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.

Reincorporated plasma membrane Ca2+-ATPase can mediate B-Type Ca2+ channels observed in native membrane of human red blood cells.

PubMed

Pinet, C; Antoine, S; Filoteo, A G; Penniston, J T; Coulombe, A

2002-06-01

Recently, we reported indirect evidence that plasma membrane Ca2+-ATPase (PMCA) can mediate B-type Ca2+ channels of cardiac myocytes. In the present study, in order to bring more direct evidence, purified PMCA from human red blood cells (RBC) was reconstituted into giant azolectin liposomes amenable to the patch-clamp technique. Purified RBC PMCA was used because it is available pure in larger quantity than cardiac PMCA. The presence of B-type Ca2+ channels was first investigated in native membranes of human RBC. They were detected and share the characteristics of cardiac myocytes. They spontaneously appeared in scarce short bursts of activity, they were activated by chlorpromazine (CPZ) with an EC50 of 149 mmole/l or 1 mmole/l vanadate, and then switched off by 10 mmole/l eosin or dose-dependently blocked by 1-5 mmole/l ATP. Independent of membrane potential, the channel gating exhibited complex patterns of many conductance levels, with three most often observed conductance levels of 22, 47 and 80 pS. The activation by vanadate suggests that these channels could play a role in the influx of extracellular Ca2+ involved in the vanadate-induced Gardos effect. In PMCA-reconstituted proteoliposomes, nearly half of the ATPase activity was retained and clear "channel-like" openings of Ba2+- or Ca2+-conducting channels were detected. Channel activity could be spontaneously present, lasting the patch lifetime or, when previously quiescent, activity could be induced by application of 50 mmole/l CPZ only in presence of 25 U/ml calmodulin (CaM), or by application of 1 mmole/l vanadate alone. Eosin (10 mmole/l) and ATP (5 mmole/l) significantly reduced spontaneous activity. Channel gating characteristics were similar to those of RBC, with main conductance levels of 21, 40 and 72 pS. The lack of direct activation by CPZ alone might be attributed to a purification-induced modification or absence of unidentified regulatory component(s) of PMCA. Despite a few differences in

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

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.

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.

Glucose supplements increase human muscle in vitro Na+-K+-ATPase activity during prolonged exercise.

PubMed

Green, H J; Duhamel, T A; Foley, K P; Ouyang, J; Smith, I C; Stewart, R D

2007-07-01

Regulation of maximal Na(+)-K(+)-ATPase activity in vastus lateralis muscle was investigated in response to prolonged exercise with (G) and without (NG) oral glucose supplements. Fifteen untrained volunteers (14 males and 1 female) with a peak aerobic power (Vo(2)(peak)) of 44.8 +/- 1.9 ml.kg(-1).min(-1); mean +/- SE cycled at approximately 57% Vo(2)(peak) to fatigue during both NG (artificial sweeteners) and G (6.13 +/- 0.09% glucose) in randomized order. Consumption of beverage began at 30 min and continued every 15 min until fatigue. Time to fatigue was increased (P < 0.05) in G compared with NG (137 +/- 7 vs. 115 +/- 6 min). Maximal Na(+)-K(+)-ATPase activity (V(max)) as measured by the 3-O-methylfluorescein phosphatase assay (nmol.mg(-1).h(-1)) was not different between conditions prior to exercise (85.2 +/- 3.3 or 86.0 +/- 3.9), at 30 min (91.4 +/- 4.7 vs. 91.9 +/- 4.1) and at fatigue (92.8 +/- 4.3 vs. 100 +/- 5.0) but was higher (P < 0.05) in G at 90 min (86.7 +/- 4.2 vs. 109 +/- 4.1). Na(+)-K(+)-ATPase content (beta(max)) measured by the vanadate facilitated [(3)H]ouabain-binding technique (pmol/g wet wt) although elevated (P < 0.05) by exercise (0<30, 90, and fatigue) was not different between NG and G. At 60 and 90 min of exercise, blood glucose was higher (P < 0.05) in G compared with NG. The G condition also resulted in higher (P < 0.05) serum insulin at similar time points to glucose and lower (P < 0.05) plasma epinephrine and norepinephrine at 90 min of exercise and at fatigue. These results suggest that G results in an increase in V(max) by mechanisms that are unclear.

Basis for changes in the auxin-sensitivity of Avena sativa (oat) leaf-sheath pulvini during the gravitropic response

NASA Technical Reports Server (NTRS)

Kim, D.; Kaufman, P. B.

1995-01-01

During the gravitropic response, auxin-sensitivity of the lower flanks of leaf-sheath pulvini of Avena sativa (oat) is at least 1000-fold higher than those of the upper flanks and non-gravistimulated pulvini. When the pulvini are treated with 1 mM Ca2+, a 10-fold increase in auxin-sensitivity of the pulvini is observed. Related to this difference in auxin-sensitivity, in vitro activation of the vanadate-sensitive H(-)-ATPase by IAA was observed. Results show that the activation of the H(+)-ATPase by IAA is probably mediated by soluble protein factors and that the H(+)-ATPase prepared from the lower flanks is activated by IAA with a 1000-fold higher auxin-sensitivity as compared with that from the upper flanks of the graviresponding pulvini. Ammonium sulfate fractionation experiments show that these soluble protein factors are in the 30 to 60% fraction. Auxin-binding assays reveal that lower flanks contain more high-affinity soluble auxin-binding sites (kD; on the order of 10(-9) M) and less low-affinity soluble auxin-binding sites (kD; on the order of 10(-6) M) than upper flanks. It is concluded that differential auxin-sensitivity of graviresponding oat-shoot pulvini is achieved by the modulation of affinities of auxin-binding sites in upper and lower flanks of the pulvini, that Ca2+ is involved in such modulation, and that one of the probable cellular functions of these auxin binding sites is the activation of the proton pump on the plasma membranes.

Vanadate monomers and dimers both inhibit the human prostatic acid phosphatase.

PubMed

Crans, D C; Simone, C M; Saha, A K; Glew, R H

1989-11-30

A combination of enzyme kinetics and 51V NMR spectroscopy was used to identify the species of vanadate that inhibits acid phosphatases. Monomeric vanadate was shown to inhibit wheat germ and potato acid phosphatases. At pH 5.5, the vanadate dimer inhibits the human prostatic acid phosphatase whereas at pH 7.0 it is the vanadate monomer that inhibits this enzyme. The pH-dependent shift in the affinity of the prostatic phosphatase for vanadate is presumably due to deprotonation of an amino acid side chain in or near the binding site resulting in a conformational change in the protein. pH may be a subtle effector of the insulin-like vanadate activity in biological systems and may explain some of the differences in selectivity observed with the protein phosphatases.

Quantitation of Na+, K+-atpase Enzymatic Activity in Tissues of the Mammalian Vestibular System

NASA Technical Reports Server (NTRS)

Kerr, T. P.

1985-01-01

In order to quantify vestibular Na(+), K(+)-ATPase, a microassay technique was developed which is sufficiently sensitive to measure the enzymatic activity in tissue from a single animal. The assay was used to characterize ATPase in he vestibular apparatus of the Mongolian gerbil. The quantitative procedure employs NPP (5 mM) as synthetic enzyme substrate. The assay relies upon spectrophotometric measurement (410 nm) of nitrophenol (NP) released by enzymatic hydrolysis of the substrate. Product formation in the absence of ouabain reflects both specific (Na(+), K(+)-ATPase) and non-specific (Mg(++)-ATPase) enzymatic activity. By measuring the accumulation of reaction product (NP) at three-minute intervals during the course of incubation, it is found that the overall enzymatic reaction proceeds linearly for at least 45 minutes. It is therefore possible to determine two separate reaction rates from a single set of tissues. Initial results indicate that total activity amounts to 53.3 + or - 11.2 (S.E.M.) nmol/hr/mg dry tissue, of which approximately 20% is ouabain-sensitive.

Silencing of secretory clusterin sensitizes NSCLC cells to V-ATPase inhibitors by downregulating survivin.

PubMed

Kim, Young-Sun; Jin, Hyeon-Ok; Hong, Sung-Eun; Song, Jie-Young; Hwang, Chang-Sun; Park, In-Chul

2018-01-08

Secretory clusterin (sCLU) is a stress-associated protein that confers resistance to therapy when overexpressed. In this study, we observed that the V-ATPase inhibitors bafilomycin A1 and concanamycin A significantly stimulated sCLU protein expression. Knockdown of sCLU with siRNA sensitized non-small cell lung cancer (NSCLC) cells to bafilomycin A1, suggesting that sCLU expression renders cells resistant to V-ATPase inhibitors. The dual PI3K/AKT and mTOR inhibitor BEZ235 suppressed sCLU expression and enhanced cell sensitivity induced by bafilomycin A1. Notably, sCLU knockdown further decreased the expression of the survivin protein by bafilomycin A1, and the ectopic expression of survivin alleviated the cell sensitivity by bafilomycin A1 and sCLU depletion, suggesting that increased sensitivity to sCLU depletion in the cells with V-ATPase inhibitors is due, at least in part, to the down-regulation of survivin. Taken together, we demonstrated that the depletion of sCLU expression enhances the sensitivity of NSCLC cells to V-ATPase inhibitors by decreasing survivin expression. Inhibition of the PI3K/AKT/mTOR pathway enhances the sensitivity of NSCLC cells to V-ATPase inhibitors, leading to decreased sCLU and survivin expression. Thus, we suggest that a combination of PI3K/AKT/mTOR inhibitors with V-ATPase inhibitors might be an effective approach for NSCLC treatment. Copyright © 2017 Elsevier Inc. All rights reserved.

