological activity of drugs is the replacement of Gln5 with pyroGlu6 and conformation of Phe residues. 5. SP and shorter analogues of fragments SP C-terminal SP regulate the active cation transport in synaptosomal membranes of cerebral cortex and hippocampus.

Arrestins and Spinophilin Competitively Regulate Na+,K+-ATPase Trafficking through Association with a Large Cytoplasmic Loop of the Na+,K+-ATPase

PubMed Central

Kimura, Tohru; Allen, Patrick B.; Nairn, Angus C.

2007-01-01

The activity and trafficking of the Na+,K+-ATPase are regulated by several hormones, including dopamine, vasopressin, and adrenergic hormones through the action of G-protein–coupled receptors (GPCRs). Arrestins, GPCR kinases (GRKs), 14-3-3 proteins, and spinophilin interact with GPCRs and modulate the duration and magnitude of receptor signaling. We have found that arrestin 2 and 3, GRK 2 and 3, 14-3-3 ε, and spinophilin directly associate with the Na+,K+-ATPase and that the associations with arrestins, GRKs, or 14-3-3 ε are blocked in the presence of spinophilin. In COS cells that overexpressed arrestin, the Na+,K+-ATPase was redistributed to intracellular compartments. This effect was not seen in mock-transfected cells or in cells expressing spinophilin. Furthermore, expression of spinophilin appeared to slow, whereas overexpression of β-arrestins accelerated internalization of the Na+,K+-ATPase endocytosis. We also find that GRKs phosphorylate the Na+,K+-ATPase in vitro on its large cytoplasmic loop. Taken together, it appears that association with arrestins, GRKs, 14-3-3 ε, and spinophilin may be important modulators of Na+,K+-ATPase trafficking. PMID:17804821

THE LOCALIZATION OF MYOFIBRILLAR ATPASE ACTIVITY IN THE FLIGHT MUSCLES OF THE BLOWFLY, CALLIPHORA ERYTHROCEPHALA

PubMed Central

Tice, Lois W.; Smith, David S.

1965-01-01

The distribution of ATPase activity in the asynchronous flight muscles of Calliphora erythrocephala (Diptera) was studied at a fine structural level, using preparations of teased fibers, both unfixed and after brief fixation in hydroxyadipaldehyde, incubated in a medium for the histochemical demonstration of myosin or actomyosin ATPase. In relaxed fibrils, activity was found confined to the A bands and was absent from the H zones as well as from the Z and I band regions. At high magnification, deposits of final product, lead phosphate, appeared primarily related to the thick filaments, or to short lateral extensions from them. Evidence was gathered which indicated that this enzyme activity was that of a triphosphatase which did not act on dinucleoside or non-nucleoside substrates. PMID:4221034

[Preparation and application of monoclonal antibodies against DR region of Na+-K+-ATPase α1 subunit].

PubMed

Yan, Xiaofei; Wu, Litao; DU, Xiaojuan; Li, Jing; Zhang, Fujun; Han, Yan; Lyu, Shemin; Li, Dongmin

2016-12-01

Objective To prepare monoclonal antibodies against DR region (897DVEDSYGQQWTYEQR911) of Na + -K + -ATPase α1 subunit and identify their properties. Methods BALB/c mice were immunized with DR-keyholelimpet hemocyanin (KLH). Splenocytes from the immunized mice were collected and subsequently fused with SP2/0 mouse myeloma cells. Positive hybridoma clones were obtained after cell fusion and selection. ELISA was used to detect DR antibody titer in the cell supernatants. DR region-specific monoclonal antibodies were analyzed by dot blotting, Western blotting and immunofluorescence assay. Na + -K + -ATPase activity was detected by SensoLyte R FDP Protein Phosphatase Assay Kit and the protective effect of the monoclonal antibody against high glucose-induced cell injury was assessed in H9c2 cells. Results Three hybridoma cell lines which secreted stable DR monoclonal antibody were obtained. The strongest positive cell line, named DRm217, was selected to prepare ascites. Dot blotting, Western blotting and immunofluorescence assay showed that DRm217 recognized specially DR region of Na + -K + -ATPase and bound on H9c2 cell membranes. DRm217 stimulated Na + -K + -ATPase activity and alleviated high glucose-induced H9c2 cells injury. Conclusion The monoclonal antibodies against DR region of Na + -K + -ATPase α1 subunit is prepared.

Effects of PKA phosphorylation on the conformation of the Na,K-ATPase regulatory protein FXYD1

PubMed Central

Teriete, Peter; Thai, Khang; Choi, Jungyuen; Marassi, Francesca M.

2009-01-01

FXYD1 (phospholemman) is a member of an evolutionarily conserved family of membrane proteins that regulate the function of the Na,K-ATPase enzyme complex in specific tissues and specific physiological states. In heart and skeletal muscle sarcolemma, FXYD1 is also the principal substrate of hormone-regulated phosphorylation by c-AMP dependent protein kinase A and by protein kinase C, which phosphorylate the protein at conserved Ser residues in its cytoplasmic domain, altering its Na,K-ATPase regulatory activity. FXYD1 adopts an L-shaped α-helical structure with the transmembrane helix loosely connected to a cytoplasmic amphipathic helix that rests on the membrane surface. In this paper we describe NMR experiments showing that neither PKA phosphorylation at Ser68 nor the physiologically relevant phosphorylation mimicking mutation Ser68Asp induces major changes in the protein conformation. The results, viewed in light of a model of FXYD1 associated with the Na,K-ATPase α and β subunits, indicate that the effects of phosphorylation on the Na,K-ATPase regulatory activity of FXYD1 could be due primarily to changes in electrostatic potential near the membrane surface and near the Na+/K+ ion binding site of the Na,K-ATPase α subunit. PMID:19761758

Tributyltin (TBT) inhibition of oligomycin-sensitive Mg-ATPase activity in mussel mitochondria.

PubMed

Pagliarani, Alessandra; Bandiera, Patrizia; Ventrella, Vittoria; Trombetti, Fabiana; Pirini, Maurizio; Nesci, Salvatore; Borgatti, Anna Rosa

2008-06-01

Tributyltin (TBT), one of the most toxic lipophilic aquatic pollutants, can be efficiently incorporated from sea water and sediments by filter-feeding molluscs. As far as we are aware TBT effects on the mitochondrial oligomycin-sensitive Mg-ATPase, the enzymatic core of energy production and a known target of TBT toxicity in mammals, have not been yet investigated in molluscs; thus the hydrolytic capability of the mitochondrial complex in the presence of micromolar concentrations of TBT was assayed in the mussel Mytilus galloprovincialis. Gill and mantle ATPase activities were progressively depressed by increasing TBT doses up to a maximal inhibition (82% in the gills and 74% in the mantle) at 0.62 microM TBT. Non-cooperative inhibition kinetics (n(H) approximately -1) and a non-competitive mechanism with respect to ATP substrate were pointed out. The mitochondrial Mg-ATPase susceptivity to TBT in the marine mussel was consistent with the formation of a TBT-Mg-ATPase complex, apparently more stable in the gills than in the mantle. The complex shape of the dose-response curve and the partial release of Mg-ATPase inhibition within the 0.6-34.4 microM TBT range suggest multiple interactions of TBT with the enzyme complex putatively related to its molecular mechanism of toxicity.

Perflurooctanoic Acid Induces Developmental Cardiotoxicity in ...

EPA Pesticide Factsheets

Perfluorooctanoic acid (PFOA) is a widespread environmental contaminant that is detectable in serum of the general U.S. population. PFOA is a known developmental toxicant that induces mortality in mammalian embryos and is thought to induce toxicity via interaction with the peroxisome proliferator activated receptor alpha (PPAR_). As the cardiovascular system is crucial for embryonic survival, PFOA-induced effects on the heart may partially explain embryonic mortality. To assess impacts of PFOA exposure on the developing heart in an avian model, we used histopathology and immunohistochemical staining for myosin to assess morphological alterations in 19-day-old chicken embryo hearts after PFOA exposure. Additionally, echocardiography and cardiac myofibril ATPase activity assays were used to assess functional alterations in 1-day-old hatchling chickens following developmental PFOA exposure. Overall thinning and thinning of a dense layer of myosin in the right ventricular wall were observed in PFOA-exposed chicken embryo hearts. Alteration of multiple cardiac structural and functional parameters, including left ventricular wall thickness, left ventricular volume, heart rate, stroke volume, and ejection fraction were detected with echocardiography in the exposed hatchling chickens. Assessment of ATPase activity indicated that the ratio of cardiac myofibril calcium-independent ATPase activity to calcium-dependent ATPase activity was not affected, which suggests that d

Relationship between Na+-dependent respiration and Na+ + K+-adenosine triphosphatase activity in the action of thyroid hormone on rat jejunal mucosa.

PubMed Central

Liberman, U A; Asano, Y; Lo, C S; Edelman, I S

1979-01-01

Administration of three successive doses of triiodothyronine (T3) (50 micrograms/100 g body wt), given on alternate days to thyroidectomized and euthyroid rats, stimulated oxygen consumption (QO2) and Na+ transport-dependent respiration (QO2 [5]) in the stripped jejunal mucosa, a preparation that consisted mostly of epithelial cells. The increase in QO2(t) accounted for 57% of the increment in QO2 in the transition from the hypothyroid to the euthyroid state and for 29% of the increment in the transition from the euthyroid to the hyperthyroid state. Administration of T3 to hypothyroid rats also increased the yield of epithelial cells. Injection of T3 into thyroidectomized and euthyroid rats increased the specific activity (at Vmax) of the (Na+ + K+)-dependent adenosine triphosphatase (NaK-ATPase) in jejunal crude membrane preparations. No significant change was recorded in the activity of Mg-ATPase in the same preparation. The ratio of QO2/NaK-ATPase and QO2(t)/NaK-ATPase in the various thyroid states remained constant, indicating proportionate increased in the respiratory and enzymatic indices. The effect of administration of T3 to thyroidectomized rats on the number of NaK-ATPase units (recovered in the crude membrane preparation) was estimated by: (a) Na+ + Mg++ + ATP-dependent binding of [3H]-ouabain to crude membrane fractions, and (b) the amount of the phosphorylated intermediate formed in the NaK-ATPase reaction from AT32P(gamma). Estimates were obtained of the maximal number of [3H]ouabain binding sites (Nm) and dissociation constants (Kd). Nm for [3H]ouabain and Nak-ATPase specific activity increased to about the same extent after T3 administration to thyroidectomized rats, with no change in the apparent Kd values. The amount of phosphorylated intermediate formed in jejunal crude membrane preparations also increased significantly. Thus, thyroid hormone administration may increase the number of active Na+pump sites in the plasma membrane. The apparent increase in the number of Na+ pump sites also correlated with the hormone dependent increases in QO2 and QO2(t). Images FIGURE 1 PMID:233567

Relationship between Na+-dependent respiration and Na+ + K+-adenosine triphosphatase activity in the action of thyroid hormone on rat jejunal mucosa.

PubMed

Liberman, U A; Asano, Y; Lo, C S; Edelman, I S

1979-07-01

Administration of three successive doses of triiodothyronine (T3) (50 micrograms/100 g body wt), given on alternate days to thyroidectomized and euthyroid rats, stimulated oxygen consumption (QO2) and Na+ transport-dependent respiration (QO2 [5]) in the stripped jejunal mucosa, a preparation that consisted mostly of epithelial cells. The increase in QO2(t) accounted for 57% of the increment in QO2 in the transition from the hypothyroid to the euthyroid state and for 29% of the increment in the transition from the euthyroid to the hyperthyroid state. Administration of T3 to hypothyroid rats also increased the yield of epithelial cells. Injection of T3 into thyroidectomized and euthyroid rats increased the specific activity (at Vmax) of the (Na+ + K+)-dependent adenosine triphosphatase (NaK-ATPase) in jejunal crude membrane preparations. No significant change was recorded in the activity of Mg-ATPase in the same preparation. The ratio of QO2/NaK-ATPase and QO2(t)/NaK-ATPase in the various thyroid states remained constant, indicating proportionate increased in the respiratory and enzymatic indices. The effect of administration of T3 to thyroidectomized rats on the number of NaK-ATPase units (recovered in the crude membrane preparation) was estimated by: (a) Na+ + Mg++ + ATP-dependent binding of [3H]-ouabain to crude membrane fractions, and (b) the amount of the phosphorylated intermediate formed in the NaK-ATPase reaction from AT32P(gamma). Estimates were obtained of the maximal number of [3H]ouabain binding sites (Nm) and dissociation constants (Kd). Nm for [3H]ouabain and Nak-ATPase specific activity increased to about the same extent after T3 administration to thyroidectomized rats, with no change in the apparent Kd values. The amount of phosphorylated intermediate formed in jejunal crude membrane preparations also increased significantly. Thus, thyroid hormone administration may increase the number of active Na+pump sites in the plasma membrane. The apparent increase in the number of Na+ pump sites also correlated with the hormone dependent increases in QO2 and QO2(t).

The hydrophilic domain of phospholamban inhibits the Ca2+ transport step of the Ca(2+)-ATPase.

PubMed Central

Hughes, G; East, J M; Lee, A G

1994-01-01

The peptide MEKVQYLTRSAIRRASTIEMPQQAR-Cys corresponding to residues 1-25 of phospholamban was found to inhibit the ATPase activity of skeletal muscle Ca(2+)-ATPase, but to have no effect on the Ca(2+)-dependence of its activity. The peptide was found to decrease the rate of the Ca2+ transport step (E1PCa2-->E2P) by a factor of 2.4. The rate of this same step was decreased by poly(L-Arg) by a factor of 2.2. The peptide shifted the E2-E1 equilibrium of the ATPase towards E1 by a factor of 4 due to stronger binding to the E1 than to the E2 conformation of the ATPase; dissociation constants for binding to E1 and E2 were estimated as 3 and 10 microM respectively. The peptide had no effect on the level of phosphorylation by Pi in the absence of Ca2+ or on the rate of phosphorylation by ATP in the presence of Ca2+. PMID:7980411

Engagement of Arginine Finger to ATP Triggers Large Conformational Changes in NtrC1 AAA+ ATPase for Remodeling Bacterial RNA Polymerase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chen, Baoyu; Sysoeva, Tatyana A.; Chowdhury, Saikat

The NtrC-like AAA+ ATPases control virulence and other important bacterial activities through delivering mechanical work to {sigma}54-RNA polymerase to activate transcription from {sigma}54-dependent genes. We report the first crystal structure for such an ATPase, NtrC1 of Aquifex aeolicus, in which the catalytic arginine engages the {gamma}-phosphate of ATP. Comparing the new structure with those previously known for apo and ADP-bound states supports a rigid-body displacement model that is consistent with large-scale conformational changes observed by low-resolution methods. First, the arginine finger induces rigid-body roll, extending surface loops above the plane of the ATPase ring to bind {sigma}54. Second, ATP hydrolysismore » permits Pi release and retraction of the arginine with a reversed roll, remodeling {sigma}54-RNAP. This model provides a fresh perspective on how ATPase subunits interact within the ring-ensemble to promote transcription, directing attention to structural changes on the arginine-finger side of an ATP-bound interface.« less

Residues of organochlorine pesticides in fish, crab and sediment from El Temsah Lake, Suez Canal, Egypt and their effect on mitochondrial ATPase of the New Zealand white rabbit.

PubMed

Ahmed, M T; Ismail, S M

1991-01-01

Residues of organochlorine pesticides were monitored in the muscles of Bolti fish Tilapia zillii, the crab Lupa pelagicus and sediment samples collected from El Temsah lake around Ismailia using gas liquid chromatography. The beta isomer of hexachlorocyclohexane (beta.HCH) was the most dominant compound detected in all samples, followed by P, P-DDE and P, P-DDT. Results showed the crab to contain higher concentrations of organochlorine in comparison to concentrations detected in fish muscles. The In-vitro effect of the residues extracted from fish, and crab on the mitochondrial brain and liver ATPase of the New Zealand white rabbit Orcytolagus cuniculus was also studied. Residues of organochlorine pesticides have induced activation in the ATPase enzyme system of both brain and liver. The mixtures of organochlorine residues of both fish and crab were able to activate liver ATPase more than brain ATPase. The present study was conducted to extrapolate possible effects incurred on man if consumed such food.

Molecular cloning of the plasma membrane H(+)-ATPase from Kluyveromyces lactis: a single nucleotide substitution in the gene confers ethidium bromide resistance and deficiency in K+ uptake.

PubMed Central

Miranda, M; Ramírez, J; Peña, A; Coria, R

1995-01-01

A Kluyveromyces lactis strain resistant to ethidium bromide and deficient in potassium uptake was isolated. Studies on the proton-pumping activity of the mutant strain showed that a decreased H(+)-ATPase specific activity was responsible for the observed phenotypes. The putative K. lactis PMA1 gene encoding the plasma membrane H(+)-ATPase was cloned by its ability to relieve the potassium transport defect of this mutant and by reversing its resistance to ethidium bromide. Its deduced amino acid sequence predicts a protein 899 residues long that is structurally colinear in its full length to H(+)-ATPases cloned from different yeasts, except for the presence of a variable N-terminal domain. By PCR-mediated amplification, we identified a transition from G to A that rendered the substitution of the fully conserved methionine at position 699 by isoleucine. We attribute to this amino acid change the low capacity of the mutant H(+)-ATPase to pump out protons. PMID:7730265

Gill area, permeability and Na+ ,K+ -ATPase activity as a function of size and salinity in the blue crab, Callinectes sapidus.

PubMed

Li, Tiandao; Roer, Robert; Vana, Matthew; Pate, Susan; Check, Jennifer

2006-03-01

Juvenile blue crabs, Callinectes sapidus, extensively utilize oligohaline and freshwater regions of the estuary. With a presumptively larger surface-area-to-body weight ratio, juvenile crabs could experience osmo- and ionoregulatory costs well in excess of that of adults. To test this hypothesis, crabs ranging over three orders of magnitude in body weight were acclimated to either sea water (1,000 mOsm) or dilute sea water (150 mOsm), and gill surface area, water and sodium permeabilities (calculated from the passive efflux of 3H2O and 22Na+), gill Na+, K+ -ATPase activity and expression were measured. Juveniles had a relatively larger gill surface area; weight-specific gill surface area decreased with body weight. Weight-specific water and sodium fluxes also decreased with weight, but not to the same extent as gill surface area; thus juveniles were able to decrease gill permeability slightly more than adults upon acclimation to dilute media. Crabs < 5 g in body weight had markedly higher activities of gill Na+ ,K+ -ATPase than crabs > 5 g in both posterior and anterior gills. Acclimation to dilute medium induced increased expression of Na+, K+ -ATPase and enzyme activity, but the increase was not as great in juveniles as in larger crabs. The increased weight-specific surface area for water gain and salt loss for small crabs in dilute media presents a challenge that is incompletely compensated by reduced permeability and increased affinity of gill Na+, K+ -ATPase for Na+. Juveniles maintain osmotic and ionic homeostasis by the expression and utilization of extremely high levels of gill Na+, K+ -ATPase, in posterior, as well as in anterior, gills. Copyright 2006 Wiley-Liss, Inc.

SH2 Ligand-Like Effects of Second Cytosolic Domain of Na/K-ATPase α1 Subunit on Src Kinase.

PubMed

Banerjee, Moumita; Duan, Qiming; Xie, Zijian

2015-01-01

Our previous studies have suggested that the α1 Na/K-ATPase interacts with Src to form a receptor complex. In vitro binding assays indicate an interaction between second cytosolic domain (CD2) of Na/K-ATPase α1 subunit and Src SH2 domain. Since SH2 domain targets Src to specific signaling complexes, we expressed CD2 as a cytosolic protein and studied whether it could act as a Src SH2 ligand in LLC-PK1 cells. Co-immunoprecipitation analyses indicated a direct binding of CD2 to Src, consistent with the in vitro binding data. Functionally, CD2 expression increased basal Src activity, suggesting a Src SH2 ligand-like property of CD2. Consistently, we found that CD2 expression attenuated several signaling pathways where Src plays an important role. For instance, although it increased surface expression of Na/K-ATPase, it decreased ouabain-induced activation of Src and ERK by blocking the formation of Na/K-ATPase/Src complex. Moreover, it also attenuated cell attachment-induced activation of Src/FAK. Consequently, CD2 delayed cell spreading, and inhibited cell proliferation. Furthermore, these effects appear to be Src-specific because CD2 expression had no effect on EGF-induced activation of EGF receptor and ERK. Hence, the new findings indicate the importance of Na/K-ATPase/Src interaction in ouabain-induced signal transduction, and support the proposition that the CD2 peptide may be utilized as a Src SH2 ligand capable of blocking Src-dependent signaling pathways via a different mechanism from a general Src kinase inhibitor.

Modification of erythrocyte membrane proteins, enzymes and transport mechanisms in chronic alcoholics: an in vivo and in vitro study.

PubMed

Maturu, Paramahamsa; Vaddi, Damodara Reddy; Pannuru, Padmavathi; Nallanchakravarthula, Varadacharyulu

2013-01-01

The aim of the study was to elucidate the molecular mechanisms underlying the alcohol perturbation leading to deleterious effects on erythrocyte membrane transport in chronic alcoholics. Membrane bound enzyme activities such as Na(+), K(+)-ATPase, Ca(2+),Mg(2+)-ATPase and acetylcholine esterase and membrane transport analysis by in vitro and erythrocyte membrane profile analysis in controls and chronic alcoholic red cells were analyzed. It was observed that decreased Na(+), K(+)-ATPase enzyme activity and increased activities of Ca(2+),Mg(2+)-ATPase and acetylcholine esterase in chronic alcoholics compared to controls. The in vitro studies of erythrocytes suggested that there is an increased uptake of glucose through chronic alcoholic red cells. However, glucose utilization by chronic alcoholic red cells was decreased. An increased sensitivity of ouabain for its binding site on Na(+), K(+)-ATPase in chronic alcoholic erythrocyte membrane was evident from this study. Though there appears to be an increased Na(+) influx in chronic alcoholic cells, the status of Na(+) transport is not altered much. However, ouabain caused slight disturbances in the transport of sodium, similar disturbances in the potassium transport resulting in much accumulation of potassium in red cells. It was concluded that chronic alcohol consumption modified certain membrane bound proteins, enzymes and transport mechanisms in chronic alcoholics.

Change in the activity of Cl-,HCO3(-)-ATPase in microsome fraction during early development of the sea urchin, Hemicentrotus pulcherrimus.

PubMed

Mitsunaga, K; Fujino, Y; Yasumasu, I

1986-12-01

In sea urchin embryos, primary mesenchyme cells, descendants from micromeres produced at the 16-cell stage, form spicules or CaCO3 deposits in their skeletal vacuoles, at the post-gastrula stage. Micromeres isolated at the 16-cell stage also differentiate into spicule-forming cells during their culture at the same time schedule as in the embryos. The present study was planned to observe change in the activity of Cl-,HCO3(-)-ATPase, which was expected to contribute to the carbonate supply for CaCO3 deposition, during development. ATP-hydrolysis in the microsome fraction, obtained from embryos of the sea urchin, Hemicentrotus pulcherrimus, and from micromere-derived cells in culture was stimulated by Cl- and HCO3- in the presence of ouabain and EGTA. The ATP-hydrolysis was inhibited by ethacrynic acid, an inhibitor of Cl-,HCO3(-)-ATPase. The activity of Cl-,HCO3(-)-ATPase in embryos and in micromere-derived cells increased during development, keeping pace with the rate of calcium deposition in spicules. Formation of calcified spicules in the cultured micromere-derived cells was inhibited by ethacrynic acid. These results indicate that Cl-,HCO3(-)-ATPase plays an important role in the mechanism of CaCO3 deposition in the primary mesenchyme cells.

Inhibitors of V-ATPase proton transport reveal uncoupling functions of tether linking cytosolic and membrane domains of V0 subunit a (Vph1p).

PubMed

Chan, Chun-Yuan; Prudom, Catherine; Raines, Summer M; Charkhzarrin, Sahba; Melman, Sandra D; De Haro, Leyma P; Allen, Chris; Lee, Samuel A; Sklar, Larry A; Parra, Karlett J

2012-03-23

Vacuolar ATPases (V-ATPases) are important for many cellular processes, as they regulate pH by pumping cytosolic protons into intracellular organelles. The cytoplasm is acidified when V-ATPase is inhibited; thus we conducted a high-throughput screen of a chemical library to search for compounds that acidify the yeast cytosol in vivo using pHluorin-based flow cytometry. Two inhibitors, alexidine dihydrochloride (EC(50) = 39 μM) and thonzonium bromide (EC(50) = 69 μM), prevented ATP-dependent proton transport in purified vacuolar membranes. They acidified the yeast cytosol and caused pH-sensitive growth defects typical of V-ATPase mutants (vma phenotype). At concentrations greater than 10 μM the inhibitors were cytotoxic, even at the permissive pH (pH 5.0). Membrane fractions treated with alexidine dihydrochloride and thonzonium bromide fully retained concanamycin A-sensitive ATPase activity despite the fact that proton translocation was inhibited by 80-90%, indicating that V-ATPases were uncoupled. Mutant V-ATPase membranes lacking residues 362-407 of the tether of Vph1p subunit a of V(0) were resistant to thonzonium bromide but not to alexidine dihydrochloride, suggesting that this conserved sequence confers uncoupling potential to V(1)V(0) complexes and that alexidine dihydrochloride uncouples the enzyme by a different mechanism. The inhibitors also uncoupled the Candida albicans enzyme and prevented cell growth, showing further specificity for V-ATPases. Thus, a new class of V-ATPase inhibitors (uncouplers), which are not simply ionophores, provided new insights into the enzyme mechanism and original evidence supporting the hypothesis that V-ATPases may not be optimally coupled in vivo. The consequences of uncoupling V-ATPases in vivo as potential drug targets are discussed.

Deinhibition of cardiac Na/sup +/-K/sup +/-ATPase after exposure to exogenous phospholipase A/sub 2/

DOE Office of Scientific and Technical Information (OSTI.GOV)

Colvin, R.A.

1987-01-01

After 2 h of exogenous phospholipase A/sub 2/ (PLA/sub 2/) exposure, membrane phospholipid decreased from 3.22 +/- 0.31 to 1.06 +/- 0.13 ..mu..mol/mg (33% of control). All classes of phospholipid, except sphingomyelin, were hydrolyzed, whereas total cholesterol content was unaffected. Increases in nonesterified fatty acids (NEFA) were reflected primarily in oleic (18:1), linoleic (18:2), and arachidonic (20:4). Na/sup +/-K/sup +/-adenosinetriphosphatase (ATPase) activity was inhibited to 29% of control by 2 h of PLA/sub 2/ treatment, and this inhibition was reversed (albeit, not completely after 5 min of PLA/sub 2/ treatment) by removal of the hydrolysis products with 0.1% bovine serummore » albumin (BSA). In contrast, the apparent binding capacity for (/sup 3/H)ouabain was not affected by PLA/sub 2/ treatment. Unmasking of latent (/sup 3/H)ouabain binding by alamethicin was utilized to estimate changes in the proportion of sealed vesicles present before and after PLA/sub 2/ treatment. PLA/sub 2/ treatment resulted in a time-dependent loss of sealed vesicles that paralleled the time course of phospholipid hydrolysis and was not reversed by washing with BSA. These studies demonstrate that cardiac Na/sup +/-K/sup +/-ATPase activity is inhibited by accumulation of endogenously produced lysophospholipids and NEFA. In contrast, loss of vesicle integrity may result from both accumulation of endogenously produced hydrolysis products and membrane phospholipid depletion.« less

Structure of the 26S proteasome with ATP-γS bound provides insights into the mechanism of nucleotide-dependent substrate translocation

PubMed Central

Śledź, Paweł; Unverdorben, Pia; Beck, Florian; Pfeifer, Günter; Schweitzer, Andreas; Förster, Friedrich; Baumeister, Wolfgang

2013-01-01

The 26S proteasome is a 2.5-MDa, ATP-dependent multisubunit proteolytic complex that processively destroys proteins carrying a degradation signal. The proteasomal ATPase heterohexamer is a key module of the 19S regulatory particle; it unfolds substrates and translocates them into the 20S core particle where degradation takes place. We used cryoelectron microscopy single-particle analysis to obtain insights into the structural changes of 26S proteasome upon the binding and hydrolysis of ATP. The ATPase ring adopts at least two distinct helical staircase conformations dependent on the nucleotide state. The transition from the conformation observed in the presence of ATP to the predominant conformation in the presence of ATP-γS induces a sliding motion of the ATPase ring over the 20S core particle ring leading to an alignment of the translocation channels of the ATPase and the core particle gate, a conformational state likely to facilitate substrate translocation. Two types of intersubunit modules formed by the large ATPase domain of one ATPase subunit and the small ATPase domain of its neighbor exist. They resemble the contacts observed in the crystal structures of ClpX and proteasome-activating nucleotidase, respectively. The ClpX-like contacts are positioned consecutively and give rise to helical shape in the hexamer, whereas the proteasome-activating nucleotidase-like contact is required to close the ring. Conformational switching between these forms allows adopting different helical conformations in different nucleotide states. We postulate that ATP hydrolysis by the regulatory particle ATPase (Rpt) 5 subunit initiates a cascade of conformational changes, leading to pulling of the substrate, which is primarily executed by Rpt1, Rpt2, and Rpt6. PMID:23589842

Steroid-like compounds in Chinese medicines promote blood circulation via inhibition of Na+/K+-ATPase

PubMed Central

Chen, Ronald JY; Chung, Tse-yu; Li, Feng-yin; Yang, Wei-hung; Jinn, Tzyy-rong; Tzen, Jason TC

2010-01-01

Aim: To examine if steroid-like compounds found in many Chinese medicinal products conventionally used for the promotion of blood circulation may act as active components via the same molecular mechanism triggered by cardiac glycosides, such as ouabain. Methods: The inhibitory potency of ouabain and the identified steroid-like compounds on Na+/K+-ATPase activity was examined and compared. Molecular modeling was exhibited for the docking of these compounds to Na+/K+-ATPase. Results: All the examined steroid-like compounds displayed more or less inhibition on Na+/K+-ATPase, with bufalin (structurally almost equivalent to ouabain) exhibiting significantly higher inhibitory potency than the others. In the pentacyclic triterpenoids examined, ursolic acid and oleanolic acid were moderate inhibitors of Na+/K+-ATPase, and their inhibitory potency was comparable to that of ginsenoside Rh2. The relatively high inhibitory potency of ursolic acid or oleanolic acid was due to the formation of a hydrogen bond between its carboxyl group and the Ile322 residue in the deep cavity close to two K+ binding sites of Na+/K+-ATPase. Moreover, the drastic difference observed in the inhibitory potency of ouabain, bufalin, ginsenoside Rh2, and pentacyclic triterpenoids is ascribed mainly to the number of hydrogen bonds and partially to the strength of hydrophobic interaction between the compounds and residues around the deep cavity of Na+/K+-ATPase. Conclusion: Steroid-like compounds seem to contribute to therapeutic effects of many cardioactive Chinese medicinal products. Chinese herbs, such as Prunella vulgaris L, rich in ursolic acid, oleanolic acid and their glycoside derivatives may be adequate sources for cardiac therapy via effective inhibition on Na+/K+-ATPase. PMID:20523340

Multi-site TBT binding skews the inhibition of oligomycin on the mitochondrial Mg-ATPase in Mytilus galloprovincialis.

PubMed

Nesci, Salvatore; Ventrella, Vittoria; Trombetti, Fabiana; Pirini, Maurizio; Pagliarani, Alessandra

2011-07-01

Tributyltin (TBT), a persistent lipophilic contaminant found especially in the aquatic environment, is known to be toxic to mitochondria with the F(1)F(0)-ATPase as main target. Recently our research group pointed out that in mussel digestive gland mitochondria TBT, apart from decreasing the catalytic efficiency of Mg-ATPase activity, at concentrations ≥1.0 μM in the ATPase reaction medium lessens the enzyme inhibition promoted by the specific inhibitor oligomycin. The present work aims at casting light on the mechanisms involved in the TBT-driven enzyme desensitization to inhibitors, a poorly explored field. The mitochondrial Mg-ATPase desensitization is shown to be confined to inhibitors of transmembrane domain F(0), namely oligomycin and N,N'-dicyclohexylcarbodiimide (DCCD). Accordingly, quercetin, which binds to catalytic portion F(1), maintains its inhibitory efficiency in the presence of TBT. Among the possible mechanisms involved in the Mg-ATPase desensitization to oligomycin by ≥1.0 μM TBT concentrations, a structural detachment of the two F(1) and F(0) domains does not occur according to experimental data. On the other hand TBT covalently binds to thiol groups on the enzyme structure, which are apparently only available at TBT concentrations approaching 20 μM. TBT is able to interact with multiple sites on the enzyme structure by bonds of different nature. While electrostatic interactions with F(0) proton channel are likely to be responsible for the ATPase activity inhibition, possible changes in the redox state of thiol groups on the protein structure due to TBT binding may promote structural changes in the enzyme structure leading to the observed F(1)F(0)-ATPase oligomycin sensitivity loss. Copyright © 2011 Elsevier Masson SAS. All rights reserved.

Human Hsp70 molecular chaperone binds two calcium ions within the ATPase domain.

PubMed

Sriram, M; Osipiuk, J; Freeman, B; Morimoto, R; Joachimiak, A

1997-03-15

The 70 kDa heat shock proteins (Hsp70) are a family of molecular chaperones, which promote protein folding and participate in many cellular functions. The Hsp70 chaperones are composed of two major domains. The N-terminal ATPase domain binds to and hydrolyzes ATP, whereas the C-terminal domain is required for polypeptide binding. Cooperation of both domains is needed for protein folding. The crystal structure of bovine Hsc70 ATPase domain (bATPase) has been determined and, more recently, the crystal structure of the peptide-binding domain of a related chaperone, DnaK, in complex with peptide substrate has been obtained. The molecular chaperone activity and conformational switch are functionally linked with ATP hydrolysis. A high-resolution structure of the ATPase domain is required to provide an understanding of the mechanism of ATP hydrolysis and how it affects communication between C- and N-terminal domains. The crystal structure of the human Hsp70 ATPase domain (hATPase) has been determined and refined at 1. 84 A, using synchrotron radiation at 120K. Two calcium sites were identified: the first calcium binds within the catalytic pocket, bridging ADP and inorganic phosphate, and the second calcium is tightly coordinated on the protein surface by Glu231, Asp232 and the carbonyl of His227. Overall, the structure of hATPase is similar to bATPase. Differences between them are found in the loops, the sites of amino acid substitution and the calcium-binding sites. Human Hsp70 chaperone is phosphorylated in vitro in the presence of divalent ions, calcium being the most effective. The structural similarity of hATPase and bATPase and the sequence similarity within the Hsp70 chaperone family suggest a universal mechanism of ATP hydrolysis among all Hsp70 molecular chaperones. Two calcium ions have been found in the hATPase structure. One corresponds to the magnesium site in bATPase and appears to be important for ATP hydrolysis and in vitro phosphorylation. Local changes in protein structure as a result of calcium binding may facilitate phosphorylation. A small, but significant, movement of metal ions and sidechains could position catalytically important threonine residues for phosphorylation. The second calcium site represents a new calcium-binding motif that can play a role in the stabilization of protein structure. We discuss how the information about catalytic events in the active site could be transmitted to the peptide-binding domain.

Migratory urge and gll Na+,K+-ATPase activity of hatchery-reared Atlantic salmon smolts from the Dennys and Penobscot River stocks, Maine

USGS Publications Warehouse

Spencer, Randall C.; Zydlewski, Joseph D.; Zydlewski, Gayle B.

2010-01-01

Hatchery-reared Atlantic salmon Salmo salar smolts produced from captive-reared Dennys River and sea-run Penobscot River broodstock are released into their source rivers in Maine. The adult return rate of Dennys smolts is comparatively low, and disparity in smolt quality between stocks resulting from genetic or broodstock rearing effects is plausible. Smolt behavior and physiology were assessed during sequential 14-d trials conducted in seminatural annular tanks with circular flow. “Migratory urge” (downstream movement) was monitored remotely using passive integrated transponder tags, and gill Na+,K+-ATPase activity was measured at the beginning and end of the trials to provide an index of smolt development. The migratory urge of both stocks was low in early April, increased 20-fold through late May, and declined by the end of June. The frequency and seasonal distribution of downstream movement were independent of stock. In March and April, initial gill Na+,K+-ATPase activities of Penobscot River smolts were lower than those of Dennys River smolts. For these trials, however, Penobscot River smolts increased enzyme activity after exposure to the tank, whereas Dennys River smolts did not, resulting in similar activities between stocks at the end of all trials. There was no clear relationship between migratory urge and gill Na+,K+-ATPase activity. Gill Na+,K+-ATPase activity of both stocks increased in advance of migratory urge and then declined while migratory urge was increasing. Maximum movement was observed from 2 h after sunset through 1 h after sunrise but varied seasonally. Dennys River smolts were slightly more nocturnal than Penobscot River smolts. These data suggest that Dennys and Penobscot River stocks are not markedly different in either physiological or behavioral expression of smolting.

ε Subunit of Bacillus subtilis F1-ATPase Relieves MgADP Inhibition

PubMed Central

Mizumoto, Junya; Kikuchi, Yuka; Nakanishi, Yo-Hei; Mouri, Naoto; Cai, Anrong; Ohta, Tokushiro; Haruyama, Takamitsu; Kato-Yamada, Yasuyuki

2013-01-01

MgADP inhibition, which is considered as a part of the regulatory system of ATP synthase, is a well-known process common to all F1-ATPases, a soluble component of ATP synthase. The entrapment of inhibitory MgADP at catalytic sites terminates catalysis. Regulation by the ε subunit is a common mechanism among F1-ATPases from bacteria and plants. The relationship between these two forms of regulatory mechanisms is obscure because it is difficult to distinguish which is active at a particular moment. Here, using F1-ATPase from Bacillus subtilis (BF1), which is strongly affected by MgADP inhibition, we can distinguish MgADP inhibition from regulation by the ε subunit. The ε subunit did not inhibit but activated BF1. We conclude that the ε subunit relieves BF1 from MgADP inhibition. PMID:23967352

Palytoxin isolated from marine coelenterates. The inhibitory action on (Na,K)-ATPase.

PubMed

Ishida, Y; Takagi, K; Takahashi, M; Satake, N; Shibata, S

1983-07-10

Palytoxin (PTX), C129H223N3O54, a highly toxic substance isolated from zoanthids of Palythoa tuberculosa, inhibited (Na,K)-ATPase (ATP phosphohydrolase, EC 3.6.1.3) prepared from guinea pig heart and hog cerebral cortex in a dose-dependent manner at concentrations greater than 10(-8) M. In the presence of Na (100 mM) and K (20 mM), PTX showed potency nearly equal to that of ouabain. When the ATPase was activated by the various Na concentrations at a constant K concentration, both PTX and ouabain inhibited the ATPase activity noncompetitively. On the other hand, when K concentration was changed at a constant Na concentration, PTX caused a competitive inhibition in all ranges of K concentrations employed, whereas ouabain caused a competitive inhibition at low concentrations and a noncompetitive inhibition at high concentrations.

The prokaryotic enhancer binding protein NTRC has an ATPase activity which is phosphorylation and DNA dependent.

PubMed Central

Austin, S; Dixon, R

1992-01-01

The prokaryotic activator protein NTRC binds to enhancer-like elements and activates transcription in response to nitrogen limitation by catalysing open complex formation by sigma 54 RNA polymerase holoenzyme. Formation of open complexes requires the phosphorylated form of NTRC and the reaction is ATP dependent. We find that NTRC has an ATPase activity which is activated by phosphorylation and is strongly stimulated by the presence of DNA containing specific NTRC binding sites. Images PMID:1534752

Gastroprotective activity of the chloroform extract of the roots from Arctium lappa L.

PubMed

Dos Santos, Ana C; Baggio, Cristiane H; Freitas, Cristina S; Lepieszynski, Juliana; Mayer, Bárbara; Twardowschy, André; Missau, Fabiana C; dos Santos, Elide P; Pizzolatti, Moacir G; Marques, Maria C A

2008-06-01

Arctium lappa L. is used in folk medicine as a diuretic, depurative and digestive stimulant and in dermatological conditions. The objective of this study was to evaluate the effect and the possible mechanisms involved in the gastroprotective effects of a chloroform extract (CE) of the roots from A. lappa and its fractions. Oral pretreatment with CE (10, 30 and 100 mg kg(-1)) significantly reduced gastric lesions induced by ethanol by 61%, 70% and 76%, respectively. Oral administration of CE (100 mg kg(-1) per day for 7 days) reduced the chronic gastric ulceration induced by acetic acid by 52%. Intraduodenal CE (100, 300 and 600 mg kg(-1)) reduced the total acidity of gastric secretion by 22%, 22% and 33%, respectively, while i.p. administration (10, 30 and 100 mg kg(-1)) inhibited total acidity by 50%, 60% and 67%, respectively. In-vitro, CE inhibited H+, K+ -ATPase activity with an EC50 of 53 microgmL(-1) and fraction A (30 and 100 microgmL(-1)) reduced this by 48% and 89%, respectively. CE had no effect on gastrointestinal motility. CE (250 microgmL(-1)) and fraction B (100 and 250 microgmL(-1)) had free-radical scavenging ability, inhibiting 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical activity by 50%, 20% and 55%, respectively. Collectively, the results show that the CE protects animals from gastric lesions by reducing gastric acid secretion via inhibition of gastric H+, K+ -ATPase.

Effect of yeast biomass with high content of carotenoids on erythrocyte deformability, NO production and Na,K-ATPase activity in healthy and LPS treated rats.

PubMed

Radosinska, J; Mezesova, L; Okruhlicova, L; Frimmel, K; Breierova, E; Bartekova, M; Vrbjar, N

2016-11-25

Measurements of red blood cell (RBC) deformability together with estimation of NO-synthase activity and Na,K-ATPase activity were used for characterization of RBC functionality in rats subjected to single dose of Escherichia coli lipopolysaccharides (LPS) at a dose of 1 mg/kg. We hypothesized that LPS might initiate a malfunction of RBC. We also investigated the potential effect of carotenoids (10 mg/kg/day) produced in red yeast biomass of Rhodotorula glutinis on RBC in LPS-challenged rats. LPS significantly reduced the deformability of RBC (by 14%) together with decrease of NO-synthase activity by 20%. Daily supplementation of carotenoids for 10 days attenuated the LPS-induced injury, as observed by 22% increase of RBC deformability and 23% increase of NO-synthase activity. The activity of Na,K-ATPase was also improved probably due to increased number of active enzyme molecules as indicated by 66% enhancement of Vmax value, hence maintaining the activity of erythrocyte Na,K-ATPase to the level even higher as compared with healthy control animals. It may be concluded that administration of yeast biomass with high content of carotenoids resulted in advanced function of erythrocytes as concerns their ability to squeeze through narrow capillaries of the circulation, better intrinsic production of NO and improvement of intracellular homeostasis of sodium.

Regulation of branchial V-H(+)-ATPase, Na(+)/K(+)-ATPase and NHE2 in response to acid and base infusions in the Pacific spiny dogfish (Squalus acanthias).

PubMed

Tresguerres, Martin; Katoh, Fumi; Fenton, Heather; Jasinska, Edyta; Goss, Greg G

2005-01-01

To study the mechanisms of branchial acid-base regulation, Pacific spiny dogfish were infused intravenously for 24 h with either HCl (495+/- 79 micromol kg(-1) h(-1)) or NaHCO(3) (981+/-235 micromol kg(-1) h(-1)). Infusion of HCl produced a transient reduction in blood pH. Despite continued infusion of acid, pH returned to normal by 12 h. Infusion of NaHCO(3) resulted in a new steady-state acid-base status at approximately 0.3 pH units higher than the controls. Immunostained serial sections of gill revealed the presence of separate vacuolar proton ATPase (V-H(+)-ATPase)-rich or sodium-potassium ATPase (Na(+)/K(+)-ATPase)-rich cells in all fish examined. A minority of the cells also labeled positive for both transporters. Gill cell membranes prepared from NaHCO(3)-infused fish showed significant increases in both V-H(+)-ATPase abundance (300+/-81%) and activity. In addition, we found that V-H(+)-ATPase subcellular localization was mainly cytoplasmic in control and HCl-infused fish, while NaHCO(3)-infused fish demonstrated a distinctly basolateral staining pattern. Western analysis in gill membranes from HCl-infused fish also revealed increased abundance of Na(+)/H(+) exchanger 2 (213+/-5%) and Na(+)/K(+)-ATPase (315+/-88%) compared to the control.

The use of antioxidants to prevent glutamate-induced derangement of calcium ion metabolism in rat cerebral cortex synaptosomes.

PubMed

Avrova, N F; Shestak, K I; Zakharova, I O; Sokolova, T V; Tyurina, Y Y; Tyurin, V A

2000-01-01

Glutamate is shown to induce increases in intracellular Ca2+ concentrations ([Ca2+]i), increases in 45Ca2+ influx, decreases in the activity of Na+,K+-ATPase activity, and activation of the Na+/Ca2+ exchanger in rat cerebral cortex synaptosomes. NMDA receptor antagonists virtually prevented these effects. Preincubation of synaptosomes with alpha-tocopherol, superoxide dismutase, and ganglioside GM1 normalized [Ca2+]i, 45Ca2+ influx, and Na+,K+-ATPase activity in rat cerebral cortex synaptosomes exposed to glutamate. Glutamate and GM1 activated the Na+/K+ exchanger, and their effects were additive. Calcium ions entering cerebral cortex nerve cells via NMDA receptors during exposure to high glutamate concentrations appeared to be only the trigger for the processes activating free-radical reactions. Activation of these reactions led to increases in Ca2+ influx into cells, decreases in Na+,K+-ATPase activity, and significant increases in [Ca2+]i, though this could be prevented by antioxidants and gangliosides.

ATP synthesis in Halobacterium saccharovorum: evidence that synthesis may be catalysed by an F0F1-ATP synthase

NASA Technical Reports Server (NTRS)

Hochstein, L. I.

1992-01-01

Halobacterium saccharovorum synthesized ATP in response to a pH shift from 8 to 6.2. Synthesis was inhibited by carbonyl cyanide m-chloro-phenylhydrazone, dicyclohexylcarbodiimide, and azide. Nitrate, an inhibitor of the membrane-bound ATPase previously isolated from this organism, did not inhibit ATP synthesis. N-Ethymaleimide, which also inhibited this ATPase, stimulated the production of ATP. These observations suggested that H. saccharovorum synthesized and hydrolysed ATP using different enzymes and that the vacuolar-like ATPase activity previously described in H. saccharovorum was an ATPase whose function is yet to be identified.

Lassomycin, a ribosomally synthesized cyclic peptide, kills Mycobacterium tuberculosis by targeting the ATP-dependent protease ClpC1P1P2

PubMed Central

Gavrish, Ekaterina; Sit, Clarissa S.; Cao, Shugeng; Kandror, Olga; Spoering, Amy; Peoples, Aaron; Ling, Losee; Fetterman, Ashley; Hughes, Dallas; Bissell, Anthony; Torrey, Heather; Akopian, Tatos; Mueller, Andreas; Epstein, Slava; Goldberg, Alfred; Clardy, Jon; Lewis, Kim

2014-01-01

Summary Languishing antibiotic discovery and flourishing antibiotic resistance have prompted development of alternative untapped sources for antibiotic discovery, including previously uncultured bacteria. Here, we screen extracts from uncultured species against M. tuberculosis and identify lassomycin, an antibiotic that exhibits potent bactericidal activity against both growing and dormant mycobacteria, including drug-resistant forms of M. tuberculosis, but little activity against other bacteria or mammalian cells. Lassomycin is a highly basic, ribosomally-encoded cyclic peptide with an unusual structural fold that only partially resembles that of other lasso peptides. We show that lassomycin binds to a highly acidic region of the ClpC1 ATPase complex and markedly stimulates its ATPase activity without stimulating ClpP1P2 catalyzed protein breakdown, which is essential for viability of mycobacteria. This mechanism, uncoupling ATPase from proteolytic activity, accounts for lassomycin's bacteriocidal activity. PMID:24684906

A novel calmodulin-regulated Ca2+-ATPase (ACA2) from Arabidopsis with an N-terminal autoinhibitory domain

NASA Technical Reports Server (NTRS)

Harper, J. F.; Hong, B.; Hwang, I.; Guo, H. Q.; Stoddard, R.; Huang, J. F.; Palmgren, M. G.; Sze, H.; Evans, M. L. (Principal Investigator)

1998-01-01

To study transporters involved in regulating intracellular Ca2+, we isolated a full-length cDNA encoding a Ca2+-ATPase from a model plant, Arabidopsis, and named it ACA2 (Arabidopsis Ca2+-ATPase, isoform 2). ACA2p is most similar to a "plasma membrane-type" Ca2+-ATPase, but is smaller (110 kDa), contains a unique N-terminal domain, and is missing a long C-terminal calmodulin-binding regulatory domain. In addition, ACA2p is localized to an endomembrane system and not the plasma membrane, as shown by aqueous-two phase fractionation of microsomal membranes. ACA2p was expressed in yeast as both a full-length protein (ACA2-1p) and an N-terminal truncation mutant (ACA2-2p; Delta residues 2-80). Only the truncation mutant restored the growth on Ca2+-depleted medium of a yeast mutant defective in both endogenous Ca2+ pumps, PMR1 and PMC1. Although basal Ca2+-ATPase activity of the full-length protein was low, it was stimulated 5-fold by calmodulin (50% activation around 30 nM). In contrast, the truncated pump was fully active and insensitive to calmodulin. A calmodulin-binding sequence was identified within the first 36 residues of the N-terminal domain, as shown by calmodulin gel overlays on fusion proteins. Thus, ACA2 encodes a novel calmodulin-regulated Ca2+-ATPase distinguished by a unique N-terminal regulatory domain and a non-plasma membrane localization.

ALUMINUM CHLORIDE EFFECT ON Ca2+,Mg(2+)-ATPase ACTIVITY AND DYNAMIC PARAMETERS OF SKELETAL MUSCLE CONTRACTION.

PubMed

Nozdrenko, D M; Abramchuk, O M; Soroca, V M; Miroshnichenko, N S

2015-01-01

We studied enzymatic activity and measured strain-gauge contraction properties of the frog Rana temporaria m. tibialis anterior muscle fascicles during the action of aluminum chloride solution. It was shown that AlCl3 solutions did not affect the dynamic properties of skeletal muscle preparation in concentrations less than 10(-4) M Increasing the concentration of AlCl3 to 10(-2) M induce complete inhibition of muscle contraction. A linear correlation between decrease in Ca2+,Mg(2+)-ATPase activity of sarcoplasmic reticulum and the investigated concentrations range of aluminum chloride was observed. The reduction in the dynamic contraction performance and the decrease Ca2+,Mg(2+)-ATPase activity of the sarcoplasmic reticulum under the effect of the investigated AlCl3 solution were minimal in pre-tetanus period of contraction.

Oxidative stress mediated aldehyde adduction of Grp78 in a mouse model of alcoholic liver disease: Functional independence of ATPase activity and chaperone function

PubMed Central

Galligan, James J.; Fritz, Kristofer S.; Backos, Donald S.; Shearn, Colin T.; Smathers, Rebecca L.; Jiang, Hua; MacLean, Kenneth N.; Reigan, Philip R.; Petersen, Dennis R.