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.

Rapid regulation of the plasma membrane H+-ATPase activity is essential to salinity tolerance in two halophyte species, Atriplex lentiformis and Chenopodium quinoa

PubMed Central

Bose, Jayakumar; Rodrigo-Moreno, Ana; Lai, Diwen; Xie, Yanjie; Shen, Wenbiao; Shabala, Sergey

2015-01-01

Background and Aims The activity of H+-ATPase is essential for energizing the plasma membrane. It provides the driving force for potassium retention and uptake through voltage-gated channels and for Na+ exclusion via Na+/H+ exchangers. Both of these traits are central to plant salinity tolerance; however, whether the increased activity of H+-ATPase is a constitutive trait in halophyte species and whether this activity is upregulated at either the transcriptional or post-translation level remain disputed. Methods The kinetics of salt-induced net H+, Na+ and K+ fluxes, membrane potential and AHA1/2/3 expression changes in the roots of two halophyte species, Atriplex lentiformis (saltbush) and Chenopodium quinoa (quinoa), were compared with data obtained from Arabidopsis thaliana roots. Key Results Intrinsic (steady-state) membrane potential values were more negative in A. lentiformis and C. quinoa compared with arabidopsis (−144 ± 3·3, −138 ± 5·4 and −128 ± 3·3 mV, respectively). Treatment with 100 mm NaCl depolarized the root plasma membrane, an effect that was much stronger in arabidopsis. The extent of plasma membrane depolarization positively correlated with NaCl-induced stimulation of vanadate-sensitive H+ efflux, Na+ efflux and K+ retention in roots (quinoa > saltbush > arabidopsis). NaCl-induced stimulation of H+ efflux was most pronounced in the root elongation zone. In contrast, H+-ATPase AHA transcript levels were much higher in arabidopsis compared with quinoa plants, and 100 mm NaCl treatment led to a further 3-fold increase in AHA1 and AHA2 transcripts in arabidopsis but not in quinoa. Conclusions Enhanced salinity tolerance in the halophyte species studied here is not related to the constitutively higher AHA transcript levels in the root epidermis, but to the plant’s ability to rapidly upregulate plasma membrane H+-ATPase upon salinity treatment. This is necessary for assisting plants to maintain highly negative

E2P phosphoforms of Na,K-ATPase. I. Comparison of phosphointermediates formed from ATP and Pi by their reactivity toward hydroxylamine and vanadate.

PubMed

Fedosova, N U; Cornelius, F; Klodos, I

1998-09-29

The properties of Na,K-ATPase phosphoenzymes formed either from ATP in the presence of Mg2+ and Na+ or from Pi in the absence of alkali cations were investigated by biochemical methods and spectrofluorometry employing the styryl dye RH421. We characterized the phosphoenzyme species by their reaction to N-methyl hydroxylamine, which attacks specifically the protein-phosphate bond. We studied reactions of the phospho- and dephospho-enzymes with vanadate, which is a transition-state analogue of phosphate in this enzyme. On the basis of substantial differences in the properties of the phosphoenzyme species formed either from ATP or Pi, especially in their reactivity to N-methyl hydroxylamine, it is suggested that the two phosphoenzyme species are two subconformations of the E2P phosphoform. Analysis of the RH421 fluorescence responses under a variety of experimental conditions and comparing different enzyme sources suggested that the increase of RH421 fluorescence induced by inorganic phosphate in the absence of alkali cations is associated with the formation of the covalent acyl-phosphate bond.

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.

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.

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.

Effect of antioxidants on vanadate-induced toxicity towards isolated perfused rat livers.

PubMed

Younes, M; Kayser, E; Strubelt, O

1991-01-01

The effect of trolox C, a water soluble vitamin E analogue, propyl gallate and ascorbate on vanadate hepatotoxicity was investigated in vitro. In isolated perfused livers from fasted rats, sodium orthovanadate (2 mmol/l) led to toxic responses including reduction of oxygen consumption, release of cytosolic (glutamate-pyruvate-transaminase (GPT) and lactate dehydrogenase (LDH)) and mitochondrial (glutamate-dehydrogenase (GLDH)) enzymes, intracellular accumulation of calcium, a marked depletion of glutathione (GSH) and an enhanced formation and release of thiobarbituric acid- (TBA) reactive material. Trolox C and propyl gallate inhibited the release of GPT and LDH partially and that of GLDH totally, but had no influence on vanadate-induced calcium accumulation or on the reduction of oxygen consumption. Both agents suppressed vanadate-induced lipid peroxidation (LPO) and partially prevented GSH depletion. Ascorbate failed to provide any protection probably due to the interference of its pro-oxidant potential with its antioxidant activity. The protection, mainly of mitochondria, afforded by those agents which also inhibited LPO substantiates our previous findings that the pro-oxidant activity of vanadate is mainly responsible for its direct hepatotoxic actions [2]. Besides, reduction of organ perfusion rate due to vasoconstriction also contributes to vanadate toxicity, but oxidative stress is not involved in this indirect toxic activity.

High-Affinity Vanadate Transport System in the Cyanobacterium Anabaena variabilis ATCC 29413

PubMed Central

Pratte, Brenda S.; Thiel, Teresa

2006-01-01

High-affinity vanadate transport systems have not heretofore been identified in any organism. Anabaena variabilis, which can fix nitrogen by using an alternative V-dependent nitrogenase, transported vanadate well. The concentration of vanadate giving half-maximum V-nitrogenase activity when added to V-starved cells was about 3 × 10−9 M. The genes for an ABC-type vanadate transport system, vupABC, were found in A. variabilis about 5 kb from the major cluster of genes encoding the V-nitrogenase, and like those genes, the vupABC genes were repressed by molybdate; however, unlike the V-nitrogenase genes the vanadate transport genes were expressed in vegetative cells. A vupB mutant failed to grow by using V-nitrogenase unless high levels of vanadate were provided, suggesting that there was also a low-affinity vanadate transport system that functioned in the vupB mutant. The vupABC genes belong to a family of putative metal transport genes that include only one other characterized transport system, the tungstate transport genes of Eubacterium acidaminophilum. Similar genes are not present in the complete genomes of other bacterial strains that have a V-nitrogenase, including Azotobacter vinelandii, Rhodopseudomonas palustris, and Methanosarcina barkeri. PMID:16385036

Snakes exhibit tissue-specific variation in cardiotonic steroid sensitivity of Na+/K+-ATPase.

PubMed

Mohammadi, Shabnam; Petschenka, Georg; French, Susannah S; Mori, Akira; Savitzky, Alan H

2018-03-01

Toads are among several groups of organisms chemically defended with lethal concentrations of cardiotonic steroids. As a result, most predators that prey on amphibians avoid toads. However, several species of snakes have gained resistance-conferring mutations of Na + /K + -ATPase, the molecular target of cardiotonic steroids, and can feed on toads readily. Despite recent advances in our understanding of this adaptation at the genetic level, we have lacked functional evidence for how mutations of Na + /K + -ATPase account for cardiotonic steroid resistance in snake tissues. To address this issue, it is necessary to determine how the Na + /K + -ATPases of snakes react to the toxins. Some tissues might have Na + /K + -ATPases that are more susceptible than others and can thus provide clues about how the toxins influence organismal function. Here we provide a mechanistic link between observed Na + /K + -ATPase substitutions and observed resistance using actual snake Na + /K + -ATPases. We used an in vitro approach to determine the tissue-specific levels of sensitivity to cardiotonic steroids in select resistant and non-resistant snakes. We compared the sensitivities of select tissues within and between species. Our results suggest that resistant snakes contain highly resistant Na + /K + -ATPases in their heart and kidney, both of which rely heavily on the enzymes to function, whereas tissues that do not rely as heavily on Na + /K + -ATPases or might be protected from cardiotonic steroids by other means (liver, gut, and brain) contain non-resistant forms of the enzyme. This study reveals functional evidence that tissue-level target-site insensitivity to cardiotonic steroids varies not only among species but also across tissues within resistant taxa. Copyright © 2017 Elsevier Inc. All rights reserved.

The Arabidopsis cax3 mutants display altered salt tolerance, pH sensitivity and reduced plasma membrane H+-ATPase activity.

PubMed

Zhao, Jian; Barkla, Bronwyn J; Marshall, Joy; Pittman, Jon K; Hirschi, Kendal D

2008-02-01

Perturbing CAX1, an Arabidopsis vacuolar H+/Ca2+ antiporter, and the related vacuolar transporter CAX3, has been previously shown to cause severe growth defects; however, the specific function of CAX3 has remained elusive. Here, we describe plant phenotypes that are shared among cax1 and cax3 including an increased sensitivity to both abscisic acid (ABA) and sugar during germination, and an increased tolerance to ethylene during early seedling development. We have also identified phenotypes unique to cax3, namely salt, lithium and low pH sensitivity. We used biochemical measurements to ascribe these cax3 sensitivities to a reduction in vacuolar H+/Ca2+ transport during salt stress and decreased plasma membrane H+-ATPase activity. These findings catalog an array of CAX phenotypes and assign a specific role for CAX3 in response to salt tolerance.

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.

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.

Studies on H+-Translocating ATPases in Plants of Varying Resistance to Salinity 1

PubMed Central

Braun, Yael; Hassidim, Miriam; Lerner, Henri R.; Reinhold, Leonora

1986-01-01

Membrane vesicles were isolated from the roots of the halophyte Atriplex nummularia Lindl. H+-translocating Mg2+-ATPase activity was manifested by the establishment of a positive membrane potential (measured as SCN− accumulation); and also by the establishment of a transmembrane pH gradient (measured by quinacrine fluorescence quenching). H+-translocation was highly specific to ATP and was stable to oligomycin. Growing the plants in the presence of 400 millimolar NaCl doubled the proton-translocating activity per milligram of membrane protein and otherwise modulated it in the following ways. First, the flat pH profile observed in non-salt-grown plants was transformed to one showing a peak at about pH 6.2. Second, the lag effect observed at low ATP concentration in curves relating SCN− accumulation to ATP concentration was abolished; the concave curvature shown in the double reciprocal plot was diminished. Third, sensitivity to K-2 (N-morpholino)ethanesulfonic acid stimulation was shown in salt-grown plants (about 40% stimulation) but was absent in non-salt-grown plants. Fourth, the KCl concentration bringing about 50% dissipation of ATP-dependent SCN− accumulation was 20 millimolar for salt-grown plants and 50 millimolar for non-salt-grown plants. Vanadate sensitivity was shown in both cases. No clear NO3− inhibition was observed. Images Fig. 3 PMID:16664942

Optical Properties of Nd Doped Rare Earth Vanadates (Preprint)

DTIC Science & Technology

2010-07-01

Rare earth orthovanadates are being used as substitute for traditional solid state laser hosts such as yttrium aluminium garnet (YAG). While the most...common of these is yttrium orthovanadate, other rare earth vanadates such as lutetium vanadate and gadolinium vanadate are being used for their... gadolinium vanadate are being used for their special properties in certain applications. We report new measurements of the refractive indices and thermo

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.

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.

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.

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.

Plasma Membrane ATPase Activity following Reversible and Irreversible Freezing Injury 1

PubMed Central

Iswari, S.; Palta, Jiwan P.

1989-01-01

Plasma membrane ATPase has been proposed as a site of functional alteration during early stages of freezing injury. To test this, plasma membrane was purified from Solanum leaflets by a single step partitioning of microsomes in a dextran-polyethylene glycol two phase system. Addition of lysolecithin in the ATPase assay produced up to 10-fold increase in ATPase activity. ATPase activity was specific for ATP with a Km around 0.4 millimolar. Presence of the ATPase enzyme was identified by immunoblotting with oat ATPase antibodies. Using the phase partitioning method, plasma membrane was isolated from Solanum commersonii leaflets which had four different degrees of freezing damage, namely, slight (reversible), partial (partially reversible), substantial and total (irreversible). With slight (reversible) damage the plasma membrane ATPase specific activity increased 1.5- to 2-fold and its Km was decreased by about 3-fold, whereas the specific activity of cytochrome c reductase and cytochrome c oxidase in the microsomes were not different from the control. However, with substantial (lethal, irreversible) damage, there was a loss of membrane protein, decrease in plasma membrane ATPase specific activity and decrease in Km, while cytochrome c oxidase and cytochrome c reductase were unaffected. These results support the hypothesis that plasma membrane ATPase is altered by slight freeze-thaw stress. Images Figure 1 Figure 2 PMID:16666856

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

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

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.

[Properties and localization of Mg- and Ca-ATpase activities in wheat embryo cell nuclei].

PubMed

Vasil'eva, N A; Belkina, G G; Stepanenko, S Y; Atalykova, F I; Oparin, A I

1978-05-01

The isolated nuclei of wheat embryo possess the ATPase activity. The addition of Mg2+ and Ca2+ significantly increases the activities of nuclear ATPases, whereas Hg2+, Cu2+ and Mn2+ inhibit the activity. The activating effect of Mg2+ is enhanced by an addition of Na and K ions. The activity of wheat embryo nuclear Mg-ATPase is higher than its Ca-ATPase activity; both ATPases also differ in their pH optima. Separation of total nuclear protein according to the solubility of its individual protein components in wheat and strong salt solutions, using the detergents, as well as ammonium sulfate precipitation and dialysis do not result in separation of Mg-activated and Ca-activated ATPases, although their levels of activities and ratios change in the course of fractionation. The Mg- and Ca-ATPase activities of the wheat embryo nuclei were found in the nuclear fraction of albumin, in nonhistone proteins and nuclear membranes. In the albumin nuclear fraction and subfractions of non-histone proteins the higher level of activity is observed in Ca-ATPase, whereas in the nuclei and soluble fractions of residual proteins in Mg-ATPase.

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

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.

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.

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

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.

Effects of aqueous extract of Hibiscus sabdariffa on renal Na(+)-K(+)-ATPase and Ca(2+)-Mg(2+)-ATPase activities in Wistar rats.

PubMed

Olatunji, Lawrence A; Usman, Taofeek O; Adebayo, Joseph O; Olatunji, Victoria A

2012-09-01

To investigate the effects of oral administration of aqueous extract of Hibiscus sabdariffa on renal Na(+)-K(+)-ATPase and Ca(2+)-Mg(2+)-ATPase activities in rats. The 25 and 50 mg/(kg·d) of aqueous extracts of H. sabdariffa were respectively given to rats in the experimental groups for 28 d, and rats in the control group received an appropriate volume of distilled water as vehicle. Na(+)-K(+)-ATPase and Ca(2+)-Mg(2+)-ATPase activities in the kidney were assayed by spectrophotometric method. Administrations of 25 and 50 mg/(kg·d) of aqueous extract of H. sabdariffa significantly decreased the Ca(2+)-Mg(2+)-ATPase activity in the kidney of rats (P<0.05). However, the renal Na(+)-K(+)-ATPase activity of the experimental rats was not affected by either dose of the extract. And the plasma Na(+), K(+) and Ca(2+) levels of the experimental rats had no significant changes. Administration of either dose of the extract did not result in any significant changes in body and kidney weights, the concentrations of plasma albumin and total protein, and alkaline phosphatase, aspartate aminotransferase and alanine aminotransferase activities. However, concentrations of creatinine and urea were significantly reduced by 50 mg/kg of the extract (P<0.05). The present study indicates that oral administration of aqueous extract of H. sabdariffa may preserve the renal function despite a decreased renal Ca(2+)-Mg(2+)-ATPase activity.

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.

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

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.

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.

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.

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

Phorbol esters inhibit smooth muscle contractions through activation of Na(+)-K(+)-ATPase.

PubMed Central

Sasaguri, T.; Watson, S. P.

1990-01-01

1. The role of protein kinase C (PKC) in agonist-induced contractions of guinea-pig ileum longitudinal smooth muscle has been investigated. 2. The phorbol esters, phorbol 12,13-dibutyrate (PDBu), phorbol 12,13-diacetate (PDA) and phorbol 12-myristate 13-acetate (PMA), relaxed tissues precontracted by submaximal concentrations of carbachol, histamine or substance P. 3. This inhibitory action of the phorbol esters was reversed following the application of ouabain, a specific inhibitor of Na(+)-K(+)-ATPase. Similarly, pretreatment with ouabain inhibited the ability of phorbol esters to relax tissues precontracted by the above agonists. 4. The slow relaxation of the tonic component of contraction induced by submaximal concentrations of carbachol and histamine, and all concentrations of substance P, was abolished in the presence of ouabain. 5. In Na(+)-loaded tissues, PDBu and carbachol caused a concentration-dependent increase of Na(+)-K(+)-ATPase activity, assessed by ouabain-sensitive 86Rb(+)-uptake. Extrusion of Na+, assessed by the cellular content of the ion, was also stimulated by PDBu (the effect of carbachol was not investigated). 6. We conclude that phorbol esters inhibit the tonic component of contractions induced by submaximal concentrations of these agonists through activation of Na(+)-K(+)-ATPase. We suggest that PKC may exert feedback control over the tonic component of agonist contractions through stimulation of the pump. PMID:1691673

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

Dietary selenium increases the antioxidant levels and ATPase activity in the arteries and veins of poultry.