2015-01-01

Pathogenesis in alcoholic liver disease (ALD) is complicated and multifactorial but clearly involves oxidative stress and inflammation. Currently, conflicting reports exist regarding the role of endoplasmic reticulum (ER) stress in the etiology of ALD. The glucose regulated protein 78 (GRP78) is the ER homologue of HSP70 and plays a critical role in the cellular response to ER stress by serving as a chaperone assisting protein folding and by regulating the signaling of the unfolded protein response (UPR). Comprised of three functional domains, an ATPase, peptide-binding, and lid domain, GRP78 folds nascent polypeptides via the substrate-binding domain. Earlier work has indicated that the ATPase function of GRP78 is intrinsically linked and essential to its chaperone activity. Previous work in our laboratory has indicated that Grp78 and the UPR are not induced in a mouse model of ALD but that Grp78 is adducted by the lipid electrophiles 4-hydroxynonenal (4-HNE) and 4-oxononenal (4-ONE) in vivo. As impairment of Grp78 has the potential to contribute to pathogenesis in ALD, we investigated the functional consequences of aldehyde adduction upon Grp78 function. Identification of 4-HNE and 4-ONE target residues in purified human GRP78 revealed a marked propensity for Lys and His adduction within the ATPase domain and a relative paucity of adduct formation within the peptide-binding domain. Consistent with these findings, we observed a concomitant dose-dependent decrease in ATP-binding and ATPase activity without any discernible impairment of chaperone function. Collectively, our data indicate that ATPase activity is not essential for Grp78 mediated chaperone activity and is consistent with the hypothesis that ER stress does not play a primary initiating role in the early stages of ALD. PMID:24924946

Spa47 is an oligomerization-activated type three secretion system (T3SS) ATPase from Shigella flexneri.

PubMed

Burgess, Jamie L; Jones, Heather B; Kumar, Prashant; Toth, Ronald T; Middaugh, C Russell; Antony, Edwin; Dickenson, Nicholas E

2016-05-01

Gram-negative pathogens often use conserved type three secretion systems (T3SS) for virulence. The Shigella type three secretion apparatus (T3SA) penetrates the host cell membrane and provides a unidirectional conduit for injection of effectors into host cells. The protein Spa47 localizes to the base of the apparatus and is speculated to be an ATPase that provides the energy for T3SA formation and secretion. Here, we developed an expression and purification protocol, producing active Spa47 and providing the first direct evidence that Spa47 is a bona fide ATPase. Additionally, size exclusion chromatography and analytical ultracentrifugation identified multiple oligomeric species of Spa47 with the largest greater than 8 fold more active for ATP hydrolysis than the monomer. An ATPase inactive Spa47 point mutant was then engineered by targeting a conserved Lysine within the predicted Walker A motif of Spa47. Interestingly, the mutant maintained a similar oligomerization pattern as active Spa47, but was unable to restore invasion phenotype when used to complement a spa47 null S. flexneri strain. Together, these results identify Spa47 as a Shigella T3SS ATPase and suggest that its activity is linked to oligomerization, perhaps as a regulatory mechanism as seen in some related pathogens. Additionally, Spa47 catalyzed ATP hydrolysis appears to be essential for host cell invasion, providing a strong platform for additional studies dissecting its role in virulence and providing an attractive target for anti-infective agents. © 2016 The Protein Society.

Biolayer interferometry of lipid nanodisc‐reconstituted yeast vacuolar H+‐ATPase

PubMed Central

Sharma, Stuti

2017-01-01

Abstract Vacuolar H+‐ATPase (V‐ATPase) is a large, multisubunit membrane protein complex responsible for the acidification of subcellular compartments and the extracellular space. V‐ATPase activity is regulated by reversible disassembly, resulting in cytosolic V 1‐ATPase and membrane‐integral V 0 proton channel sectors. Reversible disassembly is accompanied by transient interaction with cellular factors and assembly chaperones. Quantifying protein‐protein interactions involving membrane proteins, however, is challenging. Here we present a novel method to determine kinetic constants of membrane protein–protein interactions using biolayer interferometry (BLI). Yeast vacuoles are solubilized, vacuolar proteins are reconstituted into lipid nanodiscs with native vacuolar lipids and biotinylated membrane scaffold protein (MSP) followed by affinity purification of nanodisc‐reconstituted V‐ATPase (V 1 V 0ND). We show that V 1 V 0ND can be immobilized on streptavidin‐coated BLI sensors to quantitate binding of a pathogen derived inhibitor and to measure the kinetics of nucleotide dependent enzyme dissociation. PMID:28241399

Biolayer interferometry of lipid nanodisc-reconstituted yeast vacuolar H+ -ATPase.

PubMed

Sharma, Stuti; Wilkens, Stephan

2017-05-01

Vacuolar H + -ATPase (V-ATPase) is a large, multisubunit membrane protein complex responsible for the acidification of subcellular compartments and the extracellular space. V-ATPase activity is regulated by reversible disassembly, resulting in cytosolic V 1 -ATPase and membrane-integral V 0 proton channel sectors. Reversible disassembly is accompanied by transient interaction with cellular factors and assembly chaperones. Quantifying protein-protein interactions involving membrane proteins, however, is challenging. Here we present a novel method to determine kinetic constants of membrane protein-protein interactions using biolayer interferometry (BLI). Yeast vacuoles are solubilized, vacuolar proteins are reconstituted into lipid nanodiscs with native vacuolar lipids and biotinylated membrane scaffold protein (MSP) followed by affinity purification of nanodisc-reconstituted V-ATPase (V 1 V 0 ND). We show that V 1 V 0 ND can be immobilized on streptavidin-coated BLI sensors to quantitate binding of a pathogen derived inhibitor and to measure the kinetics of nucleotide dependent enzyme dissociation. © 2017 The Protein Society.

Regulatory assembly of the vacuolar proton pump VoV1-ATPase in yeast cells by FLIM-FRET

NASA Astrophysics Data System (ADS)

Ernst, Stefan; Batisse, Claire; Zarrabi, Nawid; Böttcher, Bettina; Börsch, Michael

2010-02-01

We investigate the reversible disassembly of VOV1-ATPase in life yeast cells by time resolved confocal FRET imaging. VOV1-ATPase in the vacuolar membrane pumps protons from the cytosol into the vacuole. VOV1-ATPase is a rotary biological nanomotor driven by ATP hydrolysis. The emerging proton gradient is used for secondary transport processes as well as for pH and Ca2+ homoeostasis in the cell. The activity of the VOV1-ATPase is regulated through assembly / disassembly processes. During starvation the two parts of VOV1-ATPase start to disassemble. This process is reversed after addition of glucose. The exact mechanisms are unknown. To follow the disassembly / reassembly in vivo we tagged two subunits C and E with different fluorescent proteins. Cellular distributions of C and E were monitored using a duty cycle-optimized alternating laser excitation scheme (DCO-ALEX) for time resolved confocal FRET-FLIM measurements.

The yeast protein kinase Sch9 adjusts V-ATPase assembly/disassembly to control pH homeostasis and longevity in response to glucose availability.

PubMed

Wilms, Tobias; Swinnen, Erwin; Eskes, Elja; Dolz-Edo, Laura; Uwineza, Alice; Van Essche, Ruben; Rosseels, Joëlle; Zabrocki, Piotr; Cameroni, Elisabetta; Franssens, Vanessa; De Virgilio, Claudio; Smits, Gertien J; Winderickx, Joris

2017-06-01

The conserved protein kinase Sch9 is a central player in the nutrient-induced signaling network in yeast, although only few of its direct substrates are known. We now provide evidence that Sch9 controls the vacuolar proton pump (V-ATPase) to maintain cellular pH homeostasis and ageing. A synthetic sick phenotype arises when deletion of SCH9 is combined with a dysfunctional V-ATPase, and the lack of Sch9 has a significant impact on cytosolic pH (pHc) homeostasis. Sch9 physically interacts with, and influences glucose-dependent assembly/disassembly of the V-ATPase, thereby integrating input from TORC1. Moreover, we show that the role of Sch9 in regulating ageing is tightly connected with V-ATPase activity and vacuolar acidity. As both Sch9 and the V-ATPase are highly conserved in higher eukaryotes, it will be interesting to further clarify their cooperative action on the cellular processes that influence growth and ageing.

Freshwater to seawater acclimation of juvenile bull sharks (Carcharhinus leucas): plasma osmolytes and Na+/K+-ATPase activity in gill, rectal gland, kidney and intestine.

PubMed

Pillans, Richard D; Good, Jonathan P; Anderson, W Gary; Hazon, Neil; Franklin, Craig E

2005-01-01

This study examined the osmoregulatory status of the euryhaline elasmobranch Carcharhinus leucas acclimated to freshwater (FW) and seawater (SW). Juvenile C. leucas captured in FW (3 mOsm l(-1) kg(-1)) were acclimated to SW (980-1,000 mOsm l(-1) kg(-1)) over 16 days. A FW group was maintained in captivity over a similar time period. In FW, bull sharks were hyper-osmotic regulators, having a plasma osmolarity of 595 mOsm l(-1) kg(-1). In SW, bull sharks had significantly higher plasma osmolarities (940 mOsm l(-1) kg(-1)) than FW-acclimated animals and were slightly hypo-osmotic to the environment. Plasma Na(+), Cl(-), K(+), Mg(2+), Ca(2+), urea and trimethylamine oxide (TMAO) concentrations were all significantly higher in bull sharks acclimated to SW, with urea and TMAO showing the greatest increase. Gill, rectal gland, kidney and intestinal tissue were taken from animals acclimated to FW and SW and analysed for maximal Na(+)/K(+)-ATPase activity. Na(+)/K(+)-ATPase activity in the gills and intestine was less than 1 mmol Pi mg(-1) protein h(-1) and there was no difference in activity between FW- and SW-acclimated animals. In contrast Na(+)/K(+)-ATPase activity in the rectal gland and kidney were significantly higher than gill and intestine and showed significant differences between the FW- and SW-acclimated groups. In FW and SW, rectal gland Na(+)/K(+)-ATPase activity was 5.6+/-0.8 and 9.2+/-0.6 mmol Pi mg(-1) protein h(-1), respectively. Na(+)/K(+)-ATPase activity in the kidney of FW and SW acclimated animals was 8.4+/-1.1 and 3.3+/-1.1 Pi mg(-1) protein h(-1), respectively. Thus juvenile bull sharks have the osmoregulatory plasticity to acclimate to SW; their preference for the upper reaches of rivers where salinity is low is therefore likely to be for predator avoidance and/or increased food abundance rather than because of a physiological constraint.

NO Metabolites Levels in Human Red Blood Cells are Affected by Palytoxin, an Inhibitor of Na(+)/K(+)-ATPase Pump.

PubMed

Carelli-Alinovi, Cristiana; Tellone, Ester; Russo, Anna Maria; Ficarra, Silvana; Pirolli, Davide; Galtieri, Antonio; Giardina, Bruno; Misiti, Francesco

2014-01-01

Palytoxin (PTX), a marine toxin, represents an increasing hazard for human health. Despite its high toxicity for biological systems, the mechanisms triggered by PTX, are not well understood. The high affinity of PTX for erythrocyte Na(+)/K(+)-ATPase pump is largely known, and it indicates PTX as a sensitive tool to characterize the signal transducer role for Na(+)/K(+)-ATPase pump. Previously, it has been reported that in red blood cells (RBC), probably via a signal transduction generated by the formation of a PTX-Na(+)/K(+)-ATPase complex, PTX alters band 3 functions and glucose metabolism. The present study addresses the question of which other signaling pathways are regulated by Na(+)/K(+)-ATPase in RBC. Here it has been evidenced that PTX following its interaction with Na(+)/K(+)-ATPase pump, alters RBC morphology and this event is correlated to decreases by 30% in nitrites and nitrates levels, known as markers of plasma membrane eNOS activity. Orthovanadate (OV), an antagonist of PTX binding to Na(+)/K(+)-ATPase pump, was able to reverse the effects elicited by PTX. Finally, current investigation firstly suggests that Na(+)/K(+)-ATPase pump, following its interaction with PTX, triggers a signal transduction involved in NO metabolism regulation.

Ouabain-sensitive Rb+ uptake in mouse eggs and preimplantation conceptuses

DOE Office of Scientific and Technical Information (OSTI.GOV)

Van Winkle, L.J.; Campione, A.L.

1991-07-01

The results of histochemical and immunocytochemical studies have been used elsewhere to support the hypothesis that Na+/K(+)-ATPase expression is initiated or increases dramatically in preimplantation mouse conceptuses just before they begin to cavitate. Moreover, localization of the enzyme in the inner membrane of the mural trophoblast is thought to be involved directly in formation and maintenance of the blastocyst cavity. Presumably, Na+/K(+)-ATPase extrudes the cation, Na+, and therefore water into the cavity. The cation transporting activity of the enzyme can be determined by measuring ouabain-sensitive Rb+ uptake by cells. Therefore, we measured Rb+ uptake in mouse eggs and preimplantation conceptusesmore » at various stages of development. 86Rb+ uptake by conceptuses increased linearly with time for at least 60 min in medium containing 0.7 mM total Rb+ plus K+ in the absence or presence of 1.0 mM ouabain, and ouabain inhibited more than 70% of 86Rb+ uptake. The ouabain concentration at 1/2 of maximum inhibition of the ouabain-sensitive component of 86Rb+ uptake was about 10-20 microM in eggs and conceptuses at all stages of preimplantation development. Moreover, ouabain-sensitive Rb+ uptake had a twofold higher Vmax value in blastocysts than in eggs or conceptuses at earlier stages of development (i.e., approximately 173 vs 70-100 fmole.conceptus-1.min-1), although the total cell surface area also was probably about two times greater in blastocysts than in eggs or other conceptuses. Ouabain-sensitive Rb+ transport in eggs and conceptuses may have occurred via a single ouabain-sensitive Rb+ transporter with a Hill coefficient of 1.5-1.8 (Hill plots). When it was assumed that the Hill coefficient had a value of 2.0, however, eggs and conceptuses appeared to contain at least two forms of Na+/K(+)-ATPase activity.« less

The 3-(bromoacetamido)-propylamine hydrochloride: A novel sulfhydryl reagent and its future potential in the configurational study of S1-myosin

NASA Technical Reports Server (NTRS)

Sharma, Prasanta; Cheung, Herbert C.

1989-01-01

Configurational study of S1-Myosin is an important step towards understanding force generation in muscle contraction. Previously reported NMR studies were corroborated. A new compound was synthesized, 3-(Bromoacetamido)-propylamine hydrochloride. Its potential as a sulfhydryl reagent provides an indirect but elegant approach towards future structural elucidation of S1-Myosin. The preliminary investigation has shown that this compound, BAAP, reacted with S1 in the absence of MgADP. The modified enzyme had a 2-fold increase in CaATPase activity and no detectable K-EDTA ATPase activity. Reaction of BAAP with S1 in the presence of MgADP resulted in a modified enzyme which retained a Ca-ATPase activity that was about 60 percent of the unmodified S1 and had essentially zero K-EDTA ATPase activity. Sulfhydryl titration indicated that about 1.5 and 3.5 SH groups per S1 molecule were blocked by BAAP in the absence and presence of MgADP, respectively. When coupled to a carboxyl group of EDTA, the resulting reagent could become a useful SH reagent in which chelated paramagnetic or luminescent lanthanide ions can be exploited to probe S1 conformation.

Impact of gasoline inhalation on some neurobehavioural characteristics of male rats.

PubMed

Kinawy, Amal A

2009-11-24

This paper examines closely and compares the potential hazards of inhalation of two types of gasoline (car fuel). The first type is the commonly use leaded gasoline and the second is the unleaded type enriched with oxygenate additives as lead substituent in order to raise the octane number. The impacts of gasoline exposure on Na+, K+-ATPase, superoxide dismutase (SOD), acetylcholinesterase (AChE), total protein, reduced glutathione (GSH), and lipid peroxidation (TBARS) in the cerebral cortex, and monoamine neurotransmitters dopamine (DA), norepinephrine (NE) and serotonin (5-HT) in the cerebral cortex, hippocampus, cerebellum and hypothalamus were evaluated. The effect of gasoline exposure on the aggressive behaviour tests was also studied. The present results revealed that gasoline inhalation induced significant fluctuations in the levels of the monoamine neurotransmitters in the studied brain regions. This was concomitant with a decrease in Na+, K+-ATPase activity and total protein content. Moreover, the group exposed to the unleaded gasoline exhibited an increase in lipid peroxidation and a decrease in AChE and superoxide dismutase activities. These physiological impairments were accompanied with a higher tendency towards aggressive behaviour as a consequence to gasoline inhalation. It is concluded from the present work that chronic exposure to either the leaded or the unleaded gasoline vapours impaired the levels of monoamine neurotransmitters and other biochemical parameters in different brain areas and modulated several behavioural aspects related to aggression in rats.

Inhibition of (Na(+)/K(+))-ATPase by Cibacron Blue 3G-A and its analogues.

PubMed

Breier, A; Bohácová, V; Docolomanský, P

2006-12-01

A specific feature of anthraquinone dyes (AD) is to mimic the adenine nucleotides ATP, ADP, NAD and NADH, enabling them to act as ligands in interaction with nucleotide-binding sites of several enzymes and receptors. In the present study, the interactions and/or inhibitory effects of eight AD, including Cibacron Blue 3G-A (Reactive Blue 2), Procion Blue MX-R (Reactive Blue 4) and Remazol Brilliant Blue R (Reactive Blue 19) on the activity of (Na(+)/K(+))-ATPase were investigated. The AD used in this paper could be divided into two groups: i) AD1-AD4 that do not contain the triazine moiety; ii) AD5-AD8 that contain the triazine moiety. Interaction affinity between the respective dye and (Na+/K+)-ATPase was characterized by means of enzyme kinetics. All AD, excluding AD1 and AD2 (which were practically ineffective) exerted effective competitive inhibition to the (Na(+)/K(+))-ATPase activity. Present study is devoted to elucidation of relationship between the inhibitory efficacy of AD against (Na(+)/K(+))-ATPase activity, their acid-basic properties and their three dimensional structure. From the results obtained, the following conclusions could be driven: 1. Similarities in the mutual position of positively and negatively charged parts of ATP and AD are responsible for their interaction with ATP-binding site of (Na(+)/K(+))-ATPase. This may be documented by fact that mutual position of 1-aminogroup of anthraquinone and -SO3(-) group of benzenesulphonate part of respective AD plays crucial role for inhibition of this enzyme. Distances of these two groups on all effective AD were found to be similar as the distance of the 6-aminogroup of adenine and the second phosphate group on ATP molecule. This similarity could be responsible for biomimetic recognition of AD in ATP-binding loci of (Na(+)/K(+))-ATPase. 2. The affinity of AD to ATP binding site of (Na(+)/K(+))-ATPase increases with increasing values of molar refractivity, i. e., with increasing molecular volume and polarizability.

Molecular Insights into the Potential Insecticidal Interaction of β-Dihydroagarofuran Derivatives with the H Subunit of V-ATPase.

PubMed

Wei, Jielu; Li, Ding; Xi, Xin; Liu, Lulu; Zhao, Ximei; Wu, Wenjun; Zhang, Jiwen

2017-10-11

Celangulin V (CV), one of dihydroagarofuran sesquiterpene polyesters isolated from Chinese bittersweet ( Celastrus angulatus Maxim), is famous natural botanical insecticide. Decades of research suggests that is displays excellent insecticidal activity against some insects, such as Mythimna separata Walker. Recently, it has been validated that the H subunit of V-ATPase is one of the target proteins of the insecticidal dihydroagarofuran sesquiterpene polyesters. As a continuation of the development of new pesticides from these natural products, a series of β-dihydroagarofuran derivatives have been designed and synthesized. The compound JW-3, an insecticidal derivative of CV with a p -fluorobenzyl group, exhibits higher insecticidal activity than CV. In this study, the potential inhibitory effect aused by the interaction of JW-3 with the H subunit of V-ATPase c was verified by confirmatory experiments at the molecular level. Both spectroscopic techniques and isothermal titration calorimetry measurements showed the binding of JW-3 to the subunit H of V-ATPase was specific and spontaneous. In addition, the possible mechanism of action of the compound was discussed. Docking results indicated compound JW-3 could bind well in 'the interdomain cleft' of the V-ATPase subunit H by the hydrogen bonding and make conformation of the ligand-protein complex become more stable. All results are the further validations of the hypothesis, that the target protein of insecticidal dihydroagarofuran sesquiterpene polyesters and their β-dihydroagarofuran derivatives is the subunit H of V-ATPase. The results also provide new ideas for developing pesticides acting on V-ATPase of insects.

Damage to lens fiber cells causes TRPV4-dependent Src family kinase activation in the epithelium.

PubMed

Shahidullah, M; Mandal, A; Delamere, N A

2015-11-01

The bulk of the lens consists of tightly packed fiber cells. Because mature lens fibers lack mitochondria and other organelles, lens homeostasis relies on a monolayer of epithelial cells at the anterior surface. The detection of various signaling pathways in lens epithelial cells suggests they respond to stimuli that influence lens function. Focusing on Src Family Kinases (SFKs) and Transient Receptor Potential Vanilloid 4 (TRPV4), we tested whether the epithelium can sense and respond to an event that occurs in fiber mass. The pig lens was subjected to localized freeze-thaw (FT) damage to fibers at posterior pole then the lens was incubated for 1-10 min in Krebs solution at 37 °C. Transient SFK activation in the epithelium was detectable at 1 min. Using a western blot approach, the ion channel TRPV4 was detected in the epithelium but was sparse or absent in fiber cells. Even though TRPV4 expression appears low at the actual site of FT damage to the fibers, SFK activation in the epithelium was suppressed in lenses subjected to FT damage then incubated with the TRPV4 antagonist HC067047 (10 μM). Na,K-ATPase activity was examined because previous studies report changes of Na,K-ATPase activity associated with SFK activation. Na,K-ATPase activity doubled in the epithelium removed from FT-damaged lenses and the response was prevented by HC067047 or the SFK inhibitor PP2 (10 μM). Similar changes were observed in response to fiber damage caused by injection of 5 μl hyperosmotic NaCl or mannitol solution beneath the surface of the posterior pole. The findings point to a TRPV4-dependent mechanism that enables the epithelial cells to detect remote damage in the fiber mass and respond within minutes by activating SFK and increasing Na,K-ATPase activity. Because TRPV4 channels are mechanosensitive, we speculate they may be stimulated by swelling of the lens structure caused by damage to the fibers. Increased Na,K-ATPase activity gives the lens greater capacity to control ion concentrations in the fiber mass and the Na,K-ATPase response may reflect the critical contribution of the epithelium to lens ion homeostasis. Published by Elsevier Ltd.

Slow and fast fatigable frog muscle fibres: electrophysiological and histochemical characteristics.

PubMed

Vydevska-Chichova, M; Mileva, K; Todorova, R; Dimitrova, M; Radicheva, N

2005-12-01

Continuous activity of isolated frog gastrocnemius muscle fibres provoked by repetitive stimulation of 5 Hz was used as an experimental model for fatigue development in different fibre types. Parameter changes of the elicited intracellular action potentials and mechanical twitches during the period of uninterrupted activity were used as criteria for fatigue evaluation. Slow fatigable muscle fibre (SMF) and fast fatigable muscle fibre (FMF) types were distinguished depending on the duration of their uninterrupted activity, which was significantly longer in SMFs than in FMFs. The normalized changes of action potential amplitude and duration were significantly smaller in FMFs than in SMFs. The average twitch force and velocity of contraction and relaxation were significantly higher in FMFs than in SMFs. Myosin ATPase (mATPase) and succinate dehydrogenase activity were studied by histochemical assessment in order to validate the fibre type classification based on their electrophysiological characteristics. Based on the relative mATPase reactivity, the fibres of the studied muscle were classified as one of five different types (1-2, 2, 2-3, 3 and tonic). Smaller sized fibres (tonic and type 3) expressed higher succinate dehydrogenase activity than larger sized fibres (type 1-2, 2), which is related to the fatigue resistance. The differences between fatigue development in SMFs and FMFs during continuous activity were associated with fibre-type specific mATPase and succinate dehydrogenase activity.

Aluminum Trichloride Induces Hypertension and Disturbs the Function of Erythrocyte Membrane in Male Rats.

PubMed

Zhang, Qiuyue; Cao, Zheng; Sun, Xudong; Zuang, Cuicui; Huang, Wanyue; Li, Yanfei

2016-05-01

Aluminum (Al) is the most abundant metal in the earth's crust. Al accumulates in erythrocyte and causes toxicity on erythrocyte membrane. The dysfunction of erythrocyte membrane is a potential risk to hypertension. The high Al content in plasma was associated with hypertension. To investigate the effect of AlCl3 on blood pressure and the function of erythrocyte membrane, the rats were intragastrically exposed to 0, 64(1/20 LD50), 128(1/10 LD50), and 256(1/5 LD50) mg/kg body weight AlCl3 in double distilled water for 120 days, respectively. Then, we determined the systolic and mean arterial blood pressures of rats, the osmotic fragility, the percentage of membrane proteins, the activities of Na(+)/K(+)-ATPase, Mg(2+)-ATPase, Ca(2+)-ATPase, catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GSH-pX), and malondialdehyde (MDA) content of the erythrocyte membrane in this experiment. The results showed that AlCl3 elevated the systolic and mean arterial blood pressure of rats, increased the osmotic fragility, decreased the percentage of membrane protein, inhibited the activities of Na(+)/K(+)-ATPase, Mg(2+)-ATPase, Ca(2+)-ATPase, CAT, SOD and GSH-pX, and increased the MDA content of erythrocyte membrane. These results indicate that AlCl3 may induce hypertension by disturbing the function of erythrocyte membrane.

Nucleotide-induced asymmetry within ATPase activator ring drives σ54-RNAP interaction and ATP hydrolysis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sysoeva, Tatyana A.; Chowdhury, Saikat; Guo, Liang

2013-12-10

It is largely unknown how the typical homomeric ring geometry of ATPases associated with various cellular activities enables them to perform mechanical work. Small-angle solution X-ray scattering, crystallography, and electron microscopy (EM) reconstructions revealed that partial ATP occupancy caused the heptameric closed ring of the bacterial enhancer-binding protein (bEBP) NtrC1 to rearrange into a hexameric split ring of striking asymmetry. The highly conserved and functionally crucial GAFTGA loops responsible for interacting with σ54–RNA polymerase formed a spiral staircase. We propose that splitting of the ensemble directs ATP hydrolysis within the oligomer, and the ring's asymmetry guides interaction between ATPase andmore » the complex of σ54 and promoter DNA. Similarity between the structure of the transcriptional activator NtrC1 and those of distantly related helicases Rho and E1 reveals a general mechanism in homomeric ATPases whereby complex allostery within the ring geometry forms asymmetric functional states that allow these biological motors to exert directional forces on their target macromolecules.« less

Pterocarpus santalinus: a traditional herbal drug as a protectant against ibuprofen induced gastric ulcers.