PubMed

Cao, Changyu; Zhao, Xia; Fan, Ruifeng; Zhao, Jinxin; Luan, Yilin; Zhang, Ziwei; Xu, Shiwen

2016-07-01

Selenium (Se) deficiency is associated with the pathogenesis of vascular diseases. It has been shown that oxidative levels and ATPase activity were involved in Se deficiency diseases in humans and mammals; however, the mechanism by how Se influences the oxidative levels and ATPase activity in the poultry vasculature is unclear. We assessed the effects of dietary Se deficiency on the oxidative stress parameters (superoxide dismutase, catalase, and hydroxyl radical) and ATPase (Na(+)K(+)-ATPase, Ca(++)-ATPase, Mg(++)-ATPase, and Ca(++)Mg(++)-ATPase) activity in broiler poultry. A total of 40 broilers (1-day old) were randomly divided into a Se-deficient group (L group, fed a Se-deficient diet containing 0.08 mg/kg Se) and a control group (C group, fed a diet containing sodium selenite at 0.20 mg/kg Se). Then, arteries and veins were collected following euthanasia when typical symptoms of Se deficiency appeared. Antioxidant indexes and ATPase activity were evaluated using standard assays in arteries and veins. The results indicated that superoxide dismutase activity in the artery according to dietary Se deficiency was significantly lower (p < 0.05) compared with the C group. The catalase activity in the veins and hydroxyl radical inhibition in the arteries and veins by dietary Se deficiency were significantly higher (p < 0.05) compared with the C group. The Se-deficient group showed a significantly lower (p < 0.05) tendency in Na(+)K(+)-ATPase activity, Ca(++)-ATPase activity, and Ca(++)Mg(++)-ATPase activity. There were strong correlations between antioxidant indexes and Ca(++)-ATPase activity. Thus, these results indicate that antioxidant indexes and ATPases may have special roles in broiler artery and vein injuries under Se deficiency.

Studies on H-Translocating ATPases in Plants of Varying Resistance to Salinity : I. Salinity during Growth Modulates the Proton Pump in the Halophyte Atriplex nummularia.

PubMed

Braun, Y; Hassidim, M; Lerner, H R; Reinhold, L

1986-08-01

Membrane vesicles were isolated from the roots of the halophyte Atriplex nummularia Lindl. H(+)-translocating Mg(2+)-ATPase activity was manifested by the establishment of a positive membrane potential (measured as SCN(-) accumulation); and also by the establishment of a transmembrane pH gradient (measured by quinacrine fluorescence quenching). H(+)-translocation was highly specific to ATP and was stable to oligomycin. Growing the plants in the presence of 400 millimolar NaCl doubled the proton-translocating activity per milligram of membrane protein and otherwise modulated it in the following ways. First, the flat pH profile observed in non-salt-grown plants was transformed to one showing a peak at about pH 6.2. Second, the lag effect observed at low ATP concentration in curves relating SCN(-) accumulation to ATP concentration was abolished; the concave curvature shown in the double reciprocal plot was diminished. Third, sensitivity to K-2 (N-morpholino)ethanesulfonic acid stimulation was shown in salt-grown plants (about 40% stimulation) but was absent in non-salt-grown plants. Fourth, the KCl concentration bringing about 50% dissipation of ATP-dependent SCN(-) accumulation was 20 millimolar for salt-grown plants and 50 millimolar for non-salt-grown plants. Vanadate sensitivity was shown in both cases. No clear NO(3) (-) inhibition was observed.

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.

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.

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

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

Silver vanadate nanoribbons: A label-free bioindicator in the conversion between human serum transferrin and apotransferrin via surface-enhanced Raman scattering

NASA Astrophysics Data System (ADS)

Zhou, Qing; Shao, Mingwang; Que, Ronghui; Cheng, Liang; Zhuo, Shujuan; Tong, Yanhua; Lee, Shuit-Tong

2011-05-01

Silver vanadate nanoribbons were synthesized via a hydrothermal process, which exhibited surface-enhanced Raman scattering effect. This surface-enhanced substrate was stable and reproducible for identifying human serum transferrin and human serum apotransferrin in the concentration of 1×10-5 M, which further exhibited significant sensitivity in monitoring the conversion of these two proteins in turn. This result showed that the silver vanadate nanoribbon might be employed as biomonitor in such systems.

Activation of multiple pH-regulatory pathways in granulocytes by a phosphotyrosine phosphatase antagonist.

PubMed Central

Bianchini, L; Nanda, A; Wasan, S; Grinstein, S

1994-01-01

Activated phagocytes undergo a massive burst of metabolic acid generation, yet must be able to maintain their cytosolic pH (pHi) within physiological limits. Peroxides of vanadate (V(4+)-OOH), potent inhibitors of phosphotyrosine phosphatases, have recently been shown to produce activation of the respiratory burst in HL60 granulocytes. We therefore investigated the effects of V(4+)-OOH on pHi homoeostasis in HL60 granulocytes, using a pH-sensitive fluorescent dye. V(4+)-OOH stimulation induced a biphasic pH change: a transient cytosolic acidification followed by a significant alkalinization. The initial acidification was prevented by inhibition of the NADPH oxidase and was absent in undifferentiated cells lacking oxidase activity. Analysis of the alkalinization phase demonstrated the involvement of the Na+/H+ antiporter, and also provided evidence for activation of two alternative H(+)-extrusion pathways: a bafilomycin-sensitive component, likely reflecting vacuolar-type H(+)-ATPase activity, and a Zn(2+)-sensitive H(+)-conductive pathway. Our results indicate that V(4+)-OOH stimulation not only activated the NADPH oxidase but concomitantly stimulated H(+)-extrusion pathways, enabling the cells to compensate for the massive production of intracellular H+ associated with the respiratory burst. PMID:8043000

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

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.

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

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

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.

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.

Association between erythrocyte Na+K+-ATPase activity and some blood lipids in type 1 diabetic patients from Lagos, Nigeria

PubMed Central

Iwalokun, Bamidele A; Iwalokun, Senapon O

2007-01-01

Background Altered levels of erythrocyte Na+K+-ATPase, atherogenic and anti-atherogenic lipid metabolites have been implicated in diabetic complications but their pattern of interactions remains poorly understood. This study evaluated this relationship in Nigerian patients with Type 1 diabetes mellitus. Methods A total of 34 consented Type 1 diabetic patients and age -matched 27 non-diabetic controls were enrolled. Fasting plasma levels of total cholesterol, triglycerides and HDL-cholesterol were determined spectrophotometrically and LDL-cholesterol estimated using Friedewald formula. Total protein content and Na+K+-ATPase activity were also determined spectrophotometrically from ghost erythrocyte membrane prepared by osmotic lysis. Results Results indicate significant (P < 0.05) reduction in Na+K+-ATPase activity in the Type 1 diabetic patients (0.38 ± 0.08 vs. 0.59 ± 0.07 uM Pi/mgprotein/h) compared to the control but with greater reduction in the diabetic subgroup with poor glycemic control (n = 20) and in whom cases of hypercholesterolemia (8.8%), hypertriglyceridemia (2.9%) and elevated LDL-cholesterol (5.9% each) were found. Correlation analyses further revealed significant (P < 0.05) inverse correlations [r = -(0.708-0.797] between all the atherogenic lipid metabolites measured and Na+K+-ATPase in this subgroup contrary to group with good glycemic control or non-diabetic subjects in which significant (P < 0.05) Na+K+-ATPase and HDL-C association were found (r = 0.427 - 0.489). The Na+K+-ATPase from the diabetic patients also exhibited increased sensitivity to digoxin and alterations in kinetic constants Vmax and Km determined by glycemic status of the patients. Conclusion It can be concluded that poor glycemic control evokes greater reduction in erythrocyte Na+K+-ATPase activity and promote enzyme-blood atherogenic lipid relationships in Type 1 diabetic Nigerian patients. PMID:17908327

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.

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

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.

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.

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

sup 1 H and sup 51 V NMR studies of the interaction of vanadate and 2-vanadio-3-phosphoglycerate with phosphoglycerate mutase

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

Liu, S.; Gresser, M.J.; Tracey, A.S.

1992-03-17

The formation of complexes of vanadate with 2-phosphoglycerate and 3-phosphoglycerate have been studied using {sup 51}V nuclear magnetic resonance spectroscopy. Signals attributed to two 2,3-diphosphoglycerate analogues, 2-vanadio-3-phosphoglycerate and 2-phospho-3-vanadioglycerate, were detected but were not fully resolved from signals of inorganic vanadate and the anhydride formed between vanadate and the phosphate ester moieties of the individual phosphoglycerates. Equilibrium constants for formation of the two 2,3-bisphosphate analogues were estimated as 2.5 M{sup {minus}1} for 2-vanadio-3-phosphoglycerate and 0.2 M{sup {minus}1} for 2-phospho-3-vanadioglycerate. The results of the binding study are fully consistent with noncooperativity in the binding of vanadiophosphoglycerate to the two active sitesmore » of phosphoglycerate mutase (PGM). The results obtained here are in accord with these vanadate-phosphoglycerate complexes being much more potent inhibitors of phosphoglycerate mutase than either monomeric or dimeric vanadate. These results strongly support the view that phosphoryl transfer in this enzyme involves a pentacoordinate phosphate intermediate and suggests that the two active sites operate independently of each other.« less

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.

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.

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

Increased erythrocyte Na+ pump and NaK-ATPase activity during lithium therapy.

PubMed

Hokin-Neaverson, M; Burckhardt, W A; Jefferson, J W

1976-05-01

A significant mean increase of 18% in erythrocyte sodium pump activity (p less than 0.01, t test) was observed during lithium treatment, as compared with the activity before lithium treatment was started, in a group of 20 patients who were treated with lithium therapy for a variety of psychiatric conditions. The mean level of erythrocyte membrane ouabain-sensitive ATPase activity in a group of 35 subjects who were receiving lithium therapy was significantly higher than that of a different group of 38 subjects who were not receiving lithium therapy (p less than 0.005, t test). These observations may offer a biochemical mode of action for lithium in the treatment of bipolar affective disorder, since a deficiency of sodium pump activity has been shown to be associated with that disorder.

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.

Tributyltin (TBT) and dibutyltin (DBT) differently inhibit the mitochondrial Mg-ATPase activity in mussel digestive gland.

PubMed

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

2011-02-01

Tri-n-butyltin (TBT) has long been considered as the most toxic among organotins, especially to membrane systems. The partially dealkylated derivative di-n-butyltin (DBT) has up to now received poor attention and, whenever considered, shown to be less toxic than TBT except on the immune system. The present kinetic approach evidences that both TBT and DBT in vitro inhibit the Mg-ATPase in mussel digestive gland mitochondria by a different mechanism. DBT even displays a higher efficiency than TBT (IC(50)=0.32 μM for TBT vs. 0.19 μM for DBT) in inhibiting the enzyme hydrolytic activity. Differently from TBT which at high concentrations (>1 μM) apparently decreases the oligomycin-sensitivity of the Mg-ATPase, DBT at any concentration tested does not affect the oligomycin sensitivity. TBT probably binds to F(0), either in the form of free enzyme or of enzyme-substrate complex (Ki=K'i), acting as non-competitive inhibitor with respect to the ATP substrate. Conversely DBT, which acts as uncompetitive inhibitor of ATP and as competitive inhibitor of Mg(2+) cofactor, may bind strongly to F(1) subunit, thus preventing ATP hydrolysis. The Mg-ATPase inhibition by both organotins warns against a potential threat to crucial cell energy metabolism processes even after years from contamination and partial TBT debutylation. Copyright © 2010 Elsevier Ltd. All rights reserved.

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.

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.

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.

Electrodeposition of high-density lithium vanadate nanowires for lithium-ion battery

NASA Astrophysics Data System (ADS)

Hua, Kang; Li, Xiujuan; Fang, Dong; Yi, Jianhong; Bao, Rui; Luo, Zhiping

2018-07-01

Lithium vanadate nanowires have been electrodeposited onto a titanium (Ti) foil by a direct current electrodeposition without template. The morphology, crystal structure, and the effects of deposition voltage, temperature and time on the prepared samples were tested and presented. The as-prepared lithium vanadate nanowires/Ti composite can be used as electrode for lithium-ion battery. Electrochemical measurements showed that the electrode displayed a specific discharge capacitance as high as 235.1 mAh g-1 after 100 cycles at a current density of 30 mA g-1. This research provides a new pathway to explore high tap density vanadates nanowires on metals with enhanced electrochemical performance.

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.

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

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

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 (ENTPD

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.

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

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.

Temperature and frequency dependent conductivity of bismuth zinc vanadate semiconducting glassy system

NASA Astrophysics Data System (ADS)

Punia, R.; Kundu, R. S.; Dult, Meenakshi; Murugavel, S.; Kishore, N.

2012-10-01

The ac conductivity of bismuth zinc vanadate glasses with compositions 50V2O5. xBi2O3. (50-x) ZnO has been studied in the frequency range 10-1 Hz to 2 MHz and in temperature range 333.16 K to 533.16 K. The temperature and frequency dependent conductivity is found to obey Jonscher's universal power law for all the compositions of bismuth zinc vanadate glass system. The dc conductivity (σdc), crossover frequency (ωH), and frequency exponent (s) have been estimated from the fitting of experimental data of ac conductivity with Jonscher's universal power law. Enthalpy to dissociate the cation from its original site next to a charge compensating center (Hf) and enthalpy of migration (Hm) have also been estimated. It has been observed that mobility of charge carriers and ac conductivity in case of zinc vanadate glass system increases with increase in Bi2O3 content. In order to determine the conduction mechanism, the ac conductivity and its frequency exponent have been analyzed in the frame work of various theoretical models based on classical hopping over barriers and quantum mechanical tunneling. The ac conduction takes place via tunneling of overlapping large polarons in all the compositions of presently studied vanadate glasses. The fitting of experimental data of ac conductivity with overlapping large polarons tunneling model has also been done. The parameters; density of states at Fermi level (N(EF)), activation energy associated with charge transfer between the overlapping sites (WHO), inverse localization length (α) and polaron radius (rp) obtained from fitting of this model with experimental data are reasonable.

Vanadate, molybdate and tungstate for orthomolecular medicine.

PubMed

Matsumoto, J

1994-09-01

Recent studies indicate that oxyanions, such as vanadate (V) or vanadyl (IV), cause insulin-like effects on rats by stimulating the insulin receptor tyrosine kinase. Tungstate (VI) and molybdate (VI) show the same effects on rat adipocytes and hepatocytes. Results of uncontrolled trials on volunteers accumulated in Japan also suggest that tungstate effectively regulates diabetes mellitus without detectable side effects. Since these oxyanions naturally exist in organisms, oxyanion therapy, the oral administration of vanadate, vanadyl, molybdate, or tungstate, can be considered to be orthomolecular medicine. Therefore, these oxyanions may provide a viable alternative to chemotherapy. Many diseases in addition to diabetes mellitus might also be treated since the implication of these results is that tyrosine kinases are involved in a variety of diseases.

Latent nitrate reductase activity is associated with the plasma membrane of corn roots

NASA Technical Reports Server (NTRS)

Ward, M. R.; Grimes, H. D.; Huffaker, R. C.

1989-01-01

Latent nitrate reductase activity (NRA) was detected in corn (Zea mays L., Golden Jubilee) root microsome fractions. Microsome-associated NRA was stimulated up to 20-fold by Triton X-100 (octylphenoxy polyethoxyethanol) whereas soluble NRA was only increased up to 1.2-fold. Microsome-associated NRA represented up to 19% of the total root NRA. Analysis of microsomal fractions by aqueous two-phase partitioning showed that the membrane-associated NRA was localized in the second upper phase (U2). Analysis with marker enzymes indicated that the U2 fraction was plasma membrane (PM). The PM-associated NRA was not removed by washing vesicles with up to 1.0 M NACl but was solubilized from the PM with 0.05% Triton X-100. In contrast, vanadate-sensitive ATPase activity was not solubilized from the PM by treatment with 0.1% Triton X-100. The results show that a protein capable of reducing nitrate is embedded in the hydrophobic region of the PM of corn roots.

Different Forms of Vanadate on Sugar Transport in Insulin Target and Nontarget Cells

PubMed Central

2002-01-01

The effects of several vanadates (ie, orthovanadate, pervanadate, and two stable peroxovanadium compounds) on basal and insulin-stimulated 2-DG transport in insulin target and nontarget cell lines are reported, herein. In nontarget cells, exposure to vanadates (5 × 10−6 to 10−4 mol/L) resulted in 2-DG transport stimulatory responses similar to those observed in 2-DG transport post exposure to 667 nmol/L insulin alone, or insulin in combination with vanadates. In 3T3-L1 adipocytes and L6 myotubes, exposure to a vanadate compound or 67 nmol/L insulin, stimulated 2-DG transport dramatically. Again, this effect on stimulated transport was similar to 2-DG transport post-treatment with the effective vanadates in combination with insulin. While pervanadate or stable peroxovanadates stimulated 2-DG transport at 10−5 to 10−6 mol/L, orthovanadate up to 10−4 mol/L was not effective in stimulating 2-DG transport in any of the cell lines tested. The data indicate that the various peroxovanadates are clearly superior insulin mimetics while a more limited insulin mimesis is observed with orthovanadate over a wide variety of cell types. PMID:12488596

Different Forms of Vanadate on Sugar Transport in Insulin Target and Nontarget Cells.

PubMed

Germinario, Ralph J.; Colby-Germinario, Susan P.; Posner, Barry I.; Nahm, K.

2002-01-01

The effects of several vanadates (ie, orthovanadate, pervanadate, and two stable peroxovanadium compounds) on basal and insulin-stimulated 2-DG transport in insulin target and nontarget cell lines are reported, herein. In nontarget cells, exposure to vanadates (5 x 10(-6) to 10(-4) mol/L) resulted in 2-DG transport stimulatory responses similar to those observed in 2-DG transport post exposure to 667 nmol/L insulin alone, or insulin in combination with vanadates. In 3T3-L1 adipocytes and L6 myotubes, exposure to a vanadate compound or 67 nmol/L insulin, stimulated 2-DG transport dramatically. Again, this effect on stimulated transport was similar to 2-DG transport post-treatment with the effective vanadates in combination with insulin. While pervanadate or stable peroxovanadates stimulated 2-DG transport at 10(-5) to 10(-6) mol/L, orthovanadate up to 10(-4) mol/L was not effective in stimulating 2-DG transport in any of the cell lines tested. The data indicate that the various peroxovanadates are clearly superior insulin mimetics while a more limited insulin mimesis is observed with orthovanadate over a wide variety of cell types.