PubMed

Narayan, Shoba; Devi, R S; Srinivasan, P; Shyamala Devi, C S

2005-11-01

The ethanol extract of Pterocarpus santalinus (PS) was evaluated for gastroprotection in rats using ibuprofen as the induction model. Rats treated with PS (100-400 mg/kg) showed a significant reduction in gastric lesions. PS at a dose of 200 mg/kg was found to be the minimum effective dose and hence further studies with that dose were carried out. PS treatment increased the LDH activity and decreased the lipid peroxidation levels. The extract had the ability to increase the antioxidant enzymes SOD, CAT and GPx when compared with the untreated but induced rats. The membrane bound ATPases - H(+)K(+)ATPase, Na(+)K(+)ATPase and Ca(2+)ATPases were increased upon the induction with ulcerogen. The treated group showed a decrease in the activities of these enzymes and also had the ability to restore the sodium and potassium ion concentrations to near normal levels, which were altered by ibuprofen mediated acid stimulation. The results suggest that the antiulcer properties of PS could traced to its acid inhibiting potential, antioxidant activity and the ability to maintain functional integrity of the cell membranes.

A lysosomal switch triggers proteostasis renewal in the immortal C. elegans germ lineage.

PubMed

Bohnert, K Adam; Kenyon, Cynthia

2017-11-30

Although individuals age and die with time, an animal species can continue indefinitely, because of its immortal germ-cell lineage. How the germline avoids transmitting damage from one generation to the next remains a fundamental question in biology. Here we identify a lysosomal switch that enhances germline proteostasis before fertilization. We find that Caenorhabditis elegans oocytes whose maturation is arrested by the absence of sperm exhibit hallmarks of proteostasis collapse, including protein aggregation. Remarkably, sperm-secreted hormones re-establish oocyte proteostasis once fertilization becomes imminent. Key to this restoration is activation of the vacuolar H + -ATPase (V-ATPase), a proton pump that acidifies lysosomes. Sperm stimulate V-ATPase activity in oocytes by signalling the degradation of GLD-1, a translational repressor that blocks V-ATPase synthesis. Activated lysosomes, in turn, promote a metabolic shift that mobilizes protein aggregates for degradation, and reset proteostasis by enveloping and clearing the aggregates. Lysosome acidification also occurs during Xenopus oocyte maturation; thus, a lysosomal switch that enhances oocyte proteostasis in anticipation of fertilization may be conserved in other species.

miR156 modulates rhizosphere acidification in response to phosphate limitation in Arabidopsis.

PubMed

Lei, Kai Jian; Lin, Ya Ming; An, Guo Yong

2016-03-01

Rhizosphere acidification is a general response to Pi deficiency, especially in dicotyledonous plants. However, the signaling pathway underlying this process is still unclear. Here, we demonstrate that miR156 is induced in the shoots and roots of wild type Arabidopsis plants during Pi starvation. The rhizosphere acidification capacity was increased in 35S:MIR156 (miR156 overexpression) plants, but was completely inhibited in 35S:MIM156 (target mimicry) plants. Both 35S:MIR156 and 35S:MIM156 plants showed altered proton efflux and H(+)-ATPase activity. In addition, significant up-regulation of H(+)-ATPase activity in 35S:MIR156 roots coupled with increased citric acid and malic acid exudates was observed. qRT-PCR results showed that most H(+)-ATPase and PPCK gene transcript levels were decreased in 35S:MIM156 plants, which may account for the decreased H(+)-ATPase activity in 35S:MIM156 plants. MiR156 also affect the root architecture system. Collectively, our results suggest that miR156 regulates the process of rhizosphere acidification in plants.

Exogenous NO depletes Cd-induced toxicity by eliminating oxidative damage, re-establishing ATPase activity, and maintaining stress-related hormone equilibrium in white clover plants.

PubMed

Liu, S L; Yang, R J; Pan, Y Z; Wang, M H; Zhao, Y; Wu, M X; Hu, J; Zhang, L L; Ma, M D

2015-11-01

Various nitric oxide (NO) regulators [including the NO donor sodium nitroprusside (SNP), the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO), the NO-synthase inhibitor N (G)-nitro-L-Arg-methyl ester (L-NAME), and the SNP analogues sodium nitrite/nitrate and sodium ferrocyanide] were investigated to elucidate the role of NO in white clover (Trifolium repens L.) plants after long-term (5 days) exposure to cadmium (Cd). A dose of 100 μM Cd stress significantly restrained plant growth and decreased the concentrations of chlorophyll and NO in vivo, whereas it disrupted the balance of stress-related hormones and enhanced the accumulation of Cd, thereby inducing reactive oxygen species (ROS) burst. However, the inhibition of plant growth was relieved by 50 μM SNP through its stimulation of ROS-scavenging compounds (ascorbic acid, ascorbate peroxidase, catalase, glutathione reductase, non-protein thiol, superoxide dismutase, and total glutathione), regulation of H(+)-ATPase activity of proton pumps, and increasing jasmonic acid and proline but decreasing ethylene in plant tissues. Even so, the alleviating effect of SNP on plant growth was counteracted by cPTIO and L-NAME and was not observed with SNP analogues, suggesting that the protective roles of SNP are related to the induction of NO. These results suggest that NO may improve the Cd tolerance of white clover plants by eliminating oxidative damage, re-establishing ATPase activity, and maintaining hormone equilibrium. Improving our understanding of the role of NO in white clover plants is key to expanding the plantations to various regions and the recovery of pasture species in the future.

Adenosinetriphosphatase site stoichiometry in sarcoplasmic reticulum vesicles and purified enzyme.

PubMed

Barrabin, H; Scofano, H M; Inesi, G

1984-03-27

The stoichiometry of phosphorylation (catalytic) sites in sarcoplasmic reticulum vesicles ( SRV ) and SR ATPase purified by differential solubilization with deoxycholate was found to be 4.77 +/- 0.4 and 6.05 +/- 0.18 nmol/mg of protein, respectively, when phosphorylation was carried out under conditions permitting 32P labeling of nearly all sites. Assuming that each site corresponds to a single 115K ATPase chain, the observed site stoichiometry accounts only for 55% and 70% of the total protein. Failure to obtain higher phosphorylation levels was due to the presence of nonspecific protein contaminants in SRV or to the presence of inactive aggregates in the ATPase purified with deoxycholate. This was demonstrated by dissolving SRV and purified ATPase with lithium dodecyl sulfate, subjecting them to molecular sieve HPLC, and collecting the elution fractions for determination of protein, measurement of 32P-labeled sites, and electrophoretic analysis. In fact, in the specific elution peak containing the 115K ATPase chains, phosphorylation levels were 6.62 +/- 0.33 and 7.03 +/- 0.18 in SRV and purified ATPase, corresponding to 68% and 86% of the protein in the specific elution peak. An alternate purification method was then developed, based on solubilization of SRV with dodecyl octaethylene glycol monoether ( C12E8 ), separation of delipidated ATPase by anion-exchange chromatography, and enzyme reactivation with phosphatidylcholine. This preparation yields 7.3 +/- 0.44 nmol of phosphorylation site/mg of protein of the SRV fraction before HPLC.(ABSTRACT TRUNCATED AT 250 WORDS)

HDAC Inhibition Improves the Sarcoendoplasmic Reticulum Ca2+-ATPase Activity in Cardiac Myocytes.

PubMed

Meraviglia, Viviana; Bocchi, Leonardo; Sacchetto, Roberta; Florio, Maria Cristina; Motta, Benedetta M; Corti, Corrado; Weichenberger, Christian X; Savi, Monia; D'Elia, Yuri; Rosato-Siri, Marcelo D; Suffredini, Silvia; Piubelli, Chiara; Pompilio, Giulio; Pramstaller, Peter P; Domingues, Francisco S; Stilli, Donatella; Rossini, Alessandra

2018-01-31

SERCA2a is the Ca 2+ ATPase playing the major contribution in cardiomyocyte (CM) calcium removal. Its activity can be regulated by both modulatory proteins and several post-translational modifications. The aim of the present work was to investigate whether the function of SERCA2 can be modulated by treating CMs with the histone deacetylase (HDAC) inhibitor suberanilohydroxamic acid (SAHA). The incubation with SAHA (2.5 µM, 90 min) of CMs isolated from rat adult hearts resulted in an increase of SERCA2 acetylation level and improved ATPase activity. This was associated with a significant improvement of calcium transient recovery time and cell contractility. Previous reports have identified K464 as an acetylation site in human SERCA2. Mutants were generated where K464 was substituted with glutamine (Q) or arginine (R), mimicking constitutive acetylation or deacetylation, respectively. The K464Q mutation ameliorated ATPase activity and calcium transient recovery time, thus indicating that constitutive K464 acetylation has a positive impact on human SERCA2a (hSERCA2a) function. In conclusion, SAHA induced deacetylation inhibition had a positive impact on CM calcium handling, that, at least in part, was due to improved SERCA2 activity. This observation can provide the basis for the development of novel pharmacological approaches to ameliorate SERCA2 efficiency.

Direct interaction of the Golgi V-ATPase a-subunit isoform with PI(4)P drives localization of Golgi V-ATPases in yeast.

PubMed

Banerjee, Subhrajit; Kane, Patricia M

2017-09-15

Luminal pH and phosphoinositide content are fundamental features of organelle identity. Vacuolar H + -ATPases (V-ATPases) drive organelle acidification in all eukaryotes, and membrane-bound a-subunit isoforms of the V-ATPase are implicated in organelle-specific targeting and regulation. Earlier work demonstrated that the endolysosomal lipid PI(3,5)P 2 activates V-ATPases containing the vacuolar a-subunit isoform in Saccharomyces cerevisiae Here we demonstrate that PI(4)P, the predominant Golgi phosphatidylinositol (PI) species, directly interacts with the cytosolic amino terminal (NT) domain of the yeast Golgi V-ATPase a-isoform Stv1. Lysine-84 of Stv1NT is essential for interaction with PI(4)P in vitro and in vivo, and interaction with PI(4)P is required for efficient localization of Stv1-containing V-ATPases. The cytosolic NT domain of the human V-ATPase a2 isoform specifically interacts with PI(4)P in vitro, consistent with its Golgi localization and function. We propose that NT domains of V o a-subunit isoforms interact specifically with PI lipids in their organelles of residence. These interactions can transmit organelle-specific targeting or regulation information to V-ATPases. © 2017 Banerjee and Kane. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

Trypanosoma cruzi H+-ATPase 1 (TcHA1) and 2 (TcHA2) genes complement yeast mutants defective in H+ pumps and encode plasma membrane P-type H+-ATPases with different enzymatic properties.

PubMed

Luo, Shuhong; Scott, David A; Docampo, Roberto

2002-11-15

Previous studies in Trypanosoma cruzi have shown that intracellular pH homeostasis requires ATP and is affected by H(+)-ATPase inhibitors, indicating a major role for ATP-driven proton pumps in intracellular pH control. In the present study, we report the cloning and sequencing of a pair of genes linked in tandem (TcHA1 and TcHA2) in T. cruzi which encode proteins with homology to fungal and plant P-type proton-pumping ATPases. The genes are expressed at the mRNA level in different developmental stages of T. cruzi: TcHA1 is expressed maximally in epimastigotes, whereas TcHA2 is expressed predominantly in trypomastigotes. The proteins predicted from the nucleotide sequence of the genes have 875 and 917 amino acids and molecular masses of 96.3 and 101.2 kDa, respectively. Full-length TcHA1 and an N-terminal truncated version of TcHA2 complemented a Saccharomyces cerevisiae strain deficient in P-type H(+)-ATPase activity, the proteins localized to the yeast plasma membrane, and ATP-driven proton pumping could be detected in proteoliposomes reconstituted from plasma membrane purified from transfected yeast. The reconstituted proton transport activity was reduced by inhibitors of P-type H(+)-ATPases. C-terminal truncation did not affect complementation of mutant yeast, suggesting the lack of C-terminal autoinhibitory domains in these proteins. ATPase activity in plasma membrane from TcHA1- and (N-terminal truncated) TcHA2-transfected yeast was inhibited to different extents by vanadate, whereas the latter yeast strain was more resistant to extremes of pH, suggesting that the native proteins may serve different functions at different stages in the T. cruzi life cycle.

Ocean acidification induces biochemical and morphological changes in the calcification process of large benthic foraminifera.

PubMed

Prazeres, Martina; Uthicke, Sven; Pandolfi, John M

2015-03-22

Large benthic foraminifera are significant contributors to sediment formation on coral reefs, yet they are vulnerable to ocean acidification. Here, we assessed the biochemical and morphological impacts of acidification on the calcification of Amphistegina lessonii and Marginopora vertebralis exposed to different pH conditions. We measured growth rates (surface area and buoyant weight) and Ca-ATPase and Mg-ATPase activities and calculated shell density using micro-computer tomography images. In A. lessonii, we detected a significant decrease in buoyant weight, a reduction in the density of inner skeletal chambers, and an increase of Ca-ATPase and Mg-ATPase activities at pH 7.6 when compared with ambient conditions of pH 8.1. By contrast, M. vertebralis showed an inhibition in Mg-ATPase activity under lowered pH, with growth rate and skeletal density remaining constant. While M. vertebralis is considered to be more sensitive than A. lessonii owing to its high-Mg-calcite skeleton, it appears to be less affected by changes in pH, based on the parameters assessed in this study. We suggest difference in biochemical pathways of calcification as the main factor influencing response to changes in pH levels, and that A. lessonii and M. vertebralis have the ability to regulate biochemical functions to cope with short-term increases in acidity. © 2015 The Author(s) Published by the Royal Society. All rights reserved.

Cercospora beticola Toxins (X. Inhibition of Plasma Membrane H+-ATPase by Beticolin-1).

PubMed Central

Simon-Plas, F.; Gomes, E.; Milat, M. L.; Pugin, A.; Blein, J. P.

1996-01-01

Beticolin-1 is a toxin produced by the fungus Cercospora beticola. The chemical structure of this toxin was previously elucidated. The effects of beticolin-1 on purified corn root plasma membrane H+-ATPase were studied in a solubilized form or were reconstituted into liposome membranes. The ATP hydrolysis activity of the purified solubilized enzyme was inhibited by micromolar concentrations of beticolin-1, and this inhibition was noncompetitive with respect to ATP. When this purified enzyme was inserted into liposome membranes, a competitive inhibition of the H+-ATPase hydrolysis activity by beticolin-1 was observed. The effect of beticolin-1 on the formation of H+-ATPase-phosphorylated intermediate was also studied. With the purified enzyme in its solubilized form, the level of phosphorylated intermediate was not affected by the presence of beticolin-1, whereas micromolar concentrations of the toxin led to a marked inhibition of its formation when the enzyme was reconstituted into liposomes. These data suggest that (a) the plasma membrane H+-ATPase is a direct target for beticolin-1, and (b) the kinetics of inhibition and the effect on the phosphorylated intermediate are linked and both depend on the lipid environment of the enzyme. PMID:12226329

The P-type ATPase CtpG preferentially transports Cd2+ across the Mycobacterium tuberculosis plasma membrane.

PubMed

López, Marcela; Quitian, Laudy-Viviana; Calderón, Martha-Nancy; Soto, Carlos-Y

2018-04-01

P 1B -type ATPases are involved in heavy metal transport across the plasma membrane. Some Mycobacterium tuberculosis P-type ATPases are induced during infection, suggesting that this type of transporter could play a critical role in mycobacterial survival. To date, the ion specificity of M. tuberculosis heavy metal-transporting P 1B -ATPases is not well understood. In this work, we observed that, although divalent heavy metal cations such as Cu 2+ , Co 2+ , Ni 2+ , Zn 2+ Cd 2+ and Pb 2+ stimulate the ATPase activity of the putative P 1B -type ATPase CtpG in the plasma membrane, whole cells of M. smegmatis expressing CtpG only tolerate high levels of Cd 2+ and Cu 2+ . As indicator of the catalytic constant, Michaelis-Menten kinetics showed that CtpG embedded in the mycobacterial cell membrane has a V max /K m ratio 7.4-fold higher for Cd 2+ than for Cu 2+ ions. Thus, although CtpG can accept different substrates in vitro, this P-type ATPase transports Cd 2+ more efficiently than other heavy metal cations across the mycobacterial plasma membrane.

Effects of solubilization on the inhibition of the p-type ATPase from maize roots by N-(ethoxycarbonyl)-2-ethoxy-1,2-dihydroquinoline.

PubMed

Brauer, D K; Gurriel, M; Tu, S I

1992-12-01

The biochemical events utilized by transport proteins to convert the chemical energy from the hydrolysis of ATP into an electro-chemical gradient are poorly understood. The inhibition of the plasma membrane ATPase from corn (Zea mays L.) roots by N-(ethoxycarbonyl)-2-ethoxy-1,2-dihydroquinoline (EEDQ) was compared to that of ATPase solubilized with N-tetradecyl-N,N-dimethyl-3-ammonio-1-propane-sulfonate (3-14) to provide insight into the minimal functional unit. The chromatographic behavior of the 3-14-solubilized ATPase activity during size exclusion chromatography and glycerol gradient centrifugation indicated that the solubilized enzyme was in a monomeric form. Both plasma membrane-bound and solubilized ATPase were inhibited by EEDQ in a time- and concentration-dependent manner consistent with a first-order reaction. When the log of the reciprocal of the half-time for inhibition was plotted as a function of the log of the EEDQ concentration, straight lines were obtained with slopes of approximately 0.5 and 1.0 for membrane-bound and 3-14-solubilized ATPase, respectively, indicating a change in the number of polypeptides per functional ATPase complex induced by solubilization with 3-14.

The role of calcium and calmodulin in freezing-induced freezing resistance of Populus tomentosa cuttings.

PubMed

Lin, Shan-Zhi; Zhang, Zhi-Yi; Lin, Yuan-Zhen; Zhang, Qian; Guo, Huan

2004-02-01

To explore the role of calcium-calmodulin messenger system in the transduction of low temperature signal in woody plants, Populus tomentosa cuttings after being treated with CaCl(2) (10 mmol/L), Ca(2+) chelator EGTA (3 mmol/L), Ca(2+) channel inhibitor LaCl(3) (100 mmol/L) or CaM antagonist CPZ (50 mmol/L) were used for freezing acclimation at -3 degrees C. The changes in the calmodulin (CaM) and malonaldehyde (MDA) contents, the activities of superoxide dismutase (SOD), peroxidase (POD) and Ca(2+)-dependent adenosinetriphosphatase (Ca(2+)-ATPase) of mitochondrial membrane as well as freezing resistance (expressed as LT(50)) of cuttings were investigated to elucidate the physiological mechanisms by which trees adapt to freezing. The results showed that freezing acclimation increased the CaM content, the activities of SOD, POD and Ca(2+)-ATPase of mitochondrial membrane as well as freezing resistance of cuttings, and decreased the MDA content as compared with control cuttings. Treatment with CaCl(2) at the time of freezing acclimation enhanced the effect of freezing acclimation on the above-mentioned indexes, but this enhancement was abolished by Ca(2+)chelator EGTA, Ca(2+) channel inhibitor LaCl(3) or CaM antagonist CPZ, indicating that the calcium-calmodulin messenger system was involved in the course of freezing resistance development. The presence of CaCl(2) at the same time of freezing acclimation also reduced the degree of decline in CaM content, and in SOD, POD and Ca(2+)-ATPase activities caused by freezing stress at -14 degrees C, and enhanced the level of increase in CaM content, and in SOD, POD and Ca(2+)-ATPase activity in the recovery periods at 25 degrees C . The change in CaM content was found to be closely correlated to the levels of SOD, POD and Ca(2+)-ATPase, and to the degree of freezing resistance of cuttings during freezing acclimation either with or without CaCl(2) treatment. It was suggested that the increase of CaM content induced by CaCl(2) treatment promote the formation of Ca(2+)-CaM complexes, which effectively activates the activities of SOD, POD and mitochondrial Ca(2+)-ATPase and then further result in the adaptive changes associated with the development and enhancement of freezing resistance. Thus, It could be concluded that Ca(2+)-calmodulin may be involved in the regulation of the increase in SOD, POD and Ca(2+)-ATPase activities, and the induction of freezing resistance of cuttings.

[Mechanism of changes in the rate of glycolysis and levels of ATP and 2,3-diphosphoglycerate in human erythrocytes during aging].

PubMed

Bogatskaia, L N; Pisaruk, A V

1987-01-01

Reasons which have induced changes in the glycolysis rate, ATP and 2,3-diphosphoglycerate content in human erythrocytes with ageing are studied. A fall of the hexokinase activity is shown to be one of the reasons of a significant decrease in the glycolysis rate. The total ATPase activity in erythrocytes does not change with the age. At the same time the decay rate of 2,3-diphosphoglycerate increases, that, evidently, is one of the reasons of the 2,3-diphosphoglycerate content decrease in erythrocytes with ageing.

Brain Na+, K+-ATPase Activity In Aging and Disease

PubMed Central

de Lores Arnaiz, Georgina Rodríguez; Ordieres, María Graciela López

2014-01-01

Na+/K+ pump or sodium- and potassium-activated adenosine 5’-triphosphatase (Na+, K+-ATPase), its enzymatic version, is a crucial protein responsible for the electrochemical gradient across the cell membranes. It is an ion transporter, which in addition to exchange cations, is the ligand for cardenolides. This enzyme regulates the entry of K+ with the exit of Na+ from cells, being the responsible for Na+/K+ equilibrium maintenance through neuronal membranes. This transport system couples the hydrolysis of one molecule of ATP to exchange three sodium ions for two potassium ions, thus maintaining the normal gradient of these cations in animal cells. Oxidative metabolism is very active in brain, where large amounts of chemical energy as ATP molecules are consumed, mostly required for the maintenance of the ionic gradients that underlie resting and action potentials which are involved in nerve impulse propagation, neurotransmitter release and cation homeostasis. Protein phosphorylation is a key process in biological regulation. At nervous system level, protein phosphorylation is the major molecular mechanism through which the function of neural proteins is modulted in response to extracellular signals, including the response to neurotransmitter stimuli. It is the major mechanism of neural plasticity, including memory processing. The phosphorylation of Na+, K+-ATPase catalytic subunit inhibits enzyme activity whereas the inhibition of protein kinase C restores the enzyme activity. The dephosphorylation of neuronal Na+, K+-ATPase is mediated by calcineurin, a serine / threonine phosphatase. The latter enzyme is involved in a wide range of cellular responses to Ca2+ mobilizing signals, in the regulation of neuronal excitability by controlling the activity of ion channels, in the release of neurotransmitters and hormones, as well as in synaptic plasticity and gene transcription. In the present article evidence showing Na+, K+-ATPase involvement in signaling pathways, enzyme changes in diverse neurological diseases as well as during aging, have been summarized. Issues refer mainly to Na+, K+-ATPase studies in ischemia, brain injury, depression and mood disorders, mania, stress, Alzheimer´s disease, learning and memory, and neuronal hyperexcitability and epilepsy. PMID:25018677

CYP2E1 overexpression inhibits microsomal Ca2+-ATPase activity in HepG2 cells.

PubMed

Caro, Andres A; Evans, Kerry L; Cederbaum, Arthur I

2009-01-31

Cytochrome P450 2E1 (CYP2E1) is a microsomal enzyme that generates reactive oxygen species during its catalytic cycle. We previously found an important role for calcium in CYP2E1-potentiated injury in HepG2 cells. The possibility that CYP2E1 may oxidatively damage and inactivate the microsomal Ca2+-ATPase in intact liver cells was evaluated, in order to explain why calcium is elevated during CYP2E1 toxicity. Microsomes were isolated by differential centrifugation from two liver cell line: E47 cells (HepG2 cells transfected with the pCI neo expression vector containing the human CYP2E1 cDNA, which overexpress active microsomal CYP2E1), and control C34 cells (HepG2 cells transfected with the pCI neo expression vector alone, which do not express significantly any cytochrome P450). The Ca2+-dependent ATPase activity was determined by measuring the accumulation of inorganic phosphate from ATP hydrolysis. CYP2E1 overexpression produced a 45% decrease in Ca2+-dependent ATPase activity (8.6 nmol Pi/min/mg protein in C34 microsomes versus 4.7 nmol Pi/min/mg protein in microsomes). Saturation curves with Ca2+ or ATP showed that CYP2E1 overexpression produced a decrease in Vmax but did not affect the Km for either Ca2+ or ATP. The decrease in activity was not associated with a decrease in SERCA protein levels. The ATP-dependent microsomal calcium uptake was evaluated by fluorimetry using fluo-3 as the fluorogenic probe. Calcium uptake rate in E47 microsomes was 28% lower than in C34 microsomes. Treatment of E47 cells with 2mM N-acetylcysteine prevented the decrease in microsomal Ca2+-ATPase found in E47 cells. These results suggest that CYP2E1 overexpression produces a decrease in microsomal Ca2+-ATPase activity in HepG2 cells mediated by reactive oxygen species. This may contribute to elevated cytosolic calcium and to CYP2E1-potentiated injury.

P-glycoprotein substrate transport assessed by comparing cellular and vesicular ATPase activity.

PubMed

Nervi, Pierluigi; Li-Blatter, Xiaochun; Aänismaa, Päivi; Seelig, Anna

2010-03-01

We compared the P-glycoprotein ATPase activity in inside-out plasma membrane vesicles and living NIH-MDR1-G185 cells with the aim to detect substrate transport. To this purpose we used six substrates which differ significantly in their passive influx through the plasma membrane. In cells, the cytosolic membrane leaflet harboring the substrate binding site of P-glycoprotein has to be approached by passive diffusion through the lipid membrane, whereas in inside-out plasma membrane vesicles, it is accessible directly from the aqueous phase. Compounds exhibiting fast passive influx compared to active efflux by P-glycoprotein induced similar ATPase activity profiles in cells and inside-out plasma membrane vesicles, because their concentrations in the cytosolic leaflets were similar. Compounds exhibiting similar influx as efflux induced in contrast different ATPase activity profiles in cells and inside-out vesicles. Their concentration was significantly lower in the cytosolic leaflet of cells than in the cytosolic leaflet of inside-out membrane vesicles, indicating that P-glycoprotein could cope with passive influx. P-glycoprotein thus transported all compounds at a rate proportional to ATP hydrolysis (i.e. all compounds were substrates). However, it prevented substrate entry into the cytosol only if passive influx of substrates across the lipid bilayer was in a similar range as active efflux. Copyright 2009 Elsevier B.V. All rights reserved.