Novel solar light driven photocatalyst, zinc indium vanadate for photodegradation of aqueous phenol

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

Mahapure, Sonali A.; Rane, Vilas H.; Ambekar, Jalindar D.

2011-05-15

Graphical abstract: Novel photocatalyst, zinc indium vanadate (ZnIn{sub 2}V{sub 2}O{sub 9}) demonstrated and showed an excellent photocatalytic activity for phenol degradation under visible light. Research highlights: {yields} Designing and identification of a photocatalyst having prospective potential application to be used in visible light (400-800 nm). {yields} Successful synthesis of novel ZnIn{sub 2}V{sub 2}O{sub 9} by solid state route. {yields} Confirmation of the designed product using characterization techniques. {yields} Application study comprising photodegradation of aqueous phenol at visible light despite of UV radiations. -- Abstract: In the present investigation, we have demonstrated the synthesis of novel photocatalyst, zinc indium vanadate (ZIV)more » by solid-solid state route using respective oxides of zinc, indium and vanadium. This novel photocatalyst was characterized using XRD, FESEM, UV-DRS and FTIR in order to investigate its structural, morphological and optical properties. XRD clearly shows the formation of phase pure ZIV of triclinic crystal structure with good crystallinity. FESEM micrographs showed the clustered morphology having particle size between 0.5 and 1 {mu}m. Since, optical study showed the band gap around 2.8 eV, i.e. in visible region, we have performed the photocatalytic activity of phenol degradation under visible light irradiation. The photodecomposition of phenol by ZIV is studied for the first time and an excellent photocatalytic activity was obtained using this novel photocatalyst. Considering the band gap of zinc indium vanadate in visible region, it will also be the potential candidate for water splitting.« less

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

Synthesis of calcium vanadate minerals and related compounds

USGS Publications Warehouse

Marvin, Richard F.

1956-01-01

Synthesis of natural vanadates shows that most of them are stable in an acid environment. Phase studies of a portion of the system CaO-V2O5-H2O indicate that calcium vanadates are an indicator of environmental pH conditions. Some minerals, such as pascoute, indicate rapid evaporation of vanadite solutions; other minerals, such as hewettite, show that slow evaporation took place. Cursory examination of systems K2O-UO2-(NO3)2-V2O5 and CaO-UO2(NO3)2-V2O5, both in aqueous solution, has yielded information on the relationships among carnotite, tyuyamunite, and rauvite.

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.

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.

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

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.

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.

[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.

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

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

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

[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.

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

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.

Paradoxical activation of the sodium chloride cotransporter (NCC) without hypertension in kidney deficient in a regulatory subunit of Na,K-ATPase, FXYD2.

PubMed

Arystarkhova, Elena; Ralph, Donna L; Liu, Yi Bessie; Bouley, Richard; McDonough, Alicia A; Sweadner, Kathleen J

2014-12-01

Na,K-ATPase generates the driving force for sodium reabsorption in the kidney. Na,K-ATPase functional properties are regulated by small proteins belonging to the FXYD family. In kidney FXYD2 is the most abundant: it is an inhibitory subunit expressed in almost every nephron segment. Its absence should increase sodium pump activity and promote Na(+) retention, however, no obvious renal phenotype was detected in mice with global deletion of FXYD2 (Arystarkhova et al. 2013). Here, increased total cortical Na,K-ATPase activity was documented in the Fxyd2(-/-) mouse, without increased α1β1 subunit expression. We tested the hypothesis that adaptations occur in distal convoluted tubule (DCT), a major site of sodium adjustments. Na,K-ATPase immunoreactivity in DCT was unchanged, and there was no DCT hypoplasia. There was a marked activation of thiazide-sensitive sodium chloride cotransporter (NCC; Slc12a3) in DCT, predicted to increase Na(+) reabsorption in this segment. Specifically, NCC total increased 30% and NCC phosphorylated at T53 and S71, associated with activation, increased 4-6 fold. The phosphorylation of the closely related thick ascending limb (TAL) apical NKCC2 (Slc12a1) increased at least twofold. Abundance of the total and cleaved (activated) forms of ENaC α-subunit was not different between genotypes. Nonetheless, no elevation of blood pressure was evident despite the fact that NCC and NKCC2 are in states permissive for Na(+) retention. Activation of NCC and NKCC2 may reflect an intracellular linkage to elevated Na,K-ATPase activity or a compensatory response to Na(+) loss proximal to the TAL and DCT. © 2014 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of the American Physiological Society and The Physiological Society.

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

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

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

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

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

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

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 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.

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

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

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.

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

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.

Separation of large mammalian ventricular myosin differing in ATPase activity.

PubMed

Rupp, Heinz; Maisch, Bernhard

2007-01-01

To investigate a possible heterogeneity of human ventricular myosin, papillary muscles of patients with valvular dysfunction were examined using a modified native gel electrophoresis. Myosin was separated into 2 components termed VA and VB, whereby the VA to VB proportion appeared to depend on the ventricular load. The proportion of the faster migrating band VA was correlated (P<0.05) with end-diastolic pressure and the aortic pressure-cardiac index product. The regression based on these variables accounted for 67% of the variation in VA (R2=0.67). The VA proportion was, however, not significantly correlated with cardiac norepinephrine concentration. The ATPase activity of the 2 components of myosin was assessed from the Ca3(PO4)2 precipitation by incubating the gel in the presence of ATP and CaCl2. The ATPase activity of VA was 60% of that of VB. The VA and VB forms were observed also in the cat (31.4% VA), dog (32.1% VA), pig (28.5% VA), wild pig (33.7% VA), and roe deer (30.5% VA). VA and VB were not detected in the rat exhibiting the 3 isoforms V1, V2, and V3, rabbit (100% V3), and hare (86% V1). The data demonstrate a heterogeneity of large mammalian ventricular myosin, whereby an increased cardiac load appeared to be associated with a higher myosin VA proportion that exhibited a reduced ATPase activity.

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.

Membrane depolarization-induced RhoA/Rho-associated kinase activation and sustained contraction of rat caudal arterial smooth muscle involves genistein-sensitive tyrosine phosphorylation

PubMed Central

Mita, Mitsuo; Tanaka, Hitoshi; Yanagihara, Hayato; Nakagawa, Jun-ichi; Hishinuma, Shigeru; Sutherland, Cindy; Walsh, Michael P.; Shoji, Masaru

2013-01-01

Rho-associated kinase (ROK) activation plays an important role in K+-induced contraction of rat caudal arterial smooth muscle (Mita et al., Biochem J. 2002; 364: 431–40). The present study investigated a potential role for tyrosine kinase activity in K+-induced RhoA activation and contraction. The non-selective tyrosine kinase inhibitor genistein, but not the src family tyrosine kinase inhibitor PP2, inhibited K+-induced sustained contraction (IC50 = 11.3 ± 2.4 µM). Genistein (10 µM) inhibited the K+-induced increase in myosin light chain (LC20) phosphorylation without affecting the Ca2+ transient. The tyrosine phosphatase inhibitor vanadate induced contraction that was reversed by genistein (IC50 = 6.5 ± 2.3 µM) and the ROK inhibitor Y-27632 (IC50 = 0.27 ± 0.04 µM). Vanadate also increased LC20 phosphorylation in a genistein- and Y-27632-dependent manner. K+ stimulation induced translocation of RhoA to the membrane, which was inhibited by genistein. Phosphorylation of MYPT1 (myosin-targeting subunit of myosin light chain phosphatase) was significantly increased at Thr855 and Thr697 by K+ stimulation in a genistein- and Y-27632-sensitive manner. Finally, K+ stimulation induced genistein-sensitive tyrosine phosphorylation of proteins of ∼55, 70 and 113 kDa. We conclude that a genistein-sensitive tyrosine kinase, activated by the membrane depolarization-induced increase in [Ca2+]i, is involved in the RhoA/ROK activation and sustained contraction induced by K+. Ca2+ sensitization, myosin light chain phosphatase, RhoA, Rho-associated kinase, tyrosine kinase PMID:24133693

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.

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.

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.

Synthesis, characterization and electrical properties of a lead sodium vanadate apatite

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

Chakroun-Ouadhour, E.; Ternane, R.; Hassen-Chehimi, D. Ben

2008-08-04

The lacunary lead sodium vanadate apatite Pb{sub 8}Na{sub 2}(VO{sub 4}){sub 6} was synthesized by the solid-state reaction method. The compound was characterized by X-ray powder diffraction, infrared (IR) absorption spectroscopy and Raman scattering spectroscopy. By comparing the effect of vanadate and phosphate ions on electrical properties, it was concluded that Pb{sub 8}Na{sub 2}(VO{sub 4}){sub 6} apatite is better conductor than Pb{sub 8}Na{sub 2}(PO{sub 4}){sub 6} apatite.