Leucine/glutamine and v-ATPase/lysosomal acidification via mTORC1 activation are required for position-dependent regeneration.

PubMed

Takayama, Kazuya; Muto, Akihiko; Kikuchi, Yutaka

2018-05-29

In animal regeneration, control of position-dependent cell proliferation is crucial for the complete restoration of patterned appendages in terms of both, shape and size. However, detailed mechanisms of this process are largely unknown. In this study, we identified leucine/glutamine and v-ATPase/lysosomal acidification, via mechanistic target of rapamycin complex 1 (mTORC1) activation, as effectors of amputation plane-dependent zebrafish caudal fin regeneration. mTORC1 activation, which functions in cell proliferation, was regulated by lysosomal acidification possibly via v-ATPase activity at 3 h post amputation (hpa). Inhibition of lysosomal acidification resulted in reduced growth factor-related gene expression and suppression of blastema formation at 24 and 48 hpa, respectively. Along the proximal-distal axis, position-dependent lysosomal acidification and mTORC1 activation were observed from 3 hpa. We also report that Slc7a5 (L-type amino acid transporter), whose gene expression is position-dependent, is necessary for mTORC1 activation upstream of lysosomal acidification during fin regeneration. Furthermore, treatment with leucine and glutamine, for both proximal and distal fin stumps, led to an up-regulation in cell proliferation via mTORC1 activation, indicating that leucine/glutamine signaling possesses the ability to change the position-dependent regeneration. Our findings reveal that leucine/glutamine and v-ATPase/lysosomal acidification via mTORC1 activation are required for position-dependent zebrafish fin regeneration.

The human escort protein Hep binds to the ATPase domain of mitochondrial hsp70 and regulates ATP hydrolysis.

PubMed

Zhai, Peng; Stanworth, Crystal; Liu, Shirley; Silberg, Jonathan J

2008-09-19

Hsp70 escort proteins (Hep) have been implicated as essential for maintaining the function of yeast mitochondrial hsp70 molecular chaperones (mtHsp70), but the role that escort proteins play in regulating mammalian chaperone folding and function has not been established. We present evidence that human mtHsp70 exhibits limited solubility due to aggregation mediated by its ATPase domain and show that human Hep directly enhances chaperone solubility through interactions with this domain. In the absence of Hep, mtHsp70 was insoluble when expressed in Escherichia coli, as was its isolated ATPase domain and a chimera having this domain fused to the peptide-binding domain of HscA, a soluble monomeric chaperone. In contrast, these proteins all exhibited increased solubility when expressed in the presence of Hep. In vitro studies further revealed that purified Hep regulates the interaction of mtHsp70 with nucleotides. Full-length mtHsp70 exhibited slow intrinsic ATP hydrolysis activity (6.8+/-0.2 x 10(-4) s(-1)) at 25 degrees C, which was stimulated up to 49-fold by Hep. Hep also stimulated the activity of the isolated ATPase domain, albeit to a lower maximal extent (11.5-fold). In addition, gel-filtration studies showed that formation of chaperone-escort protein complexes inhibited mtHsp70 self-association, and they revealed that Hep binding to full-length mtHsp70 and its isolated ATPase domain is strongest in the absence of nucleotides. These findings provide evidence that metazoan escort proteins regulate the catalytic activity and solubility of their cognate chaperones, and they indicate that both forms of regulation arise from interactions with the mtHsp70 ATPase domain.

The Human Escort Protein Hep Binds to the ATPase Domain of Mitochondrial Hsp70 and Regulates ATP Hydrolysis*

PubMed Central

Zhai, Peng; Stanworth, Crystal; Liu, Shirley; Silberg, Jonathan J.

2008-01-01

Hsp70 escort proteins (Hep) have been implicated as essential for maintaining the function of yeast mitochondrial hsp70 molecular chaperones (mtHsp70), but the role that escort proteins play in regulating mammalian chaperone folding and function has not been established. We present evidence that human mtHsp70 exhibits limited solubility due to aggregation mediated by its ATPase domain and show that human Hep directly enhances chaperone solubility through interactions with this domain. In the absence of Hep, mtHsp70 was insoluble when expressed in Escherichia coli, as was its isolated ATPase domain and a chimera having this domain fused to the peptide-binding domain of HscA, a soluble monomeric chaperone. In contrast, these proteins all exhibited increased solubility when expressed in the presence of Hep. In vitro studies further revealed that purified Hep regulates the interaction of mtHsp70 with nucleotides. Full-length mtHsp70 exhibited slow intrinsic ATP hydrolysis activity (6.8 ± 0.2 × 10-4 s-1) at 25 °C, which was stimulated up to 49-fold by Hep. Hep also stimulated the activity of the isolated ATPase domain, albeit to a lower maximal extent (11.5-fold). In addition, gel-filtration studies showed that formation of chaperone-escort protein complexes inhibited mtHsp70 self-association, and they revealed that Hep binding to full-length mtHsp70 and its isolated ATPase domain is strongest in the absence of nucleotides. These findings provide evidence that metazoan escort proteins regulate the catalytic activity and solubility of their cognate chaperones, and they indicate that both forms of regulation arise from interactions with the mtHsp70 ATPase domain. PMID:18632665

Regulation of the thermoalkaliphilic F1-ATPase from Caldalkalibacillus thermarum

PubMed Central

Ferguson, Scott A.; Cook, Gregory M.; Montgomery, Martin G.; Leslie, Andrew G. W.

2016-01-01

The crystal structure has been determined of the F1-catalytic domain of the F-ATPase from Caldalkalibacillus thermarum, which hydrolyzes adenosine triphosphate (ATP) poorly. It is very similar to those of active mitochondrial and bacterial F1-ATPases. In the F-ATPase from Geobacillus stearothermophilus, conformational changes in the ε-subunit are influenced by intracellular ATP concentration and membrane potential. When ATP is plentiful, the ε-subunit assumes a “down” state, with an ATP molecule bound to its two C-terminal α-helices; when ATP is scarce, the α-helices are proposed to inhibit ATP hydrolysis by assuming an “up” state, where the α-helices, devoid of ATP, enter the α3β3-catalytic region. However, in the Escherichia coli enzyme, there is no evidence that such ATP binding to the ε-subunit is mechanistically important for modulating the enzyme’s hydrolytic activity. In the structure of the F1-ATPase from C. thermarum, ATP and a magnesium ion are bound to the α-helices in the down state. In a form with a mutated ε-subunit unable to bind ATP, the enzyme remains inactive and the ε-subunit is down. Therefore, neither the γ-subunit nor the regulatory ATP bound to the ε-subunit is involved in the inhibitory mechanism of this particular enzyme. The structure of the α3β3-catalytic domain is likewise closely similar to those of active F1-ATPases. However, although the βE-catalytic site is in the usual “open” conformation, it is occupied by the unique combination of an ADP molecule with no magnesium ion and a phosphate ion. These bound hydrolytic products are likely to be the basis of inhibition of ATP hydrolysis. PMID:27621435

Moyamoya disease-associated protein mysterin/RNF213 is a novel AAA+ ATPase, which dynamically changes its oligomeric state

NASA Astrophysics Data System (ADS)

Morito, Daisuke; Nishikawa, Kouki; Hoseki, Jun; Kitamura, Akira; Kotani, Yuri; Kiso, Kazumi; Kinjo, Masataka; Fujiyoshi, Yoshinori; Nagata, Kazuhiro

2014-03-01

Moyamoya disease is an idiopathic human cerebrovascular disorder that is characterized by progressive stenosis and abnormal collateral vessels. We recently identified mysterin/RNF213 as its first susceptibility gene, which encodes a 591-kDa protein containing enzymatically active P-loop ATPase and ubiquitin ligase domains and is involved in proper vascular development in zebrafish. Here we demonstrate that mysterin further contains two tandem AAA+ ATPase modules and forms huge ring-shaped oligomeric complex. AAA+ ATPases are known to generally mediate various biophysical and mechanical processes with the characteristic ring-shaped structure. Fluorescence correlation spectroscopy and biochemical evaluation suggested that mysterin dynamically changes its oligomeric forms through ATP/ADP binding and hydrolysis cycles. Thus, the moyamoya disease-associated gene product is a unique protein that functions as ubiquitin ligase and AAA+ ATPase, which possibly contributes to vascular development through mechanical processes in the cell.

Moyamoya disease-associated protein mysterin/RNF213 is a novel AAA+ ATPase, which dynamically changes its oligomeric state

PubMed Central

Morito, Daisuke; Nishikawa, Kouki; Hoseki, Jun; Kitamura, Akira; Kotani, Yuri; Kiso, Kazumi; Kinjo, Masataka; Fujiyoshi, Yoshinori; Nagata, Kazuhiro

2014-01-01

Moyamoya disease is an idiopathic human cerebrovascular disorder that is characterized by progressive stenosis and abnormal collateral vessels. We recently identified mysterin/RNF213 as its first susceptibility gene, which encodes a 591-kDa protein containing enzymatically active P-loop ATPase and ubiquitin ligase domains and is involved in proper vascular development in zebrafish. Here we demonstrate that mysterin further contains two tandem AAA+ ATPase modules and forms huge ring-shaped oligomeric complex. AAA+ ATPases are known to generally mediate various biophysical and mechanical processes with the characteristic ring-shaped structure. Fluorescence correlation spectroscopy and biochemical evaluation suggested that mysterin dynamically changes its oligomeric forms through ATP/ADP binding and hydrolysis cycles. Thus, the moyamoya disease-associated gene product is a unique protein that functions as ubiquitin ligase and AAA+ ATPase, which possibly contributes to vascular development through mechanical processes in the cell. PMID:24658080

Time-specific and population-level differences in physiological responses of fathead minnows (Pimephales promelas) and golden shiners (Notemigonus crysoleucas) exposed to copper.

PubMed

Peles, John D; Pistole, David H; Moffe, Mickey C

2012-03-01

The influence of exposure time on gill Na+/K+ ATPase activity and metabolic rate in populations of fathead minnows (Pimephales promelas) and golden shiners (Notemigonus crysoleucas) hatcheries in Ohio (OH) and Pennsylvania (PA) when exposed to sublethal concentrations of copper (Cu) was examined. The pattern of change in gill Na+/K+ ATPase activity was similar in all species/populations and results support expectations based on the concept of acclimation. In all populations, Na+/K+ ATPase activity declined significantly compared to reference values within 24 h, recovered by 48 h, and then continued to increase before exceeding reference values by 192 h. With the exception of PA fathead minnows, Na+/K+ ATPase activities returned to reference levels by 384 h. Although metabolic rates of individual fish were not strongly correlated with Na+/K+ ATPase activities, the pattern of change in mean values of these physiological parameters was very similar. However, OH populations of both fathead minnows and golden shiners demonstrated much more dramatic changes in metabolic rate compared to PA fish. At 24 h, metabolic rate of PA fathead minnows had decreased by 16% compared to the reference value whereas the OH population had decreased by 31%; metabolic rate of PA golden shiners declined by 23% compared to 59% in OH shiners at 24 h. Similar differences were observed in the maximum metabolic rates achieved at 192 h. While the increased sensitivity of OH fish to Cu is not readily explainable by genetic or environmental factors, results suggest the need for considering population level differences when evaluating the physiological effects of toxicants. Copyright © 2011 Elsevier B.V. All rights reserved.

Structural and Biochemical Characterization of Spa47 Provides Mechanistic Insight into Type III Secretion System ATPase Activation and Shigella Virulence Regulation.

PubMed

Burgess, Jamie L; Burgess, R Alan; Morales, Yalemi; Bouvang, Jenna M; Johnson, Sean J; Dickenson, Nicholas E

2016-12-09

Like many Gram-negative pathogens, Shigella rely on a complex type III secretion system (T3SS) to inject effector proteins into host cells, take over host functions, and ultimately establish infection. Despite these critical roles, the energetics and regulatory mechanisms controlling the T3SS and pathogen virulence remain largely unclear. In this study, we present a series of high resolution crystal structures of Spa47 and use the structures to model an activated Spa47 oligomer, finding that ATP hydrolysis may be supported by specific side chain contributions from adjacent protomers within the complex. Follow-up mutagenesis experiments targeting the predicted active site residues validate the oligomeric model and determined that each of the tested residues are essential for Spa47 ATPase activity, although they are not directly responsible for stable oligomer formation. Although N-terminal domain truncation was necessary for crystal formation, it resulted in strictly monomeric Spa47 that is unable to hydrolyze ATP, despite maintaining the canonical ATPase core structure and active site residues. Coupled with studies of ATPase inactive full-length Spa47 point mutants, we find that Spa47 oligomerization and ATP hydrolysis are needed for complete T3SS apparatus formation, a proper translocator secretion profile, and Shigella virulence. This work represents the first structure-function characterization of Spa47, uniquely complementing the multitude of included Shigella T3SS phenotype assays and providing a more complete understanding of T3SS ATPase-mediated pathogen virulence. Additionally, these findings provide a strong platform for follow-up studies evaluating regulation of Spa47 oligomerization in vivo as a much needed means of treating and perhaps preventing shigellosis. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Structural and Biochemical Characterization of Spa47 Provides Mechanistic Insight into Type III Secretion System ATPase Activation and Shigella Virulence Regulation*

PubMed Central

Burgess, Jamie L.; Burgess, R. Alan; Morales, Yalemi; Bouvang, Jenna M.; Johnson, Sean J.; Dickenson, Nicholas E.

2016-01-01

Like many Gram-negative pathogens, Shigella rely on a complex type III secretion system (T3SS) to inject effector proteins into host cells, take over host functions, and ultimately establish infection. Despite these critical roles, the energetics and regulatory mechanisms controlling the T3SS and pathogen virulence remain largely unclear. In this study, we present a series of high resolution crystal structures of Spa47 and use the structures to model an activated Spa47 oligomer, finding that ATP hydrolysis may be supported by specific side chain contributions from adjacent protomers within the complex. Follow-up mutagenesis experiments targeting the predicted active site residues validate the oligomeric model and determined that each of the tested residues are essential for Spa47 ATPase activity, although they are not directly responsible for stable oligomer formation. Although N-terminal domain truncation was necessary for crystal formation, it resulted in strictly monomeric Spa47 that is unable to hydrolyze ATP, despite maintaining the canonical ATPase core structure and active site residues. Coupled with studies of ATPase inactive full-length Spa47 point mutants, we find that Spa47 oligomerization and ATP hydrolysis are needed for complete T3SS apparatus formation, a proper translocator secretion profile, and Shigella virulence. This work represents the first structure-function characterization of Spa47, uniquely complementing the multitude of included Shigella T3SS phenotype assays and providing a more complete understanding of T3SS ATPase-mediated pathogen virulence. Additionally, these findings provide a strong platform for follow-up studies evaluating regulation of Spa47 oligomerization in vivo as a much needed means of treating and perhaps preventing shigellosis. PMID:27770024

Increased Expression of P-Glycoprotein Is Associated With Chlorpyrifos Resistance in the German Cockroach (Blattodea: Blattellidae).

PubMed

Hou, Weiyuan; Jiang, Chu; Zhou, Xiaojie; Qian, Kun; Wang, Lei; Shen, Yanhui; Zhao, Yan

2016-12-01

A principal method for control of the German cockroach, Blattella germanica (L.), is the broad-spectrum organophosphorus insecticide, chlorpyrifos (O,O-diethyl O-3,5,6-trichloro-2-pyridyl phosphorothioate); however, extensive and repeated application has resulted in the development of resistance to chlorpyrifos in this insect. Evidence suggests that ATP-binding cassette protein transporters, including P-glycoprotein, are involved in insecticide resistance. However, little is known of the role of P-glycoprotein in insecticide resistance in the German cockroach. Here, we developed a chlorpyrifos-resistant strain of German cockroach and investigated the relationship between P-glycoprotein and chlorpyrifos resistance using toxicity assays; inhibition studies with two P-glycoprotein inhibitors, verapamil and quinine; P-glycoprotein-ATPase activity assays; and western blotting analysis. After 23 generations of selection from susceptible strain cockroaches, we obtained animals with high resistance to chlorpyrifos. When P-glycoprotein-ATPase activity was inhibited by verapamil and quinine, we observed enhanced susceptibility to chlorpyrifos in both control and chlorpyrifos-resistant cockroaches. No significant alterations of P-glycoprotein expression or ATPase activity were observed in cockroaches acutely exposed to LD50 doses of chlorpyrifos for 24 h, while P-glycoprotein expression and ATPase activity were clearly elevated in the chlorpyrifos-resistant cockroach strain. Thus, we conclude that P-glycoprotein is associated with chlorpyrifos resistance in the German cockroach and that elevated levels of P-glycoprotein expression and ATPase activity may be an important mechanism of chlorpyrifos resistance in the German cockroach. © The Authors 2016. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Tension cost correlates with mechanical and biochemical parameters in different myocardial contractility conditions

PubMed Central

Moreira, Cleci M.; Meira, Eduardo F.; Vestena, Luis; Stefanon, Ivanita; Vassallo, Dalton V.; Padilha, Alessandra S.

2012-01-01

OBJECTIVES: Tension cost, the ratio of myosin ATPase activity to tension, reflects the economy of tension development in the myocardium. To evaluate the mechanical advantage represented by the tension cost, we studied papillary muscle contractility and the activity of myosin ATPase in the left ventricles in normal and pathophysiological conditions. METHODS: Experimental protocols were performed using rat left ventricles from: (1) streptozotocin-induced diabetic and control Wistar rats; (2) N-nitro-L-arginine methyl ester (L-NAME) hypertensive and untreated Wistar rats; (3) deoxycorticosterone acetate (DOCA) salt-treated, nephrectomized and salt- and DOCA-treated rats; (4) spontaneous hypertensive rats (SHR) and Wistar Kyoto (WKY) rats; (5) rats with myocardial infarction and sham-operated rats. The isometric force, tetanic tension, and the activity of myosin ATPase were measured. RESULTS: The results obtained from infarcted, diabetic, and deoxycorticosterone acetate-salt-treated rats showed reductions in twitch and tetanic tension compared to the control and sham-operated groups. Twitch and tetanic tension increased in the N-nitro-L-arginine methyl ester-treated rats compared with the Wistar rats. Myosin ATPase activity was depressed in the infarcted, diabetic, and deoxycorticosterone acetate salt-treated rats compared with control and sham-operated rats and was increased in N-nitro-L-arginine methyl ester-treated rats. These parameters did not differ between SHR and WKY rats. In the studied conditions (e.g., post-myocardial infarction, deoxycorticosterone acetate salt-induced hypertension, chronic N-nitro-L-arginine methyl ester treatment, and streptozotocin-induced diabetes), a positive correlation between force or plateau tetanic tension and myosin ATPase activity was observed. CONCLUSION: Our results suggest that the myocardium adapts to force generation by increasing or reducing the tension cost to maintain myocardial contractility with a better mechanical advantage. PMID:22666794

Akt Substrate of 160 kD Regulates Na+,K+-ATPase Trafficking in Response to Energy Depletion and Renal Ischemia

PubMed Central

Alves, Daiane S.; Thulin, Gunilla; Loffing, Johannes; Kashgarian, Michael

2015-01-01

Renal ischemia and reperfusion injury causes loss of renal epithelial cell polarity and perturbations in tubular solute and fluid transport. Na+,K+-ATPase, which is normally found at the basolateral plasma membrane of renal epithelial cells, is internalized and accumulates in intracellular compartments after renal ischemic injury. We previously reported that the subcellular distribution of Na+,K+-ATPase is modulated by direct binding to Akt substrate of 160 kD (AS160), a Rab GTPase-activating protein that regulates the trafficking of glucose transporter 4 in response to insulin and muscle contraction. Here, we investigated the effect of AS160 on Na+,K+-ATPase trafficking in response to energy depletion. We found that AS160 is required for the intracellular accumulation of Na+,K+-ATPase that occurs in response to energy depletion in cultured epithelial cells. Energy depletion led to dephosphorylation of AS160 at S588, which was required for the energy depletion–induced accumulation of Na,K-ATPase in intracellular compartments. In AS160-knockout mice, the effects of renal ischemia on the distribution of Na+,K+-ATPase were substantially reduced in the epithelial cells of distal segments of the renal tubules. These data demonstrate that AS160 has a direct role in linking the trafficking of Na+,K+-ATPase to the energy state of renal epithelial cells. PMID:25788531

P-type ATPases as drug targets: tools for medicine and science.

PubMed

Yatime, Laure; Buch-Pedersen, Morten J; Musgaard, Maria; Morth, J Preben; Lund Winther, Anne-Marie; Pedersen, Bjørn P; Olesen, Claus; Andersen, Jens Peter; Vilsen, Bente; Schiøtt, Birgit; Palmgren, Michael G; Møller, Jesper V; Nissen, Poul; Fedosova, Natalya

2009-04-01

P-type ATPases catalyze the selective active transport of ions like H+, Na+, K+, Ca2+, Zn2+, and Cu2+ across diverse biological membrane systems. Many members of the P-type ATPase protein family, such as the Na+,K+-, H+,K+-, Ca2+-, and H+-ATPases, are involved in the development of pathophysiological conditions or provide critical function to pathogens. Therefore, they seem to be promising targets for future drugs and novel antifungal agents and herbicides. Here, we review the current knowledge about P-type ATPase inhibitors and their present use as tools in science, medicine, and biotechnology. Recent structural information on a variety of P-type ATPase family members signifies that all P-type ATPases can be expected to share a similar basic structure and a similar basic machinery of ion transport. The ion transport pathway crossing the membrane lipid bilayer is constructed of two access channels leading from either side of the membrane to the ion binding sites at a central cavity. The selective opening and closure of the access channels allows vectorial access/release of ions from the binding sites. Recent structural information along with new homology modeling of diverse P-type ATPases in complex with known ligands demonstrate that the most proficient way for the development of efficient and selective drugs is to target their ion transport pathway.

FTIR Study of ATP-Induced Changes in Na+/K+-ATPase from Duck Supraorbital Glands

PubMed Central

Pratap, Promod R.; Dediu, Oana; Nienhaus, G. Ulrich

2003-01-01

The Na+/K+-ATPase uses energy from the hydrolysis of ATP to pump Na+ ions out of and K+ ions into the cell. ATP-induced conformational changes in the protein have been examined in the Na+/K+-ATPase isolated from duck supraorbital salt glands using Fourier transform infrared spectroscopy. Both standard transmission and attenuated total internal reflection sample geometries have been employed. Under transmission conditions, enzyme at 75 mg/ml was incubated with dimethoxybenzoin-caged ATP. ATP was released by flashing with a UV laser pulse at 355 nm, which resulted in a large change in the amide I band. The absorbance at 1659 cm−1 decreased with a concomitant increase in the absorbance at 1620 cm−1. These changes are consistent with a partial conversion of protein secondary structure from α-helix to β-sheet. The changes were ∼8% of the total absorbance, much larger than those seen with other P-type ATPases. Using attenuated total internal reflection Fourier transform infrared spectroscopy, the decrease in absorbance at ∼1650 cm−1 was titrated with ATP, and the titration midpoint K0.5 was determined under different ionic conditions. In the presence of metal ions (Na+, Na+ and K+, or Mg2+), K0.5 was on the order of a few μM. In the absence of these ions, K0.5 was an order of magnitude lower (0.1 μM), indicating a higher apparent affinity. This effect suggests that the equilibrium for the ATP-induced conformational changes is dependent on the presence of metal ions. PMID:14645062

Endogenous strychnine: description of hypo- and hyperstrychninergic state in relation to neuropsychiatric diseases.

PubMed

Kurup, Ravi Kumar; Kurup, Parameswara Achutha

2002-10-01

Previous work from our laboratory has demonstrated the presence of endogenous strychnine in the mammalian brain and human serum samples. The present study examines the role of strychnine in neuropsychiatric disorders. Strychnine is synthesized from tryptophan. The blood levels of tyrosine, tryptophan, and strychnine were studied as also RBC membrane Na(+)-K+ ATPase activity. It was found that serum tyrosine levels were reduced and that tryptophan levels were elevated in all neuropsychiatric disorders studied with a reduction in RBC Na(+)-K+ ATPase activity. Strychnine was present in significant amounts in the serum of patients with epilepsy, Parkinson's disease, and manic depressive psychosis. The presence of strychnine in significant amounts could be related to elevated tryptophan levels, suggesting the synthesis of these alkaloids from tryptophan. Na(+)-K+ ATPase inhibition present in most of the disorders could be related to increased depolarizing strychninergic transmission. The role of strychnine in the pathogenesis of these disorders, in the setting of membrane Na(+)-K+ ATPase inhibition, is discussed.

Structural Basis for Disassembly of Katanin Heterododecamers.

PubMed

Nithianantham, Stanley; McNally, Francis J; Al-Bassam, Jawdat

2018-05-11

The reorganization of microtubules in mitosis, meiosis and development requires the microtubule-severing activity of katanin.  Katanin is a heterodimer composed of an ATPase Associated with diverse cellular Activities (AAA) subunit and a regulatory subunit. Microtubule severing requires ATP hydrolysis by katanin's conserved AAA ATPase domains. Whereas other AAA ATPases form stable hexamers, we show that katanin only forms monomer or dimers of heterodimers in solution.  Katanin oligomers consistent with hexamers of heterodimers or heterododecamers were only observed for an ATP hydrolysis deficient mutant in the presence of ATP.  X-ray structures of katanin's AAA ATPase in monomeric nucleotide-free and pseudo-oligomeric ADP-bound states reveal conformational changes in AAA subdomains that explained the structural basis for instability of katanin heterododecamer.  We propose that the rapid dissociation of katanin AAA oligomers may lead to an auto-inhibited state that prevents inappropriate microtubule severing, or that cyclical disassembly into heterodimers may critically contribute to the microtubule-severing mechanism. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.

Structural Insights into the Allosteric Operation of the Lon AAA+ Protease.

PubMed

Lin, Chien-Chu; Su, Shih-Chieh; Su, Ming-Yuan; Liang, Pi-Hui; Feng, Chia-Cheng; Wu, Shih-Hsiung; Chang, Chung-I

2016-05-03

The Lon AAA+ protease (LonA) is an evolutionarily conserved protease that couples the ATPase cycle into motion to drive substrate translocation and degradation. A hallmark feature shared by AAA+ proteases is the stimulation of ATPase activity by substrates. Here we report the structure of LonA bound to three ADPs, revealing the first AAA+ protease assembly where the six protomers are arranged alternately in nucleotide-free and bound states. Nucleotide binding induces large coordinated movements of conserved pore loops from two pairs of three non-adjacent protomers and shuttling of the proteolytic groove between the ATPase site and a previously unknown Arg paddle. Structural and biochemical evidence supports the roles of the substrate-bound proteolytic groove in allosteric stimulation of ATPase activity and the conserved Arg paddle in driving substrate degradation. Altogether, this work provides a molecular framework for understanding how ATP-dependent chemomechanical movements drive allosteric processes for substrate degradation in a major protein-destruction machine. Copyright © 2016 Elsevier Ltd. All rights reserved.

Evidence that the platelet plasma membrane does not contain a (Ca2+ + Mg2+)-dependent ATPase.

PubMed

Steiner, B; Lüscher, E F

1985-09-10

The present study was designed to determine the subcellular distribution of the platelet (Ca2+ + Mg2+)-ATPase. Human platelets were surface labeled by the periodate-boro[3H]hydride method. Plasma membrane vesicles were then isolated to a purity of approx. 90% by a procedure utilizing wheat germ agglutinin affinity chromatography. These membranes were found to be 2.6-fold enriched in surface glycoproteins compared to an unfractionated vesicle fraction and almost 7-fold enriched compared to intact platelets. In contrast, the isolated plasma membranes showed a decreased specific activity of the (Ca2+ + Mg2+)-ATPase compared to the unfractionated vesicle fraction. This decrease in specific activity was found to be similar to that of an endoplasmic reticulum marker, glucose-6-phosphatase, and to that of a platelet inner membrane marker, phospholipase A2. We conclude, therefore, that the (Ca2+ + Mg2+)-ATPase is not located in the platelet plasma membrane but is restricted to membranes of intracellular origin.

Reproductive organ and vascular specific promoter of the rice plasma membrane Ca2+ATPase mediates environmental stress responses in plants.

PubMed

Huda, Kazi Md Kamrul; Banu, Mst Sufara Akhter; Pathi, Krishna Mohan; Tuteja, Narendra

2013-01-01

Plasma membrane Ca(2+)ATPase is a transport protein in the plasma membrane of cells and helps in removal of calcium (Ca(2+)) from the cell, hence regulating Ca(2+) level within cells. Though plant Ca(2+)ATPases have been shown to be involved in plant stress responses but their promoter regions have not been well studied. The 1478 bp promoter sequence of rice plasma membrane Ca(2+)ATPase contains cis-acting elements responsive to stresses and plant hormones. To identify the functional region, serial deletions of the promoter were fused with the GUS sequence and four constructs were obtained. These were differentially activated under NaCl, PEG cold, methyl viologen, abscisic acid and methyl jasmonate treatments. We demonstrated that the rice plasma membrane Ca(2+)ATPase promoter is responsible for vascular-specific and multiple stress-inducible gene expression. Only full-length promoter showed specific GUS expression under stress conditions in floral parts. High GUS activity was observed in roots with all the promoter constructs. The -1478 to -886 bp flanking region responded well upon treatment with salt and drought. Only the full-length promoter presented cold-induced GUS expression in leaves, while in shoots slight expression was observed for -1210 and -886 bp flanking region. The -1210 bp deletion significantly responded to exogenous methyl viologen and abscisic acid induction. The -1210 and -886 bp flanking region resulted in increased GUS activity in leaves under methyl jasmonate treatments, whereas in shoots the -886 bp and -519 bp deletion gave higher expression. Salicylic acid failed to induce GUS activities in leaves for all the constructs. The rice plasma membrane Ca(2+)ATPase promoter is a reproductive organ-specific as well as vascular-specific. This promoter contains drought, salt, cold, methyl viologen, abscisic acid and methyl jasmonate related cis-elements, which regulated gene expression. Overall, the tissue-specificity and inducible nature of this promoter could grant wide applicability in plant biotechnology.

Novel Sulfur Metabolites of Garlic Attenuate Cardiac Hypertrophy and Remodeling through Induction of Na+/K+-ATPase Expression

PubMed Central

Khatua, Tarak N.; Borkar, Roshan M.; Mohammed, Soheb A.; Dinda, Amit K.; Srinivas, R.; Banerjee, Sanjay K.

2017-01-01

Epidemiologic studies show an inverse correlation between garlic consumption and progression of cardiovascular disease. However, the molecular basis for the beneficial effect of garlic on the heart is not known. Therefore, the objective of the present study was to (1) investigate the effect of raw garlic on isoproterenol (Iso) induced cardiac hypertrophy (2) find the active metabolites of garlic responsible for the beneficial effect. Cardiac hypertrophy was induced in rats by subcutaneous single injection of Iso 5 mg kg-1 day-1 for 15 days and the effect of garlic (250 mg/kg/day orally) was evaluated. Garlic metabolites in in vivo were identified by LC/MS study. The effect of garlic and its metabolites were evaluated against hypertrophy in H9C2 cells. Garlic normalized cardiac oxidative stress after Iso administration. Cardiac pathology and mitochondrial enzyme activities were improved in hypertrophy heart after garlic administration. Decreased Na+/K+-ATPase protein level that observed in hypertrophy heart was increased after garlic administration. We identified three garlic metabolites in rat serum. To confirm the role of garlic metabolites on cardiac hypertrophy, Na+/K+-ATPase expression and intracellular calcium levels were measured after treating H9C2 cells with raw garlic and two of its active metabolites, allyl methyl sulfide and allyl methyl sulfoxide. Raw garlic and both metabolites increased Na+/K+-ATPase protein level and decreased intracellular calcium levels and cell size in Iso treated H9C2 cells. This antihypertrophic effect of garlic and its sulfur metabolites were lost in H9C2 cells in presence of Na+/K+-ATPase inhibitor. In conclusion, garlic and its active metabolites increased Na+/K+-ATPase in rat heart, and attenuated cardiac hypertrophy and associated remodeling. Our data suggest that identified new garlic metabolites may be useful for therapeutic intervention against cardiac hypertrophy. PMID:28194108

[Changes in ion transport through membranes, ATPase activity and antibiotics effects in Enterococcus hirae after low intensity electromagnetic irradiation of 51,8 and 53,0 GHz frequencies].

PubMed

Torgomian, É; Oganian, V; Blbulian, C; Trchunian, A

2013-01-01

It was ascertained that one-hour exposure of Enterococcus hirae ATCC9790 bacteria grown under anaerobe condition during sugar (glucose) fermentation to coherent electromagnetic irradiation (EMI) of 51,8 and 53,0 GHz frequencies or millimeter waves (5,79 and 5,66 mm wavelengths) of low-intensity (flux capacity of 0,06 mW/sm2) caused a significant decrease in energy-dependent H+ and K+ transports across the membranes of whole cells. Therewith, K+ influx into cells was appreciably less at the frequency of 53,0 GHz. Likewise, a significant decrease of total and N,N'-dicyclohexylcarbodiimide-sensitive ATPase activity of the membrane vesicles occurred after EMI of 51,8 and 53,0 GHz. These results indicated the input of membranous changes in bacterial action of low intensity extremely high frequency EMI, when the F0F1-ATPase was probably playing a key role. Additionally, the enhancement of the effects of antibiotics--ceftriaxone, kanamycin and ampicillin at their minimal inhibitory concentrations (100, 200 and 1,4 microM, correspondingly) on the bacterial growth by these irradiations was shown. Also, combined action of EMI and antibiotics depressed strongly H+ and K+ fluxes across membrane. Especially, H+ flux was more sensitive to the action of ceftriaxone, but K+ flux was sensitive to kanamycin. All these made the assumption that EMI of 51,8 and 53,0 GHz frequencies, especially 53,0 GHz, was followed by change in bacterial sensitivity toward antibiotics that was more obvious with ceftriaxone and ampicillin.

Aniseed oil increases glucose absorption and reduces urine output in the rat.

PubMed

Kreydiyyeh, Sawsan Ibrahim; Usta, Julnar; Knio, Khuzama; Markossian, Sarine; Dagher, Shawky

2003-12-19

Anise (Pimpinella anisum) has been used as a traditional aromatic herb in many drinks and baked foods because of the presence of volatile oils in its fruits commonly known as seeds. Hot water extracts of the seeds have been used also in folk medicine for their diuretic and laxative effect, expectorant and anti-spasmodic action, and their ability to ease intestinal colic and flatulence. The aim of this work was to study the effect of aniseed oil on transport processes through intestinal and renal epithelia and determine its mechanism of action. The essential oils were extracted from the seeds by hydrodistillation and analyzed by gas chromatography. Aniseed oil enhanced significantly glucose absorption from the rat jejunum and increased the Na+-K+ ATPase activity in a jejunal homogenate in a dose dependent manner. The oil, however, exerted no effect on water absorption from the colon and did not alter the activity of the colonic Na+-K+ ATPase. When added to drinking water, it reduced the volume of urine produced in the rat and increased the activity of the renal Na+-K+ ATPase even at extremely low concentrations. It was concluded that aniseed oil increases glucose absorption by increasing the activity of the Na+-K+ ATPase and consequently the sodium gradient needed for the sugar transport. Its anti-diuretic effect is also mediated through a similar mechanism in the kidney whereby a stimulation of the Na+-K+ pump increases tubular sodium reabsorption and osmotic water movement. The colonic Na+-K+ ATPase was however, resistant to the oil.

A structural and kinetic study on myofibrils prevented from shortening by chemical cross-linking.

PubMed

Herrmann, C; Sleep, J; Chaussepied, P; Travers, F; Barman, T

1993-07-20

In previous work, we studied the early steps of the Mg(2+)-ATPase activity of Ca(2+)-activated myofibrils [Houadjeto, M., Travers, F., & Barman, T. (1992) Biochemistry 31, 1564-1569]. The myofibrils were free to contract, and the results obtained refer to the ATPase cycle of myofibrils contracting with no external load. Here we studied the ATPase of myofibrils contracting isometrically. To prevent shortening, we cross-linked them with 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDC). SDS-PAGE and Western blot analyses showed that the myosin rods were extensively cross-linked and that 8% of the myosin heads were cross-linked to the thin filament. The transient kinetics of the cross-linked myofibrils were studied in 0.1 M potassium acetate, pH 7.4 and 4 degrees C, by the rapid-flow quench method. The ATP binding steps were studied by the cold ATP chase and the cleavage and release of products steps by the Pi burst method. In Pi burst experiments, the sizes of the bursts were equal within experimental error to the ATPase site concentrations (as determined by the cold ATP chase methods) for both cross-linked (isometric) and un-cross-linked (isotonic) myofibrils. This shows that in both cases the rate-limiting step is after the cleavage of ATP. When cross-linked, the kcat of Ca(2+)-activated myofibrils was reduced from 1.7 to 0.8 s-1. This is consistent with the observation that fibers shortening at moderate velocity have a higher ATPase activity than isometric fibers.(ABSTRACT TRUNCATED AT 250 WORDS)

Ovine cardiac Na,K-ATPase: isolation by means of selective solubilization in Lubrol and the effect of 1 alpha,2 alpha-epoxyscillirosidin on this enzyme.

PubMed

Venter, P A; Naudé, R J; Oelofsen, W; Swan, G E

1997-01-01

The inhibition of cardiac Na,K-ATPase by 1 alpha,2 alpha-epoxyscillirosidin is the principal cause of poisoning of cattle by the tulip, Homeria pallida. The ultimate goals of this study were to study the interaction between 1 alpha,2 alpha-epoxyscillirosidin and ovine Na,K-ATPase by means of inhibition and displacement binding studies. Ovine cardiac Na,K-ATPase was isolated in membrane-bound form by means of deoxycholate treatment, high-speed ultracentrifugation, NaI treatment and selective solubilization in Lubrol. The inhibition of ovine cardiac and commercial porcine cerebral cortex Na,K-ATPase by 1 alpha,2 alpha-epoxyscilirosidin and ouabain was studied using a discontinuous Na,K-ATPase assay. The binding of 1 alpha,2 alpha-epoxyscillirosidin, ouabain and digoxin to the above enzymes was compared using a displacement binding assay with [3H] oubain. The Lubrol-solubilized ovine cardiac Na,K-ATPase showed a specific activity of 0.3 U/mg with no ouabain insensitive activity. I50 values of 2.1 x 10(-8) and 2.7 x 10(-8) were obtained for the inhibition of this enzyme by 1 alpha,2 alpha-epoxyscillirosidin and ouabain, respectively. 1 alpha,2 alpha-Epoxyscillirosidin has a much higher KD value (1.5 x 10(-7) M), however, than ouabain (9.5 x 10(-9) M) and digoxin (1.7 x 10(-8) M) in displacement binding studies with [3H]ouabain. 1 alpha,2 alpha-Epoxyscillirosidin is a potent inhibitor of ovine cardiac Na,K-ATPase and is a slightly stronger inhibitor of the enzyme than ouabain. The anomalous result for the displacement of 1 alpha,2 alpha-epoxyscillirosidin from its receptor is either a result of different affinities that K+ has for the enzyme ouabain and enzyme-1 alpha,2 alpha-epoxyscillirosidin complexes or because of different complex stabilities of these complexes.

Identification of Zebrafish Fxyd11a Protein that is Highly Expressed in Ion-Transporting Epithelium of the Gill and Skin and its Possible Role in Ion Homeostasis

PubMed Central

Saito, Kaori; Nakamura, Nobuhiro; Ito, Yusuke; Hoshijima, Kazuyuki; Esaki, Masahiro; Zhao, Boqiang; Hirose, Shigehisa

2010-01-01

FXYD proteins, small single-transmembrane proteins, have been proposed to be auxiliary regulatory subunits of Na+–K+-ATPase and have recently been implied in ion osmoregulation of teleost fish. In freshwater (FW) fish, numerous ions are actively taken up through mitochondrion-rich cells (MRCs) of the gill and skin epithelia, using the Na+ electrochemical gradient generated by Na+–K+-ATPase. In the present study, to understand the molecular mechanism for the regulation of Na+–K+-ATPase in MRCs of FW fish, we sought to identify FXYD proteins expressed in MRCs of zebrafish. Reverse-transcriptase PCR studies of adult zebrafish tissues revealed that, out of eight fxyd genes found in zebrafish database, only zebrafish fxyd11 (zfxyd11) mRNA exhibited a gill-specific expression. Double immunofluorescence staining showed that zFxyd11 is abundantly expressed in MRCs rich in Na+–K+-ATPase (NaK-MRCs) but not in those rich in vacuolar-type H+-transporting ATPase. An in situ proximity ligation assay demonstrated its close association with Na+–K+-ATPase in NaK-MRCs. The zfxyd11 mRNA expression was detectable at 1 day postfertilization, and its expression levels in the whole larvae and adult gills were regulated in response to changes in environmental ionic concentrations. Furthermore, knockdown of zFxyd11 resulted in a significant increase in the number of Na+–K+-ATPase–positive cells in the larval skin. These results suggest that zFxyd11 may regulate the transport ability of NaK-MRCs by modulating Na+–K+-ATPase activity, and may be involved in the regulation of body fluid and electrolyte homeostasis. PMID:21423371

Selective inhibition of tumor cell associated Vacuolar-ATPase 'a2' isoform overcomes cisplatin resistance in ovarian cancer cells.

PubMed

Kulshrestha, Arpita; Katara, Gajendra K; Ginter, Jordyn; Pamarthy, Sahithi; Ibrahim, Safaa A; Jaiswal, Mukesh K; Sandulescu, Corina; Periakaruppan, Ramayee; Dolan, James; Gilman-Sachs, Alice; Beaman, Kenneth D

2016-06-01

Development of resistance to platinum compounds significantly hinders successful ovarian cancer (OVCA) treatment. In tumor cells, dysregulated pH gradient across cell membranes is a key physiological mechanism of metastasis/chemo-resistance. These pH alterations are mediated by aberrant activation of key multi-subunit proton pumps, Vacuolar-ATPases (V-ATPases). In tumor cells, its 'a2' isoform (V-ATPase-V0a2) is a component of functional plasma-membrane complex and promotes tumor invasion through tumor-acidification and immuno-modulation. Its involvement in chemo-resistance has not been studied. Here, we show that V-ATPase-V0a2 is over-expressed in acquired-cisplatin resistant OVCA cells (cis-A2780/cis-TOV112D). Of all the 'a' subunit isoforms, V-ATPase-V0a2 exhibited an elevated expression on plasma membrane of cisplatin-resistant cells compared to sensitive counterparts. Immuno-histochemistry revealed V-ATPase-V0a2 expression in both low grade (highly drug-resistant) and high grade (highly recurrent) human OVCA tissues indicating its role in a centralized mechanism of tumor resistance. In cisplatin resistant cells, shRNA mediated inhibition of V-ATPase-V0a2 enhanced sensitivity towards both cisplatin and carboplatin. This improved cytotoxicity was mediated by enhanced cisplatin-DNA-adduct formation and suppressed DNA-repair pathway, leading to enhanced apoptosis. Suppression of V0a2 activity strongly reduced cytosolic pH in resistant tumor cells, which is known to enhance platinum-associated DNA-damage. As an indicator of reduced metastasis and chemo-resistance, in contrast to plasma membrane localization, a diffused cytoplasmic localization of acidic vacuoles was observed in V0a2-knockdown resistant cells. Interestingly, pre-treatment with monoclonal V0a2-inhibitory antibody enhanced cisplatin cytotoxicity in resistant cells. Taken together, our findings suggest that the isoform specific inhibition of V-ATPase-V0a2 could serve as a therapeutic strategy for chemo-resistant ovarian carcinoma and improve efficacy of platinum drugs. Copyright © 2016 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Metabolic and morphologic properties of single muscle fibers in the rat after spaceflight, Cosmos 1887

NASA Technical Reports Server (NTRS)

Miu, B.; Martin, T. P.; Roy, R. R.; Oganov, V.; Ilyina-Kakueva, E.; Marini, J. F.; Leger, J. J.; Bodine-Fowler, S. C.; Edgerton, V. R.

1990-01-01

The adaptation of a slow (soleus, Sol) and a fast (medial gastrocnemius, MG) skeletal muscle to spaceflight was studied in five young male rats. The flight period was 12.5 days and the rats were killed approximately 48 h after returning to 1 g. Five other rats that were housed in cages similar to those used by the flight rats were maintained at 1 g for the same period of time to serve as ground-based controls. Fibers were classified as dark or light staining for myosin adenosine triphosphatase (ATPase). On the average, the fibers in the Sol of the flight rats atrophied twice as much as those in the MG. Further, the fibers located in the deep (close to the bone and having the highest percentage of light ATPase and high oxidative fibers in the muscle cross section) region of the MG atrophied more than the fibers located in the superficial (away from the bone and having the lowest percentage of light ATPase and high oxidative fibers in the muscle cross-section) region of the muscle. Based on quantitative histochemical assays of single muscle fibers, succinate dehydrogenase (SDH) activity per unit volume was unchanged in fibers of the Sol and MG. However, in the Sol, but not the MG, the total amount of SDH activity in a 10-microns-thick section of a fiber decreased significantly in response to spaceflight. Based on population distributions, it appears that the alpha-glycerophosphate dehydrogenase (GPD) activities were elevated in the dark ATPase fibers in the Sol, whereas the light fibers in the Sol and both fiber types in the MG did not appear to change. The ratio of GPD to SDH activities increased in the dark (but not light) fibers of the Sol and was unaffected in the MG. Immunohistochemical analyses indicate that approximately 40% of the fibers in the Sol of flight rats expressed a fast myosin heavy chain compared with 22% in control rats. Further, 31% of the fibers in the Sol of flight rats expressed both fast and slow myosin heavy chains compared with 8% in control rats. Immunohistochemical changes in the MG were minimal. These data suggest that the magnitude and direction of enzymatic activity and cell volume changes are dependent on the muscle, the region of the muscle, and the type of myosin expressed in the fibers. Further, the ability of fibers to maintain normal or even elevated activities per unit volume of some metabolic enzymes is remarkable considering the marked and rapid decrease in fiber volume.

Strong alkalinization in the anterior midgut of larval yellow fever mosquitoes (Aedes aegypti): involvement of luminal Na+/K+-ATPase.

PubMed

Onken, Horst; Patel, Malay; Javoroncov, Margarita; Izeirovski, Sejmir; Moffett, Stacia B; Moffett, David F

2009-03-01

Recently, Na(+)/K(+)-ATPase has been detected in the luminal membrane of the anterior midgut of larval yellow fever mosquitoes (Aedes aegypti) with immunohistochemical techniques. In this study, the possible involvement of this ATPase in strong alkalinization was investigated on the level of whole larvae, isolated and perfused midgut preparations and on the molecular level of the Na(+)/K(+)-ATPase protein. Ouabain (5 mM) did not inhibit the capability of intact larval mosquitoes to alkalinize their anterior midgut. Also in isolated and perfused midgut preparations the perfusion of the lumen with ouabain (5 mM) did not result in a significant change of the transepithelial voltage or the capacity of luminal alkalinization. Na(+)/K(+)-ATPase activity was completely abolished when KCl was substituted with choline chloride, suggesting that the enzyme cannot act as an ATP-driven Na(+)/H(+)-exchanger. Altogether the results of the present investigation indicate that apical Na(+)/K(+)-ATPase is not of direct importance for strong luminal alkalinization in the anterior midgut of larval yellow fever mosquitoes.

Truncated presequences of mitochondrial F1-ATPase beta subunit from Nicotiana plumbaginifolia transport CAT and GUS proteins into mitochondria of transgenic tobacco.

PubMed

Chaumont, F; Silva Filho, M de C; Thomas, D; Leterme, S; Boutry, M

1994-02-01

The mitochondrial F1-ATPase beta subunit (ATPase-beta) of Nicotiana plumbaginifolia is nucleus-encoded as a precursor containing an NH2-terminal extension. By sequencing the mature N. tabacum ATPase-beta, we determined the length of the presequence, viz. 54 residues. To define the essential regions of this presequence, we produced a series of 3' deletions in the sequence coding for the 90 NH2-terminal residues of ATPase-beta. The truncated sequences were fused with the chloramphenicol acetyl transferase (cat) and beta-glucuronidase (gus) genes and introduced into tobacco plants. From the observed distribution of CAT and GUS activity in the plant cells, we conclude that the first 23 amino-acid residues of ATPase-beta remain capable of specifically targeting reporter proteins into mitochondria. Immunodetection in transgenic plants and in vitro import experiments with various CAT fusion proteins show that the precursors are processed at the expected cleavage site but also at a cryptic site located in the linker region between the presequence and the first methionine of native CAT.

Kinetics and regulation of the myofibrillar adenosine triphosphatase.

PubMed Central

Goodno, C C; Wall, C M; Perry, S V

1978-01-01

1. The steady-state kinetic behaviour of the ATPase (adenosine triphosphatase) of intact myofibrils was studied in the presence of both high and low concentrations of Ca2+ (0.25 mM and less than 10 nM respectively). 2. Kinetic data were collected over the initial linear phase of the assay, which lasts for 20--60s. To obtain consistent data we found it necessary to use either fresh myofibril preparations or preparations that had been stored in the presence of thiol compounds. 3. When assayed in the presence of 0.25 mM-Ca2+, the myofibrillar ATPase obeyed Michaelis-Menten kinetics over the range 0.03--5.0 mM-MgATP (Km 16 +/- 6 micrometer, V 0.4 +/- 0.1 mumol/min per mg). 4. At low Ca2+ concentrations (less than 10 nM) the myofibrillar ATPase displayed pronounced substrate inhibition, which was not observed at high Ca2+ concentrations. Thus increasing the MgATP concentration had the net effect of decreasing the ATPase activity at low Ca2+ relative to that at high Ca2+. This preferential effect of MgATP on the low-Ca2+ ATPase may be important in Ca2+ control. 5. The substrate inhibition that was observed at low Ca2+ was lost on storage or thiol modification of the myofibrils. 6. Under physiological conditions (2 mM-MgATP, I 0.15, pH 7.0), the ATPase of fresh and thiol-protected myofibrils displayed approx. 100-fold activation by Ca2+. PMID:154323

Intracellular pH (pHin) and cytosolic calcium ([Ca2+]cyt) regulation via ATPases: studies in cell populations, single cells, and subcellular compartments

NASA Astrophysics Data System (ADS)

Rojas, Jose D.; Sanka, Shankar C.; Gyorke, Sandor; Wesson, Donald E.; Minta, Akwasi; Martinez-Zaguilan, Raul

1999-07-01

Changes in pHin and (Ca2+)cyt are important in the signal transduction mechanisms leading to many physiological responses including cell growth, motility, secretion/exocytosis, etc. The concentrations of these ions are regulated via primary and secondary ion transporting mechanisms. In diabetes, specific pH and Ca2+ regulatory mechanism might be altered. To study these ions, we employ fluorescence spectroscopy, and cell imagin spectroscopy/confocal microscopy. pH and Ca2+ indicators are loaded in the cytosol with acetoxymethyl ester forms of dyes, and in endosomal/lysosomal (E/L) compartments by overnight incubation of cells with dextran- conjugated ion fluorescent probes. We focus on specific pH and Ca2+ regulatory systems: plasmalemmal vacuolar- type H+-ATPases (pm V-ATPases) and sarcoplasmic/endoplasmic reticulum Ca2+-ATPases (SERCA). As experimental models, we employ vascular smooth muscle (VSM) and microvascular endothelial cells. We have chosen these cells because they are important in blood flow regulation and in angiogenesis. These processes are altered in diabetes. In many cell types, ion transport processes are dependent on metabolism of glucose for maximal activity. Our main findings are: (a) glycolysis coupling the activity of SERCA is required for cytosolic Ca2+ homeostasis in both VSM and microvascular endothelial cells; (b) E/L compartments are important for pH and Ca2+ regulation via H+-ATPases and SERCA, respectively; and (c) pm-V- ATPases are important for pHin regulation in microvascular endothelial cells.