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.

Sodium orthovanadate (vanadate), a potent mitigator of radiation-induced damage to the hematopoietic system in mice

PubMed Central

Wang, Bing; Tanaka, Kaoru; Morita, Akinori; Ninomiya, Yasuharu; Maruyama, Kouichi; Fujita, Kazuko; Hosoi, Yoshio; Nenoi, Mitsuru

2013-01-01

Previous in vitro and in vivo studies have shown that sodium orthovanadate (vanadate), an inorganic vanadium compound, could effectively suppress radiation-induced p53-mediated apoptosis via both transcription-dependent and transcription-independent pathways. As a potent radiation protector administered at a dose of 20 mg/kg body weight (20 mg/kg) prior to total body irradiation (TBI) by intra-peritoneal (ip) injection, it completely protected mice from hematopoietic syndrome and partially from gastrointestinal syndrome. In the present study, radiation mitigation effects from vanadate were investigated by ip injection of vanadate after TBI in mice. Results showed that a single administration of vanadate at a dose of 20 mg/kg markedly improved the 30-day survival rate and the peripheral blood hemogram, relieved bone marrow aplasia and decreased occurrence of the bone marrow micronucleated erythrocytes in the surviving animals. The dose reduction factor was 1.2 when a single dose of 20 mg/kg was administered 15 min after TBI in mice using the 30-day survival test as the endpoint. Results also showed that either doubling the vanadate dose (40 mg/kg) in a single administration or continuing the vanadate treatment (after a single administration at 20 mg/kg) from the following day at a dose of 5 mg/kg per day for 4 consecutive days further significantly improved the efficacy for rescuing bone marrow failure in the 30-day survival test. Taken together, these findings indicate that vanadate would be a potent mitigator suppressing the acute lethality (hematopoietic syndrome) and minimizing the detrimental effects (anhematopoiesis and delayed genotoxic effects) induced by TBI in mice. PMID:23349341

Effect of vanadium compounds on acid phosphatase activity.

PubMed

Vescina, C M; Sálice, V C; Cortizo, A M; Etcheverry, S B

1996-01-01

The direct effect of different vanadium compounds on acid phosphatase (ACP) activity was investigated. Vanadate and vanadyl but not pervanadate inhibited the wheat germ ACP activity. These vanadium derivatives did not alter the fibroblast Swiss 3T3 soluble fraction ACP activity. Using inhibitors of tyrosine phosphatases (PTPases), the wheat germ ACP was partially characterized as a PTPase. This study suggests that the inhibitory ability of different vanadium derivatives to modulate ACP activity seems to depend on the geometry around the vanadium atom more than on the oxidation state. Our results indicate a correlation between the PTPase activity and the sensitivity to vanadate and vanadyl cation.

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

A Role for the ATP7A Copper-transporting ATPase in Macrophage Bactericidal Activity*

PubMed Central

White, Carine; Lee, Jaekwon; Kambe, Taiho; Fritsche, Kevin; Petris, Michael J.

2009-01-01

Copper is an essential micronutrient that is necessary for healthy immune function. This requirement is underscored by an increased susceptibility to bacterial infection in copper-deficient animals; however, a molecular understanding of its importance in immune defense is unknown. In this study, we investigated the effect of proinflammatory agents on copper homeostasis in RAW264.7 macrophages. Interferon-γ was found to increase expression of the high affinity copper importer, CTR1, and stimulate copper uptake. This was accompanied by copper-stimulated trafficking of the ATP7A copper exporter from the Golgi to vesicles that partially overlapped with phagosomal compartments. Silencing of ATP7A expression attenuated bacterial killing, suggesting a role for ATP7A-dependent copper transport in the bactericidal activity of macrophages. Significantly, a copper-sensitive mutant of Escherichia coli lacking the CopA copper-transporting ATPase was hypersensitive to killing by RAW264.7 macrophages, and this phenotype was dependent on ATP7A expression. Collectively, these data suggest that copper-transporting ATPases, CopA and ATP7A, in both bacteria and macrophage are unique determinants of bacteria survival and identify an unexpected role for copper at the host-pathogen interface. PMID:19808669

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.

Atomic Layer Deposition of Bismuth Vanadates for Solar Energy Materials.

PubMed

Stefik, Morgan

2016-07-07

The fabrication of porous nanocomposites is key to the advancement of energy conversion and storage devices that interface with electrolytes. Bismuth vanadate, BiVO4 , is a promising oxide for solar water splitting where the controlled fabrication of BiVO4 layers within porous, conducting scaffolds has remained a challenge. Here, the atomic layer deposition of bismuth vanadates is reported from BiPh3 , vanadium(V) oxytriisopropoxide, and water. The resulting films have tunable stoichiometry and may be crystallized to form the photoactive scheelite structure of BiVO4 . A selective etching process was used with vanadium-rich depositions to enable the synthesis of phase-pure BiVO4 after spinodal decomposition. BiVO4 thin films were measured for photoelectrochemical performance under AM 1.5 illumination. The average photocurrents were 1.17 mA cm(-2) at 1.23 V versus the reversible hydrogen electrode using a hole-scavenging sulfite electrolyte. The capability to deposit conformal bismuth vanadates will enable a new generation of nanocomposite architectures for solar water splitting. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

The V-ATPase a2-subunit as a putative endosomal pH-sensor.

PubMed

Marshansky, V

2007-11-01

V-ATPase (vesicular H(+)-ATPase)-driven intravesicular acidification is crucial for vesicular trafficking. Defects in vesicular acidification and trafficking have recently been recognized as essential determinants of various human diseases. An important role of endosomal acidification in receptor-ligand dissociation and in activation of lysosomal hydrolytic enzymes is well established. However, the molecular mechanisms by which luminal pH information is transmitted to the cytosolic small GTPases that control trafficking events such as budding, coat formation and fusion are unknown. Here, we discuss our recent discovery that endosomal V-ATPase is a pH-sensor regulating the degradative pathway. According to our model, V-ATPase is responsible for: (i) the generation of a pH gradient between vesicular membranes; (ii) sensing of intravesicular pH; and (iii) transmitting this information to the cytosolic side of the membrane. We also propose the hypothetical molecular mechanism involved in function of the V-ATPase a2-subunit as a putative pH-sensor. Based on extensive experimental evidence on the crucial role of histidine residues in the function of PSPs (pH-sensing proteins) in eukaryotic cells, we hypothesize that pH-sensitive histidine residues within the intra-endosomal loops and/or C-terminal luminal tail of the a2-subunit could also be involved in the pH-sensing function of V-ATPase. However, in order to identify putative pH-sensitive histidine residues and to test this hypothesis, it is absolutely essential that we increase our understanding of the folding and transmembrane topology of the a-subunit isoforms of V-ATPase. Thus the crucial role of intra-endosomal histidine residues in pH-dependent conformational changes of the V-ATPase a2-isoform, its interaction with cytosolic small GTPases and ultimately in its acidification-dependent regulation of the endosomal/lysosomal protein degradative pathway remain to be determined.

Evidence of the presence of a calmodulin-sensitive plasma membrane Ca2+-ATPase in Trypanosoma equiperdum.

PubMed

Pérez-Gordones, María Carolina; Ramírez-Iglesias, José Rubén; Cervino, Vincenza; Uzcanga, Graciela L; Benaim, Gustavo; Mendoza, Marta

2017-04-01

Trypanosoma equiperdum belongs to the subgenus Trypanozoon, which has a significant socio-economic impact by limiting animal protein productivity worldwide. Proteins involved in the intracellular Ca 2+ regulation are prospective chemotherapeutic targets since several drugs used in experimental treatment against trypanosomatids exert their action through the disruption of the parasite intracellular Ca 2+ homeostasis. Therefore, the plasma membrane Ca 2+ -ATPase (PMCA) is considered as a potential drug target. This is the first study revealing the presence of a PMCA in T. equiperdum (TePMCA) showing that it is calmodulin (CaM) sensitive, revealed by ATPase activity, western-blot analysis and immuno-absorption assays. The cloning sequence for TePMCA encodes a 1080 amino acid protein which contains domains conserved in all PMCAs so far studied. Molecular modeling predicted that the protein has 10 transmembrane and three cytoplasmic loops which include the ATP-binding site, the phosphorylation domain and Ca 2+ translocation site. Like all PMCAs reported in other trypanosomatids, TePMCA lacks a classic CaM binding domain. Nevertheless, this enzyme presents in the C-terminal tail a region of 28 amino acids (TeC28), which most likely adopts a helical conformation within a 1-18 CaM binding motif. Molecular docking between Trypanosoma cruzi CaM (TcCaM) and TeC28 shows a significant similarity with the CaM-C28PMCA4b reference structure (2kne). TcCaM-TeC28 shows an anti-parallel interaction, the peptide wrapped by CaM and the anchor buried in the hydrophobic pocket, structural characteristic described for similar complexes. Our results allows to conclude that T. equiperdum possess a CaM-sensitive PMCA, which presents a non-canonical CaM binding domain that host a 1-18 motif. Copyright © 2017 Elsevier B.V. All rights reserved.