Small terminase couples viral DNA-binding to genome-packaging ATPase activity

PubMed Central

Roy, Ankoor; Bhardwaj, Anshul; Datta, Pinaki; Lander, Gabriel C.; Cingolani, Gino

2012-01-01

SUMMARY Packaging of viral genomes into empty procapsids is powered by a large DNA-packaging motor. In most viruses, this machine is composed of a large (L) and a small (S) terminase subunit complexed with a dodecamer of portal protein. Here, we describe the 1.75 Å crystal structure of the bacteriophage P22 S-terminase in a nonameric conformation. The structure presents a central channel ~23 Å in diameter, sufficiently large to accommodate hydrated B-DNA. The last 23 residues of S-terminase are essential for binding to DNA and assembly to L-terminase. Upon binding to its own DNA, S-terminase functions as a specific activator of L-terminase ATPase activity. The DNA-dependent stimulation of ATPase activity thus rationalizes the exclusive specificity of genome-packaging motors for viral DNA in the crowd of host DNA, ensuring fidelity of packaging and avoiding wasteful ATP hydrolysis. This posits a model for DNA-dependent activation of genome-packaging motors of general interest in virology. PMID:22771211

Lassomycin, a ribosomally synthesized cyclic peptide, kills mycobacterium tuberculosis by targeting the ATP-dependent protease ClpC1P1P2.

PubMed

Gavrish, Ekaterina; Sit, Clarissa S; Cao, Shugeng; Kandror, Olga; Spoering, Amy; Peoples, Aaron; Ling, Losee; Fetterman, Ashley; Hughes, Dallas; Bissell, Anthony; Torrey, Heather; Akopian, Tatos; Mueller, Andreas; Epstein, Slava; Goldberg, Alfred; Clardy, Jon; Lewis, Kim

2014-04-24

Languishing antibiotic discovery and flourishing antibiotic resistance have prompted the development of alternative untapped sources for antibiotic discovery, including previously uncultured bacteria. Here, we screen extracts from uncultured species against Mycobacterium tuberculosis and identify lassomycin, an antibiotic that exhibits potent bactericidal activity against both growing and dormant mycobacteria, including drug-resistant forms of M. tuberculosis, but little activity against other bacteria or mammalian cells. Lassomycin is a highly basic, ribosomally encoded cyclic peptide with an unusual structural fold that only partially resembles that of other lasso peptides. We show that lassomycin binds to a highly acidic region of the ClpC1 ATPase complex and markedly stimulates its ATPase activity without stimulating ClpP1P2-catalyzed protein breakdown, which is essential for viability of mycobacteria. This mechanism, uncoupling ATPase from proteolytic activity, accounts for the bactericidal activity of lassomycin. Copyright © 2014 Elsevier Ltd. All rights reserved.

Combined effects of lead and acid rain on photosynthesis in soybean seedlings.

PubMed

Hu, Huiqing; Wang, Lihong; Liao, Chenyu; Fan, Caixia; Zhou, Qing; Huang, Xiaohua

2014-10-01

To explore how lead (Pb) and acid rain simultaneously affect plants, the combined effects of Pb and acid rain on the chlorophyll content, chlorophyll fluorescence reaction, Hill reaction rate, and Mg(2+)-ATPase activity in soybean seedlings were investigated. The results indicated that, when soybean seedlings were treated with Pb or acid rain alone, the chlorophyll content, Hill reaction rate, Mg(2+)-ATPase activity, and maximal photochemical efficiency (F(v)/F(m)) were decreased, while the initial fluorescence (F 0) and maximum quantum yield (Y) were increased, compared with those of the control. The combined treatment with Pb and acid rain decreased the chlorophyll content, Hill reaction rate, Mg(2+)-ATPase activity, F(v)/F(m), and Y and increased F 0 in soybean seedlings. Under the combined treatment with Pb and acid rain, the two factors showed additive effects on the chlorophyll content in soybean seedlings and exhibited antagonistic effects on the Hill reaction rate. Under the combined treatment with high-concentration Pb and acid rain, the two factors exhibited synergistic effects on the Mg(2+)-ATPase activity, F 0, F v/F m, as well as Y. In summary, the inhibition of the photosynthetic process is an important physiological basis for the simultaneous actions of Pb and acid rain in soybean seedlings.

The electrochemical-proton-gradient-activated states of F0F1 ATPase in plant mitochondria as revealed by detergents.

PubMed

Valerio, M; Diolez, P; Haraux, F

1993-09-01

ATP hydrolysis, triggered by the addition of polyoxyethylene-9-lauryl ether (Lubrol) or lauryldimethylamine oxide (LDAO) to energized plant mitochondria was studied in some details. The membrane disruption was quasi-instantaneous (2-3 s) with both detergents, as shown by the decrease of turbidity and the stopping of respiration. In pea leaf mitochondria, Lubrol triggered ATP hydrolysis in almost the same way as valinomycin plus nigericin, except that the activity was slightly stimulated and became insensitive to carboxyatractyloside. This allowed investigations of ATP hydrolysis without any interference of the ATP/ADP antiporter or the phosphate carrier. Lubrol did not prevent the ATPase from deactivating in pea leaf mitochondria, and did not trigger any ATP hydrolysis in potato tuber mitochondria. At variance with Lubrol, LDAO changed the properties of the F0F1 ATPase. It made the enzyme oligomycin insensitive and froze it in an activated state. The activity was also 5-8-times stimulated in pea leaf mitochondria. Moreover, LDAO revealed an important ATP hydrolase activity when added to energized potato tuber mitochondria. Despite the specific effect of LDAO, the activity triggered by this detergent strongly depended on the energized state of the organelles before detergent addition. From this study, it is concluded that the electrochemical proton gradient is completely necessary to activate the F0F1-ATPase in intact plant mitochondria, as known in chloroplasts and suggested by some reports in animal mitochondria. Moreover, it is suggested that the main difference between the enzymes of pea leaf and potato tuber mitochondria is their rate of deactivation after the collapse of the transmembrane electrochemical potential difference. Finally, when properly used, detergents appear to be a powerful tool to probe the state of the ATPase in intact mitochondria, and maybe in more integrated systems.

Pharmacological inhibition of lysosomes activates the MTORC1 signaling pathway in chondrocytes in an autophagy-independent manner.

PubMed

Newton, Phillip T; Vuppalapati, Karuna K; Bouderlique, Thibault; Chagin, Andrei S

2015-01-01

Mechanistic target of rapamycin (serine/threonine kinase) complex 1 (MTORC1) is a protein-signaling complex at the fulcrum of anabolic and catabolic processes, which acts depending on wide-ranging environmental cues. It is generally accepted that lysosomes facilitate MTORC1 activation by generating an internal pool of amino acids. Amino acids activate MTORC1 by stimulating its translocation to the lysosomal membrane where it forms a super-complex involving the lysosomal-membrane-bound vacuolar-type H(+)-ATPase (v-ATPase) proton pump. This translocation and MTORC1 activation require functional lysosomes. Here we found that, in contrast to this well-accepted concept, in epiphyseal chondrocytes inhibition of lysosomal activity by v-ATPase inhibitors bafilomycin A1 or concanamycin A potently activated MTORC1 signaling. The activity of MTORC1 was visualized by phosphorylated forms of RPS6 (ribosomal protein S6) and EIF4EBP1, 2 well-known downstream targets of MTORC1. Maximal RPS6 phosphorylation was observed at 48-h treatment and reached as high as a 12-fold increase (p < 0.018). This activation of MTORC1 was further confirmed in bone organ culture and promoted potent stimulation of longitudinal growth (p < 0.001). Importantly, the same effect was observed in ATG5 (autophagy-related 5)-deficient bones suggesting a macroautophagy-independent mechanism of MTORC1 inhibition by lysosomes. Thus, our data show that in epiphyseal chondrocytes lysosomes inhibit MTORC1 in a macroautophagy-independent manner and this inhibition likely depends on v-ATPase activity.

A possible molecular mechanism of the action of digitalis: ouabain action on calcium binding to sites associated with a purified sodium-potassium-activated adenosine triphosphatase from kidney.

PubMed

Gervais, A; Lane, L K; Anner, B M; Lindenmayer, G E; Schwartz, A

1977-01-01

Calcium binding at 0 degrees C to a purified sheep kidney Na+,K+-ATPase was described by linear Scatchard plots. Binding at saturating free calcium was 65-80 nmol/mg of protein, or 30-40 mol of calcium/mol of enzyme. Aqueous emulsions of lipids extracted from Na+,K+-ATPase yielded dissociation constants and maximum calcium-binding values that were similar to those for native Na+,K+-ATPase. Phospholipase A treatment markedly reduced calcium binding. Pretreatment of native Na+,K+-ATPase with ouabain increased the dissociation constant for calcium binding from 131 +/- 7 to 192 +/- 7 muM without altering maximum calcium binding. Ouabain pretreatment did not affect calcium binding to extracted phospholipids, ouabain-insensitive ATPases, or heat denatured Na+,K+-ATPase, Na+ and K+ (5-20 mM) increased the dissociation constants for calcium, which suggests competition between the monovalent cations and calcium for the binding sites. At higher concentrations of monovalent cations, ouabain increased the apparent affinity of binding sites for calcium. Extrapolation to physiological cation concentrations revealed that the ouabain-induced increase in apparent affinity for calcium may be as much as 2- to 3-fold. These results suggest: (1) calcium binds to phospholipids associated with Na+,K+-ATPase; (2) ouabain interaction with Na+,K+-ATPase induces a perturbation that is transmitted to adjacent phospholipids, altering their affinity for calcium; and (3) at physiological concentrations of Na+ or K+, or both, ouabain interaction with Na+,K+-ATPase may lead to an increased pool of membrane-bound calcium.

Concerted action of the PHD, chromo and motor domains regulates the human chromatin remodelling ATPase CHD4.

PubMed

Morra, Rosa; Lee, Benjamin M; Shaw, Heather; Tuma, Roman; Mancini, Erika J

2012-07-30

CHD4, the core subunit of the Nucleosome Remodelling and Deacetylase (NuRD) complex, is a chromatin remodelling ATPase that, in addition to a helicase domain, harbors tandem plant homeo finger and chromo domains. By using a panel of domain constructs we dissect their roles and demonstrate that DNA binding, histone binding and ATPase activities are allosterically regulated. Molecular shape reconstruction from small-angle X-ray scattering reveals extensive domain-domain interactions, which provide a structural explanation for the regulation of CHD4 activities by intramolecular domain communication. Our results demonstrate functional interdependency between domains within a chromatin remodeller. Copyright © 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

HDAC Inhibition Improves the Sarcoendoplasmic Reticulum Ca2+-ATPase Activity in Cardiac Myocytes

PubMed Central

Meraviglia, Viviana; Sacchetto, Roberta; Motta, Benedetta M.; Corti, Corrado; D’Elia, Yuri; Rosato-Siri, Marcelo D.; Suffredini, Silvia; Pompilio, Giulio; Pramstaller, Peter P.; Stilli, Donatella

2018-01-01

SERCA2a is the Ca2+ ATPase playing the major contribution in cardiomyocyte (CM) calcium removal. Its activity can be regulated by both modulatory proteins and several post-translational modifications. The aim of the present work was to investigate whether the function of SERCA2 can be modulated by treating CMs with the histone deacetylase (HDAC) inhibitor suberanilohydroxamic acid (SAHA). The incubation with SAHA (2.5 µM, 90 min) of CMs isolated from rat adult hearts resulted in an increase of SERCA2 acetylation level and improved ATPase activity. This was associated with a significant improvement of calcium transient recovery time and cell contractility. Previous reports have identified K464 as an acetylation site in human SERCA2. Mutants were generated where K464 was substituted with glutamine (Q) or arginine (R), mimicking constitutive acetylation or deacetylation, respectively. The K464Q mutation ameliorated ATPase activity and calcium transient recovery time, thus indicating that constitutive K464 acetylation has a positive impact on human SERCA2a (hSERCA2a) function. In conclusion, SAHA induced deacetylation inhibition had a positive impact on CM calcium handling, that, at least in part, was due to improved SERCA2 activity. This observation can provide the basis for the development of novel pharmacological approaches to ameliorate SERCA2 efficiency. PMID:29385061

Root bacterial endophytes confer drought resistance and enhance expression and activity of a vacuolar H+ -pumping pyrophosphatase in pepper plants.

PubMed

Vigani, Gianpiero; Rolli, Eleonora; Marasco, Ramona; Dell'Orto, Marta; Michoud, Grégoire; Soussi, Asma; Raddadi, Noura; Borin, Sara; Sorlini, Claudia; Zocchi, Graziano; Daffonchio, Daniele

2018-05-22

It has been previously shown that the transgenic overexpression of the plant root vacuolar proton pumps H + -ATPase (V-ATPase) and H + -PPase (V-PPase) confer tolerance to drought. Since plant-root endophytic bacteria can also promote drought tolerance, we hypothesize that such promotion can be associated to the enhancement of the host vacuolar proton pumps expression and activity. To test this hypothesis, we selected two endophytic bacteria endowed with an array of in vitro plant growth promoting traits. Their genome sequences confirmed the presence of traits previously shown to confer drought resistance to plants, such as the synthesis of nitric oxide and of organic volatile organic compounds. We used the two strains on pepper (Capsicuum annuum L.) because of its high sensitivity to drought. Under drought conditions, both strains stimulated a larger root system and enhanced the leaves' photosynthetic activity. By testing the expression and activity of the vacuolar proton pumps, H + -ATPase (V-ATPase) and H + -PPase (V-PPase), we found that bacterial colonization enhanced V-PPase only. We conclude that the enhanced expression and activity of V-PPase can be favoured by the colonization of drought-tolerance-inducing bacterial endophytes. This article is protected by copyright. All rights reserved. © 2018 Society for Applied Microbiology and John Wiley & Sons Ltd.

Smolting in coastal cutthroat trout Onchorhynchus clarkii clarkii

USGS Publications Warehouse

Zydlewski, Joseph D.; Zydlewski, G.; Kennedy, B.; Gale, W.

2014-01-01

Gill Na+, K+-ATPase activity, condition factor and seawater (SW) challenges were used to assess the development of smolt characteristics in a cohort of hatchery coastal cutthroat trout Oncorhynchus clarkii clarkii from the Cowlitz River in Washington State, U.S.A. Gill Na+, K+-ATPase activity increased slightly in the spring, coinciding with an increase in hypo-osmoregulatory ability. These changes were of lesser magnitude than are observed in other salmonine species. Even at the peak of tolerance, these fish exhibited notable osmotic perturbations in full strength SW. Condition factor in these hatchery fish declined steadily through the spring. Wild captured migrants from four tributaries of the Columbia River had moderately elevated gill Na+, K+-ATPase activity, consistent with smolt development and with greater enzyme activity than autumn captured juveniles from one of the tributaries, Abernathy Creek. Migrant fish also had reduced condition factor. General linear models of 7 years of data from Abernathy Creek suggest that yearly variation, advancing photoperiod (as ordinal date) and fish size (fork length) were significant factors for predicting gill Na+, K+-ATPase activity in these wild fish. Both yearly variation and temperature were significant factors for predicting condition factor. These results suggest that coastal O. c. clarkii exhibit weakly developed characteristics of smolting. These changes are influenced by environmental conditions with great individual variation. The data suggest great physiological plasticity consistent with the variable life-history tactics observed in this species.

CALIX[4]ARENE C-99 INHIBITS MYOSIN ATPase ACTIVITY AND CHANGES THE ORGANIZATION OF CONTRACTILE FILAMENTS OF MYOMETRIUM.

PubMed

Labyntseva, R D; Bevza, A A; Lul'ko, A O; Cherenok, S O; Kalchenko, V I; Kosterin, S O

2015-01-01

Calix[4]arenes are cup-like macrocyclic (polyphenolic) compounds, they are regarded as promising molecular "platforms" for the design of new physiologically active compounds. We have earlier found that calix[4]arene C-99 inhibits the ATPase activity of actomyosin and myosin subfragment-1 of pig uterus in vitro. The aim of this study was to investigate the interaction of calix[4]arene C-99 with myosin from rat uterine myocytes. It was found that the ATPase activity of myosin prepared from pre-incubated with 100 mM of calix[4]arene C-99 myocytes was almost 50% lower than in control. Additionally, we have revealed the effect of calix[4]arene C-99 on the subcellular distribution of actin and myosin in uterus myocytes by the method of confocal microscopy. This effect can be caused by reorganization of the structure of the contractile smooth muscle cell proteins due to their interaction with calix[4]arene. The obtained results demonstrate the ability of calix[4]arene C-99 to penetrate into the uterus muscle cells and affect not only the myosin ATPase activity, but also the structure of the actin and myosin filaments in the myometrial cells. Demonstrated ability of calix[4]arene C-99 can be used for development of new pharmacological agents for efficient normalization of myometrial contractile hyperfunction.

Magnesium taurate prevents cataractogenesis via restoration of lenticular oxidative damage and ATPase function in cadmium chloride-induced hypertensive experimental animals.

PubMed

Choudhary, Rajesh; Bodakhe, Surendra H

2016-12-01

Previously we found that hypertension potentiates the risk the cataractogenesis. In the present study, we investigated the protective effects of magnesium taurate (MgT) on hypertension and associated lenticular damages against cadmium chloride (CdCl 2 )-induced hypertensive animals. Male Sprague-Dawley albino rats (150-180g) were assigned to five experimental groups (n=6). Among the five groups, normal group received 0.3% carboxymethyl cellulose (10ml/kg/day, p.o.). Hypertension control group received CdCl 2 (0.5mg/kg/day, i.p.). Tests and standard groups received MgT (3 and 6mg/kg/day, p.o.) and amlodipine (3mg/kg/day, p.o.) concurrently with CdCl 2 respectively , for six consecutive weeks. Blood pressure, heart rate, and eyes were examined biweekly, and pathophysiological parameters in serum and eye lenses were evaluated after six weeks of the experimental protocol. The chronic administration of MgT concurrently with CdCl 2 significantly restored the blood pressure, serum and lens antioxidants (CAT, SOD, GPx, and GSH), MDA level, and ions (Na + , K + , and Ca 2+ ). Additionally, MgT treatment led to significant increase in the lens proteins (total and soluble), Ca 2+ ATPase, and Na + K + ATPase activity as compared to hypertension control group. Ophthalmoscope observations indicated that MgT treatments delayed the progression of cataract against the hypertensive state. The study shows that MgT prevents the progression of cataractogenesis via restoration of blood pressure, lenticular oxidative damages, and lens ATPase functions in the hypertensive state. The results suggest that MgT supplement may play a beneficial role to manage hypertension and associated cataractogenesis. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

A Brg1 mutation that uncouples ATPase activity from chromatin remodeling reveals an essential role for SWI/SNF-related complexes in β-globin expression and erythroid development

PubMed Central

Bultman, Scott J.; Gebuhr, Thomas C.; Magnuson, Terry

2005-01-01

The Brg1 catalytic subunit of SWI/SNF-related complexes has been implicated in many developmental and physiological processes, but null homozygotes die as blastocysts prior to implantation. To circumvent this early embryonic lethality, we performed an ENU mutagenesis screen and generated a Brg1 hypomorph mutation in the ATPase domain. The mutant Brg1 protein is stable, assembles into SWI/SNF-related complexes, and exhibits normal ATPase activity but is unable to establish DNase I hypersensitivity sites characteristic of open chromatin. Mutant embryos develop normally until midgestation but then exhibit a distinct block in the development of the erythroid lineage, leading to anemia and death. The mutant Brg1 protein is recruited to the β-globin locus, but chromatin remodeling and transcription are perturbed. Histone acetylation and DNA methylation are also affected. To our knowledge, Brg1 is the first chromatin-modifying factor shown to be required for β-globin regulation and erythropoiesis in vivo. Not only does this mutation establish a role for Brg1 during organogenesis, it also demonstrates that ATPase activity can be uncoupled from chromatin remodeling. PMID:16287714

Plasma membrane H+-ATPase regulation is required for auxin gradient formation preceding phototropic growth

PubMed Central

Hohm, Tim; Demarsy, Emilie; Quan, Clément; Allenbach Petrolati, Laure; Preuten, Tobias; Vernoux, Teva; Bergmann, Sven; Fankhauser, Christian

2014-01-01

Phototropism is a growth response allowing plants to align their photosynthetic organs toward incoming light and thereby to optimize photosynthetic activity. Formation of a lateral gradient of the phytohormone auxin is a key step to trigger asymmetric growth of the shoot leading to phototropic reorientation. To identify important regulators of auxin gradient formation, we developed an auxin flux model that enabled us to test in silico the impact of different morphological and biophysical parameters on gradient formation, including the contribution of the extracellular space (cell wall) or apoplast. Our model indicates that cell size, cell distributions, and apoplast thickness are all important factors affecting gradient formation. Among all tested variables, regulation of apoplastic pH was the most important to enable the formation of a lateral auxin gradient. To test this prediction, we interfered with the activity of plasma membrane H+-ATPases that are required to control apoplastic pH. Our results show that H+-ATPases are indeed important for the establishment of a lateral auxin gradient and phototropism. Moreover, we show that during phototropism, H+-ATPase activity is regulated by the phototropin photoreceptors, providing a mechanism by which light influences apoplastic pH. PMID:25261457

Bufadienolides from parotoid gland secretions of Cuban toad Peltophryne fustiger (Bufonidae): Inhibition of human kidney Na(+)/K(+)-ATPase activity.

PubMed

Perera Córdova, Wilmer H; Leitão, Suzana Guimarães; Cunha-Filho, Geraldino; Bosch, Roberto Alonso; Alonso, Isel Pascual; Pereda-Miranda, Rogelio; Gervou, Rodrigo; Touza, Natália Araújo; Quintas, Luis Eduardo M; Noël, François

2016-02-01

Parotoid gland secretions of toad species are a vast reservoir of bioactive molecules with a wide range of biological properties. Herein, for the first time, it is described the isolation by preparative reversed-phase HPLC and the structure elucidation by NMR spectroscopy and/or mass spectrometry of nine major bufadienolides from parotoid gland secretions of the Cuban endemic toad Peltophryne fustiger: ψ-bufarenogin, gamabufotalin, bufarenogin, arenobufagin, 3-(N-suberoylargininyl) marinobufagin, bufotalinin, telocinobufagin, marinobufagin and bufalin. In addition, the secretion was analyzed by UPLC-MS/MS which also allowed the identification of azelayl arginine. The effect of arenobufagin, bufalin and ψ-bufarenogin on Na(+)/K(+)-ATPase activity in a human kidney preparation was evaluated. These bufadienolides fully inhibited the Na(+)/K(+)-ATPase in a concentration-dependent manner, although arenobufagin (IC50 = 28.3 nM) and bufalin (IC50 = 28.7 nM) were 100 times more potent than ψ-bufarenogin (IC50 = 3020 nM). These results provided evidence about the importance of the hydroxylation at position C-14 in the bufadienolide skeleton for the inhibitory activity on the Na(+)/K(+)-ATPase. Published by Elsevier Ltd.

Osmoregulation in Lilium Pollen Grains Occurs via Modulation of the Plasma Membrane H+ ATPase Activity by 14-3-3 Proteins1[C][W][OA

PubMed Central

Pertl, Heidi; Pöckl, Magdalena; Blaschke, Christian; Obermeyer, Gerhard

2010-01-01

To allow successful germination and growth of a pollen tube, mature and dehydrated pollen grains (PGs) take up water and have to adjust their turgor pressure according to the water potential of the surrounding stigma surface. The turgor pressure of PGs of lily (Lilium longiflorum) was measured with a modified pressure probe for simultaneous recordings of turgor pressure and membrane potential to investigate the relation between water and electrogenic ion transport in osmoregulation. Upon hyperosmolar shock, the turgor pressure decreased, and the plasma membrane (PM) hyperpolarizes in parallel, whereas depolarization of the PM was observed with hypoosmolar treatment. An acidification and alkalinization of the external medium was monitored after hyper- and hypoosmotic treatments, respectively, and pH changes were blocked by vanadate, indicating a putative role of the PM H+ ATPase. Indeed, an increase in PM-associated 14-3-3 proteins and an increase in PM H+ ATPase activity were detected in PGs challenged by hyperosmolar medium. We therefore suggest that in PGs the PM H+ ATPase via modulation of its activity by 14-3-3 proteins is involved in the regulation of turgor pressure. PMID:20974894

Effects of zearalenone on calcium homeostasis of splenic lymphocytes of chickens in vitro.

PubMed

Wang, Y C; Deng, J L; Xu, S W; Peng, X; Zuo, Z C; Cui, H M; Wang, Y; Ren, Z H

2012-08-01

Zearalenone (ZEA) is an estrogenic mycotoxin. It is produced by several Fusarium species and can contaminate food and feed. To investigate the role of calcium homeostasis in ZEA-induced toxicity of poultry and elucidate its cytotoxic mechanism, splenic lymphocytes isolated from chickens were exposed to ZEA (0-25 μg/mL) for 48 h. The intracellular calcium concentration ([Ca2+]i), pH, calmodulin (CaM) mRNA levels, and Na+/K+-ATPase activities and Ca2+-ATPase activities were detected by the fluorescent dyes Fluo-3/AM and BCECF/AM, quantitative real-time PCR, and chromatometry. Supernatant CaM concentrations were simultaneously detected by ELISA. As the ZEA exposure concentration increased, the [Ca2+]i and CaM mRNA levels gradually increased, while intracellular pH, CaM concentrations of supernatants, and intracellular Na+,K+-ATPase and Ca2+-ATPase activities gradually decreased in a dose-dependent manner. There were significant differences (P<0.05 or P<0.01) between the treatment groups and the control group. These results indicate that ZEA cytotoxicity arises by causing an imbalance in calcium homeostasis and intracellular acidification in lymphocytes.

The E3 ubiquitin ligase ZNRF2 is a substrate of mTORC1 and regulates its activation by amino acids

PubMed Central

Hoxhaj, Gerta; Caddye, Edward; Najafov, Ayaz; Houde, Vanessa P; Johnson, Catherine; Dissanayake, Kumara; Toth, Rachel; Campbell, David G; Prescott, Alan R; MacKintosh, Carol

2016-01-01

The mechanistic Target of Rapamycin complex 1 (mTORC1) senses intracellular amino acid levels through an intricate machinery, which includes the Rag GTPases, Ragulator and vacuolar ATPase (V-ATPase). The membrane-associated E3 ubiquitin ligase ZNRF2 is released into the cytosol upon its phosphorylation by Akt. In this study, we show that ZNRF2 interacts with mTOR on membranes, promoting the amino acid-stimulated translocation of mTORC1 to lysosomes and its activation in human cells. ZNRF2 also interacts with the V-ATPase and preserves lysosomal acidity. Moreover, knockdown of ZNRF2 decreases cell size and cell proliferation. Upon growth factor and amino acid stimulation, mTORC1 phosphorylates ZNRF2 on Ser145, and this phosphosite is dephosphorylated by protein phosphatase 6. Ser145 phosphorylation stimulates vesicle-to-cytosol translocation of ZNRF2 and forms a novel negative feedback on mTORC1. Our findings uncover ZNRF2 as a component of the amino acid sensing machinery that acts upstream of Rag-GTPases and the V-ATPase to activate mTORC1. DOI: http://dx.doi.org/10.7554/eLife.12278.001 PMID:27244671

Plant lipid environment and membrane enzymes: the case of the plasma membrane H+-ATPase.

PubMed

Morales-Cedillo, Francisco; González-Solís, Ariadna; Gutiérrez-Angoa, Lizbeth; Cano-Ramírez, Dora Luz; Gavilanes-Ruiz, Marina

2015-04-01

Several lipid classes constitute the universal matrix of the biological membranes. With their amphipathic nature, lipids not only build the continuous barrier that confers identity to every cell and organelle, but they are also active actors that modulate the activity of the proteins immersed in the lipid bilayer. The plasma membrane H(+)-ATPase, an enzyme from plant cells, is an excellent example of a transmembrane protein whose activity is influenced by the hydrophilic compartments at both sides of the membrane and by the hydrophobic domains of the lipid bilayer. As a result, an extensive documentation of the effect of numerous amphiphiles in the enzyme activity can be found. Detergents, membrane glycerolipids, and sterols can produce activation or inhibition of the enzyme activity. In some cases, these effects are associated with the lipids of the membrane bulk, but in others, a direct interaction of the lipid with the protein is involved. This review gives an account of reports related to the action of the membrane lipids on the H(+)-ATPase activity.

Redox stress in geobacilli from geothermal springs: Phenomenon and membrane-associated response mechanisms.

PubMed

Ghazaryan, Astghik; Blbulyan, Syuzanna; Poladyan, Anna; Trchounian, Armen

2015-10-01

Geobacillus toebii ArzA-8, from Armenian geothermal springs, grew well in nutrient broth. During its growth, changes in pH in opposite directions were observed depending on glucose supplementation. Accordingly, the decrease in the redox potential was determined using titanium-silicate (Eh) and platinum (Eh') electrodes: Eh decreased to -150 ± 3 mV and Eh' to -350 ± 2 mV without glucose; the decrease in these potentials was smaller with glucose. Redox stress due to an oxidizer, K3[Fe(CN)6], or a reducer, dl-dithiothreitol (DTT), inhibited bacterial growth. However, a stimulatory effect of K3[Fe(CN)6] or DTT was observed with or without glucose, respectively. With glucose, the H(+) efflux was sensitive to N,N'-dicyclohexylcarbodiimide (DCCD), an inhibitor of FoF1FOF1-ATPase and other H(+) translocation mechanisms, but the addition of an oxidizer or reducer suppressed the H(+) efflux. The ATPase activity of membrane vesicles was ~1.3-fold higher in cells grown with glucose compared with cells grown without glucose. DCCD and DTT suppressed ATPase activity in cells grown without glucose, whereas DTT stimulated FOF1-ATPase activity in cells grown with glucose. Thus, G. toebii senses redox stress; this thermophile likely presents specific membrane-associated response mechanisms involving FOF1-ATPase to overcome redox stress and survive; these mechanisms are important for adaptation to extreme environments. Copyright © 2015 Elsevier B.V. All rights reserved.

Properties of Plasma Membrane from Pea Root Seedlings under Altered Gravity

NASA Astrophysics Data System (ADS)

Klymchuk, D.; Baranenko, V.; Vorobyova, T. V.; Kurylenko, I.; Chyzhykova, O.; Dubovoy, V.

In this study, the properties of pea (Pisum sativum L.) plasma membrane were examined to determine how the membrane structure and functions are regulated in response to clinorotation (2 rev/min) conditions. Membrane preparations enriched by plasma membrane vesicles were obtained by aqueous two-phase partitioning from 6-day seedling roots. The specific characteristics of H^+-ATPase, lípid composition and peroxidation intensity as well as fluidity of lipid bilayer were analysed. ATP hydrolytic activity was inhibited by ortovanadate and was insensitive to aside and nitrate in sealed plasma membrane vesicles isolated from both clinorotated and control seedlings. Plasma membrane vesicles from clinorotated seedlings in comparison to controls were characterised by increase in the total lipid/protein ratio, ATP hydrolytic activity and intensifying of lipid peroxidation. Sitosterol and campesterol were the predominant free sterol species. Clinorotated seedlings contained a slightly higher level of unsaturated fatty acid than controls. Plasma membrane vesicles were labelled with pyrene and fluorescence originating from monomeric (I_M) molecules and excimeric (I_E) aggregates were measured. The calculated I_E/I_M values were higher in clinorotated seedlings compared with controls reflecting the reduction in membrane microviscosity. The involvement of the changes in plasma membrane lipid content and composition, fluidity and H^+-ATPase activity in response of pea seedlings to altered gravity is discussed.

Cardioprotective effect of Sida rhomboidea. Roxb extract against isoproterenol induced myocardial necrosis in rats.

PubMed

Thounaojam, Menaka C; Jadeja, Ravirajsinh N; Ansarullah; Karn, Sanjay S; Shah, Jigar D; Patel, Dipak K; Salunke, Sunita P; Padate, Geeta S; Devkar, Ranjitsinh V; Ramachandran, A V

2011-05-01

The present study investigates cardioprotective effect of Sida rhomboidea. Roxb (SR) extract on heart weight, plasma lipid profile, plasma marker enzymes, lipid peroxidation, endogenous enzymatic and non-enzymatic antioxidants and membrane bound ATPases against isoproterenol (IP) induced myocardial necrosis (MN) in rats. Rats treated with IP (85 mg/kg, s.c.) recorded significant (p<0.05) increment in heart weight, plasma lipid profile, plasma marker enzymes of cardiac damage, cardiac lipid peroxidation (LPO) and activity levels of Ca(+2) ATPase whereas there was significant (p<0.05) decrease in plasma HDL, cardiac endogenous enzymatic and non-enzymatic antioxidants, Na(+)-K(+) ATPase and Mg(+2) ATPase. Pre-treatment with SR extract (400 mg/kg per day, p.o.) for 30 consecutive days followed by IP injections on days 29th and 30th, showed significant (p<0.05) decrease in heart weight, plasma lipid profile, plasma marker enzymes of cardiac damage, cardiac lipid peroxidation, Ca(+2) ATPase and significant increase in plasma HDL, cardiac endogenous enzymatic and non-enzymatic antioxidants, Na(+)-K(+) ATPase and Mg(+2) ATPase compared to IP treated group. Hence, this study is the first scientific report on cardioprotective effect of SR against IP induced MN in rats. Copyright © 2010 Elsevier GmbH. All rights reserved.

Crystal structure of the plasma membrane proton pump.

PubMed

Pedersen, Bjørn P; Buch-Pedersen, Morten J; Morth, J Preben; Palmgren, Michael G; Nissen, Poul

2007-12-13

A prerequisite for life is the ability to maintain electrochemical imbalances across biomembranes. In all eukaryotes the plasma membrane potential and secondary transport systems are energized by the activity of P-type ATPase membrane proteins: H+-ATPase (the proton pump) in plants and fungi, and Na+,K+-ATPase (the sodium-potassium pump) in animals. The name P-type derives from the fact that these proteins exploit a phosphorylated reaction cycle intermediate of ATP hydrolysis. The plasma membrane proton pumps belong to the type III P-type ATPase subfamily, whereas Na+,K+-ATPase and Ca2+-ATPase are type II. Electron microscopy has revealed the overall shape of proton pumps, however, an atomic structure has been lacking. Here we present the first structure of a P-type proton pump determined by X-ray crystallography. Ten transmembrane helices and three cytoplasmic domains define the functional unit of ATP-coupled proton transport across the plasma membrane, and the structure is locked in a functional state not previously observed in P-type ATPases. The transmembrane domain reveals a large cavity, which is likely to be filled with water, located near the middle of the membrane plane where it is lined by conserved hydrophilic and charged residues. Proton transport against a high membrane potential is readily explained by this structural arrangement.

HIF and HOIL-1L-mediated PKCζ degradation stabilizes plasma membrane Na,K-ATPase to protect against hypoxia-induced lung injury.

PubMed

Magnani, Natalia D; Dada, Laura A; Queisser, Markus A; Brazee, Patricia L; Welch, Lynn C; Anekalla, Kishore R; Zhou, Guofei; Vagin, Olga; Misharin, Alexander V; Budinger, G R Scott; Iwai, Kazuhiro; Ciechanover, Aaron J; Sznajder, Jacob I

2017-11-21

Organisms have evolved adaptive mechanisms in response to stress for cellular survival. During acute hypoxic stress, cells down-regulate energy-consuming enzymes such as Na,K-ATPase. Within minutes of alveolar epithelial cell (AEC) exposure to hypoxia, protein kinase C zeta (PKCζ) phosphorylates the α 1 -Na,K-ATPase subunit and triggers it for endocytosis, independently of the hypoxia-inducible factor (HIF). However, the Na,K-ATPase activity is essential for cell homeostasis. HIF induces the heme-oxidized IRP2 ubiquitin ligase 1L (HOIL-1L), which leads to PKCζ degradation. Here we report a mechanism of prosurvival adaptation of AECs to prolonged hypoxia where PKCζ degradation allows plasma membrane Na,K-ATPase stabilization at ∼50% of normoxic levels, preventing its excessive down-regulation and cell death. Mice lacking HOIL-1L in lung epithelial cells ( Cre SPC /HOIL-1L fl/fl ) were sensitized to hypoxia because they express higher levels of PKCζ and, consequently, lower plasma membrane Na,K-ATPase levels, which increased cell death and worsened lung injury. In AECs, expression of an α 1 -Na,K-ATPase construct bearing an S18A (α 1 -S18A) mutation, which precludes PKCζ phosphorylation, stabilized the Na,K-ATPase at the plasma membrane and prevented hypoxia-induced cell death even in the absence of HOIL-1L. Adenoviral overexpression of the α 1 -S18A mutant Na,K-ATPase in vivo rescued the enhanced sensitivity of Cre SPC/ HOIL-1L fl/fl mice to hypoxic lung injury. These data suggest that stabilization of Na,K-ATPase during severe hypoxia is a HIF-dependent process involving PKCζ degradation. Accordingly, we provide evidence of an important adaptive mechanism to severe hypoxia, whereby halting the exaggerated down-regulation of plasma membrane Na,K-ATPase prevents cell death and lung injury.

cAMP-dependent protein kinase phosphorylates and activates nuclear Ca2+-ATPase

PubMed Central

Rogue, Patrick J.; Humbert, Jean-Paul; Meyer, Alphonse; Freyermuth, Solange; Krady, Marie-Marthe; Malviya, Anant N.

1998-01-01

A Ca2+-pump ATPase, similar to that in the endoplasmic reticulum, has been located on the outer membrane of rat liver nuclei. The effect of cAMP-dependent protein kinase (PKA) on nuclear Ca2+-ATPase (NCA) was studied by using purified rat liver nuclei. Treatment of isolated nuclei with the catalytic unit of PKA resulted in the phosphorylation of a 105-kDa band that was recognized by antibodies specific for sarcoplasmic reticulum Ca2+-ATPase type 2b. Partial purification and immunoblotting confirmed that the 105-kDa protein band phosphorylated by PKA is NCA. The stoichiometry of phosphorylation was 0.76 mol of phosphate incorporated/mol of partially purified enzyme. Measurement of ATP-dependent 45Ca2+ uptake into purified nuclei showed that PKA phosphorylation enhanced the Ca2+-pumping activity of NCA. We show that PKA phosphorylation of Ca2+-ATPase enhances the transport of 10-kDa fluorescent-labeled dextrans across the nuclear envelope. The findings reported in this paper are consistent with the notion that the crosstalk between the cAMP/PKA- and Ca2+-dependent signaling pathways identified at the cytoplasmic level extends to the nucleus. Furthermore, these data support a function for crosstalk in the regulation of calcium-dependent transport across the nuclear envelope. PMID:9689054

Inhibitory and combinatorial effect of diphyllin, a v-ATPase blocker, on influenza viruses.

PubMed

Chen, Hui-Wen; Cheng, Jenna Xiao; Liu, Ming-Tsan; King, Kevin; Peng, Ju-Yi; Zhang, Xin-Quan; Wang, Ching-Ho; Shresta, Sujan; Schooley, Robert T; Liu, Yu-Tsueng

2013-09-01

An influenza pandemic poses a serious threat to humans and animals. Conventional treatments against influenza include two classes of pathogen-targeting antivirals: M2 ion channel blockers (such as amantadine) and neuraminidase inhibitors (such as oseltamivir). Examination of the mechanism of influenza viral infection has shown that endosomal acidification plays a major role in facilitating the fusion between viral and endosomal membranes. This pathway has led to investigations on vacuolar ATPase (v-ATPase) activity, whose role as a regulating factor on influenza virus replication has been verified in extensive genome-wide screenings. Blocking v-ATPase activity thus presents the opportunity to interfere with influenza viral infection by preventing the pH-dependent membrane fusion between endosomes and virions. This study aims to apply diphyllin, a natural compound shown to be as a novel v-ATPase inhibitor, as a potential antiviral for various influenza virus strains using cell-based assays. The results show that diphyllin alters cellular susceptibility to influenza viruses through the inhibition of endosomal acidification, thus interfering with downstream virus replication, including that of known drug-resistant strains. In addition, combinatorial treatment of the host-targeting diphyllin with pathogen-targeting therapeutics (oseltamivir and amantadine) demonstrates enhanced antiviral effects and cell protection in vitro. Copyright © 2013 Elsevier B.V. All rights reserved.

Neuroendocrine regulation of osmoregulation and the evolution of air-breathing in decapod crustaceans.

PubMed

Morris, S

2001-03-01

Gills are the primary organ for salt transport, but in land crabs they are removed from water and thus ion exchanges, as well as CO(2) and ammonia excretion, are compromised. Urinary salt loss is minimised in land crabs by redirecting the urine across the gills where salt reabsorption occurs. Euryhaline marine crabs utilise apical membrane branchial Na(+)/H(+) and Cl(-)/HCO(3)(-) exchange powered by a basal membrane Na(+)/K(+)-ATPase, but in freshwater crustaceans an apical V-ATPase provides for electrogenic uptake of Cl(-) in exchange for HCO(3)(-). The HCO(3)(-) is provided by carbonic anhydrase facilitating CO(2) excretion while NH(4)(+) can substitute for K(+) in the basal ATPase and for H(+) in the apical exchange. Gecarcinid land crabs and the terrestrial anomuran Birgus latro can lower the NaCl concentration of the urine to 5 % of that of the haemolymph as it passes across the gills. This provides a filtration-reabsorption system analogous to the vertebrate kidney. Crabs exercise hormonal control over branchial transport processes. Aquatic hyper-regulators release neuroamines from the pericardial organs, including dopamine and 5-hydroxytryptamine (5-HT), which via a cAMP-mediated phosphorylation stimulate Na(+)/K(+)-ATPase activity and NaCl uptake. Freshwater species utilise a V-ATPase, and additional mechanisms of control have been suggested. Crustacean hyperglycaemic hormone (CHH) has now also been confirmed to have effects on hydromineral regulation, and a putative role for neuropeptides in salt and water balance suggests that current models for salt regulation are probably incomplete. In a terrestrial crabs there may be controls on both active uptake and diffusive loss. The land crab Gecarcoidea natalis drinking saline water for 3 weeks reduced net branchial Na(+) uptake but not Na(+)/K(+)-ATPase activity, thus implying a reduction in diffusive Na(+) loss. Further, in G. natalis Na(+) uptake and Na(+)/K(+)-ATPase were stimulated by 5-HT independently of cAMP. Conversely, in the anomuran B. latro, branchial Na(+) and Cl(-) uptake and Na(+)/K(+)-ATPase are inhibited by dopamine, mediated by cAMP. There has been a multiple evolution of a kidney-type system in terrestrial crabs capable of managing salt, CO(2) and NH(3) movements.

Quantitative Imaging of Energy Expenditure in Human Brain

PubMed Central

Zhu, Xiao-Hong; Qiao, Hongyan; Du, Fei; Xiong, Qiang; Liu, Xiao; Zhang, Xiaoliang; Ugurbil, Kamil; Chen, Wei

2012-01-01

Despite the essential role of the brain energy generated from ATP hydrolysis in supporting cortical neuronal activity and brain function, it is challenging to noninvasively image and directly quantify the energy expenditure in the human brain. In this study, we applied an advanced in vivo 31P MRS imaging approach to obtain regional cerebral metabolic rates of high-energy phosphate reactions catalyzed by ATPase (CMRATPase) and creatine kinase (CMRCK), and to determine CMRATPase and CMRCK in pure grey mater (GM) and white mater (WM), respectively. It was found that both ATPase and CK rates are three times higher in GM than WM; and CMRCK is seven times higher than CMRATPase in GM and WM. Among the total brain ATP consumption in the human cortical GM and WM, 77% of them are used by GM in which approximately 96% is by neurons. A single cortical neuron utilizes approximately 4.7 billion ATPs per second in a resting human brain. This study demonstrates the unique utility of in vivo 31P MRS imaging modality for direct imaging of brain energy generated from ATP hydrolysis, and provides new insights into the human brain energetics and its role in supporting neuronal activity and brain function. PMID:22487547

Bis-enoxacin Inhibits Bone Resorption and Orthodontic Tooth Movement

PubMed Central

Toro, E.J.; Zuo, J.; Guiterrez, A.; La Rosa, R.L.; Gawron, A.J.; Bradaschia-Correa, V.; Arana-Chavez, V.; Dolce, C.; Rivera, M.F.; Kesavalu, L.; Bhattacharyya, I.; Neubert, J.K.; Holliday, L.S.

2013-01-01

Enoxacin inhibits binding between the B-subunit of vacuolar H+-ATPase (V-ATPase) and microfilaments, and also between osteoclast formation and bone resorption in vitro. We hypothesized that a bisphosphonate derivative of enoxacin, bis-enoxacin (BE), which was previously studied as a bone-directed antibiotic, might have similar activities. BE shared a number of characteristics with enoxacin: It blocked binding between the recombinant B-subunit and microfilaments and inhibited osteoclastogenesis in cell culture with IC50s of about 10 µM in each case. BE did not alter the relative expression levels of various osteoclast-specific proteins. Even though tartrate-resistant acid phosphatase 5b was expressed, proteolytic activation of the latent pro-enzyme was inhibited. However, unlike enoxacin, BE stimulated caspase-3 activity. BE bound to bone slices and inhibited bone resorption by osteoclasts on BE-coated bone slices in cell culture. BE reduced the amount of orthodontic tooth movement achieved in rats after 28 days. Analysis of these data suggests that BE is a novel anti-resorptive molecule that is active both in vitro and in vivo and may have clinical uses. Abbreviations: BE, bis-enoxacin; V-ATPase, vacuolar H+-ATPase; TRAP, tartrate-resistant acid phosphatase; αMEM D10, minimal essential media, alpha modification with 10% fetal bovine serum; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; RANKL, receptor activator of nuclear factor kappa B-ligand; NFATc1, nuclear factor of activated T-cells; ADAM, a disintegrin and metalloprotease domain; OTM, orthodontic tooth movement. PMID:23958763

Biochar and lignite affect H+-ATPase and H+-PPase activities in root tonoplast and nutrient contents of mung bean under salt stress.

PubMed

Torabian, Shahram; Farhangi-Abriz, Salar; Rathjen, Judith

2018-05-31

This research was conducted to evaluate effects of biochar (50 and 100 g kg -1 soil) and lignite (50 and 100 g kg -1 soil) treatments on H + -ATPase and H + -PPase activity of root tonoplast, nutrient content, and performance of mung bean under salt stress. High saline conditions increased H + -ATPase and H + -PPase activities in root tonoplast, sodium (Na) content, reactive oxygen species (H 2 O 2 and O 2 - ) generation, relative electrolyte leakage (REL) and 2,2-Diphenyl-1-picrylhydrazyl (DPPH) activity in root and leaf, but decreased relative water content (RWC), chlorophyll content index, leaf area, potassium (K), calcium (Ca), magnesium (Mg), zinc (Zn) and iron (Fe) content of plant tissues, root and shoot dry weight of mung bean. Lignite and biochar treatments decreased the H + -ATPase and H + -PPase activities of root tonoplast under salt stress. Moreover, these treatments increased the cation exchange capacity of soil and nutrient values in plant tissues. Biochar and lignite diminished the generation of reactive oxygen species and DPPH activity in root and leaf cells, and these superior effects improved chlorophyll content index, leaf area and growth of mung bean under both conditions. In general, the results of this study demonstrated that biochar and lignite decreased the entry of Na ion into the cells, enriched plant cells with nutrients, and consequently improved mung bean performance under salt toxicity. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

Effect of Ginkgo biloba extract 50 on immunity and antioxidant enzyme activities in ischemia reperfusion rats.

PubMed

Lu, Shaoping; Guo, Xia; Zhao, Pinting

2011-11-02

The aim of the study was to investigate the effect of Ginkgo biloba extract 50 (GBE50), a well-known natural antioxidant, against immunity and antioxidant enzyme activities in ischemia reperfusion (IR) rats. Rats were then divided into six groups fed for 15 days with the same diet: three groups (IV, V, VI) were treated by different doses of GBE50 suspension [20, 40, or 60 mg/kg body weight by oral gavage every day at a fixed time (10.00 a.m.)] (equal to 5, 10 and 20 times, respectively, the maximum recommended human dose), and three groups (I, II, III) were untreated. At the end of the experiment, rats' hearts were subjected to 30 min of ischemia followed by 90 min of reperfusion. Results showed that IR significantly enhanced heart rate, S-T height, myocardium (myeloperoxidase) MPO activity and blood interleukin-8 (IL-8), tumor necrosis factor Alpha (TNF-a), interleukin-1β (IL-1β) levels, blood aspartate transaminase (AST), lactate dehydrogenase (LDH), and creatinine kinase (CK) activities, reduced myocardium sodium-potassium adenosine triphosphatase (Na(+)-K(+)-ATPase), calcium-magnesium adenosine triphosphatase (Ca(2+)-Mg(2+)-ATPase) activities and antioxidant enzyme activities in IR group (III) compared to sham control group (II). Pretreatment of GBE50 markedly significantly reduced heart rate, S-T height, myocardium MPO activity and blood IL-8, TNF-a, IL-1β levels, blood AST, LDH, and CK activities, enhanced myocardium Na(+)-K(+)-ATPase, Ca(2+)-Mg(2+)-ATPase activities and antioxidant enzyme activities in IR group (II) compared to IR group (III). The results suggested that the GBE50 may reduce the oxidative stress in the reperfused myocardium, and increased immunity and antioxidant activities in IR rats.

Studies on the Mechanism of Action of Dinitramine

PubMed Central

Travis, Robert L.; Woods, William G.

1977-01-01

The effect of dinitramine, a selective herbicide, on the plasma membrane of the soybean (Glycine max L.) root was studied. Used as marker systems to observe the herbicide effect were two plasma-membrane-specific enzymes, pH 6.5 ATPase and glucan synthetase. The activity of pH 6.5 ATPase decreased significantly in membrane vesicles prepared from roots harvested 15 minutes after treatment with dinitramine. Maximum inhibition occurred in roots harvested 2 hours after treatment. Glucan synthetase activity decreased similarly within 2 hours of treatment. Membrane permeability to 86Rb was rapidly increased by dinitramine. The activity of pH 6.5 ATPase returned to the control level within 8 hours of treatment with dinitramine. These results show dinitramine's initial site of action to be the plasma membrane, producing an over-all reduction in membrane function through inactivation of membrane-associated proteins. PMID:16660043

P-type proton ATPases are involved in intracellular calcium and proton uptake in the plant parasite Phytomonas francai.

PubMed

Miranda, Kildare; Vercesi, Anibal E; Catisti, Rosana; De Souza, Wanderley; Rodrigues, Claudia O; Docampo, Roberto

2005-01-01

The use of digitonin to permeabilize the plasma membrane of promastigotes of Phytomonas francai allowed the identification of two non-mitochondrial Ca(2+) compartments; one sensitive to ionomycin and vanadate (neutral or alkaline), possibly the endoplasmic reticulum, and another sensitive to the combination of nigericin plus ionomycin (acidic), possibly the acidocalcisomes. A P-type (phospho-intermediate form) Ca(2+)-ATPase activity was found to be responsible for intracellular Ca(2+) transport in these cells, with no evidence of a mitochondrial Ca(2+) transport activity. ATP-driven acidification of internal compartments in cell lysates and cells mechanically permeabilized was assayed spectrophotometrically with acridine orange. This activity was inhibited by low concentrations of vanadate and digitonin, was insensitive to bafilomycin A(1), and stimulated by Na(+) ions. Taken together, our results indicate that P-type ATPases are involved in intracellular Ca(2+) and H(+) transport in promastigotes of P. francai.