Aqueous rechargeable zinc/sodium vanadate batteries with enhanced performance from simultaneous insertion of dual carriers.

PubMed

Wan, Fang; Zhang, Linlin; Dai, Xi; Wang, Xinyu; Niu, Zhiqiang; Chen, Jun

2018-04-25

Rechargeable aqueous zinc-ion batteries are promising energy storage devices due to their high safety and low cost. However, they remain in their infancy because of the limited choice of positive electrodes with high capacity and satisfactory cycling performance. Furthermore, their energy storage mechanisms are not well established yet. Here we report a highly reversible zinc/sodium vanadate system, where sodium vanadate hydrate nanobelts serve as positive electrode and zinc sulfate aqueous solution with sodium sulfate additive is used as electrolyte. Different from conventional energy release/storage in zinc-ion batteries with only zinc-ion insertion/extraction, zinc/sodium vanadate hydrate batteries possess a simultaneous proton, and zinc-ion insertion/extraction process that is mainly responsible for their excellent performance, such as a high reversible capacity of 380 mAh g -1 and capacity retention of 82% over 1000 cycles. Moreover, the quasi-solid-state zinc/sodium vanadate hydrate battery is also a good candidate for flexible energy storage device.

V-ATPase and osmotic imbalances activate endolysosomal LC3 lipidation.

PubMed

Florey, Oliver; Gammoh, Noor; Kim, Sung Eun; Jiang, Xuejun; Overholtzer, Michael

2015-01-01

Recently a noncanonical activity of autophagy proteins has been discovered that targets lipidation of microtubule-associated protein 1 light chain 3 (LC3) onto macroendocytic vacuoles, including macropinosomes, phagosomes, and entotic vacuoles. While this pathway is distinct from canonical autophagy, the mechanism of how these nonautophagic membranes are targeted for LC3 lipidation remains unclear. Here we present evidence that this pathway requires activity of the vacuolar-type H(+)-ATPase (V-ATPase) and is induced by osmotic imbalances within endolysosomal compartments. LC3 lipidation by this mechanism is induced by treatment of cells with the lysosomotropic agent chloroquine, and through exposure to the Heliobacter pylori pore-forming toxin VacA. These data add novel mechanistic insights into the regulation of noncanonical LC3 lipidation and its associated processes, including LC3-associated phagocytosis (LAP), and demonstrate that the widely and therapeutically used drug chloroquine, which is conventionally used to inhibit autophagy flux, is an inducer of LC3 lipidation.

Inhibition of the k-stimulated ATPase of the plasmalemma of pinto bean leaves by ozone.

PubMed

Dominy, P J; Heath, R L

1985-01-01

Three varieties of Phaseolus vulgaris which differ in their sensitivity to ozone were examined for changes in some physiological and structural plasma membrane characteristics. Plasma membrane vesicles were prepared from control and ozone-treated (0.2 to 0.5 microliters per liter ozone for 5 hours) leaf tissue, and the (K(+) + Mg(2+))-ATPase activity determined and compared. No major changes were observed in the resistant varieties. The sensitive variety showed a severe inhibition of ATPase activity which was largely due to a decrease in the K(+)-stimulated component. This inhibition was completely reversed by the addition of sulfhydryl compounds.Ozone-induced plasma membrane permeability changes may be effected by damage to membrane proteins, perhaps by oxidation of amino acid sulfhydryl groups to disulfide and sulfenic moieties.

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.

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

Osmotic and thermal effects on in situ ATPase activity in permeabilized gill epithelial cells of the fish Gillichthys mirabilis

PubMed

KÜLtz; Somero

1995-01-01

Long-jawed mudsuckers (Gillichthys mirabilis) were acclimated to sea water (SW) at 7 °C, SW at 26 °C or dilute sea water (DSW) at 26 °C for 5 months. Gill cells were isolated and the proportion of mitochondria-rich (MR) cells was determined. The number of cells harvested amounted to 4.7x10(7)±0.6x10(7) to 10.6x10(7)±1.1x10(7) and the yield was between 7.1x10(8)±0.6x10(8) and 10.7x10(8)±1.4x10(8) cells g-1 gill epithelial mass. Cell viability was 96.8±0.4 to 97.8±0.6 %. The number, size and volume of MR cells decreased significantly during DSW acclimation, but did not change during thermal acclimation. The protein content was not influenced by osmotic or thermal acclimation and ranged between 20.0±1.6 and 22.1±1.5 pg cell-1. Using a new method, which is based on the formation of plasma membrane channels by alamethicin, we were able to permeabilize gill cells. For the first time, the Na+/K+-ATPase and H+-ATPase activities of fish gills were determined in intact cells in situ. The activity of both ATPases was dependent on alamethicin concentration (optimum 100 µg mg-1 protein) and on preincubation time (optimum 10 min). The in situ activity of both ATPases was influenced by osmotic, but not thermal, acclimation. A positive linear correlation was found between in situ Na+/K+-ATPase activity and total MR cell volume. However, we show, for the first time, that a negative linear correlation exists between H+-ATPase activity and total MR cell volume, suggesting a localization of H+-ATPase in pavement cells. In permeabilized cells, the activity of both ATPases was 2.6­3.9 times higher than that of crude homogenates and 1.6­2.1 times higher than that of permeabilized homogenate vesicles. We hypothesize that in crude homogenates three-quarters of Na+/K+-ATPase and two-thirds of H+-ATPase activity are not detectable both because of a mixture of inside-out and right-side-out vesicles and because

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.

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.

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.

The VPH1 gene encodes a 95-kDa integral membrane polypeptide required for in vivo assembly and activity of the yeast vacuolar H(+)-ATPase.

PubMed

Manolson, M F; Proteau, D; Preston, R A; Stenbit, A; Roberts, B T; Hoyt, M A; Preuss, D; Mulholland, J; Botstein, D; Jones, E W

1992-07-15

Yeast vacuolar acidification-defective (vph) mutants were identified using the pH-sensitive fluorescence of 6-carboxyfluorescein diacetate (Preston, R. A., Murphy, R. F., and Jones, E. W. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 7027-7031). Vacuoles purified from yeast bearing the vph1-1 mutation had no detectable bafilomycin-sensitive ATPase activity or ATP-dependent proton pumping. The peripherally bound nucleotide-binding subunits of the vacuolar H(+)-ATPase (60 and 69 kDa) were no longer associated with vacuolar membranes yet were present in wild type levels in yeast whole cell extracts. The VPH1 gene was cloned by complementation of the vph1-1 mutation and independently cloned by screening a lambda gt11 expression library with antibodies directed against a 95-kDa vacuolar integral membrane protein. Deletion disruption of the VPH1 gene revealed that the VPH1 gene is not essential for viability but is required for vacuolar H(+)-ATPase assembly and vacuolar acidification. VPH1 encodes a predicted polypeptide of 840 amino acid residues (molecular mass 95.6 kDa) and contains six putative membrane-spanning regions. Cell fractionation and immunodetection demonstrate that Vph1p is a vacuolar integral membrane protein that co-purifies with vacuolar H(+)-ATPase activity. Multiple sequence alignments show extensive homology over the entire lengths of the following four polypeptides: Vph1p, the 116-kDa polypeptide of the rat clathrin-coated vesicles/synaptic vesicle proton pump, the predicted polypeptide encoded by the yeast gene STV1 (Similar To VPH1, identified as an open reading frame next to the BUB2 gene), and the TJ6 mouse immune suppressor factor.

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.

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.

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

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.

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.

The (biological) speciation of vanadate(V) as revealed by (51)V NMR: A tribute on Lage Pettersson and his work.

PubMed

Rehder, Dieter

2015-06-01

Four decades of research carried out by Lage Pettersson, his group and his coworkers are reviewed, research that has been directed predominantly towards the speciation of vanadate and systems containing, along with vanadate and co-reactants such as phosphate and peroxide, biologically relevant organics. In particular, those organics have been addressed that either are (potential) ligands for vanadate-derived coordination compounds generated at physiological conditions and/or function as constituents in medicinally interesting oxidovanadium compounds. Examples for molecules introduced in the context of the physiological vanadate-ligand interaction include the dipeptides Pro-Ala, Ala-Gly, Ala-His and Ala-Ser, the serum constituents lactate and citrate, and the nucleobases adenosine and uridine. The speciation in the vanadate-picolinate and vanadate-maltol systems is geared towards insulin-enhancing vanadium drugs. The speciation as a function of pH, ionic strength and the concentration of vanadate and the ligand(s) is based on potentiometric and (51)V NMR investigations, a methodical combination that allows reliable access to composition, formation constants and, to some extent, also structural details for the manifold of species present in aqueous media at physiological pH and beyond. The time frame 1971 to 2014 is reviewed, emphasizing the interval 1985 to 2006, and thus focusing on biologically interesting vanadium systems. Figurative representations from the original literature have been included. Copyright © 2015 Elsevier Inc. All rights reserved.

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.

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

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.

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

P-glycoprotein ATPase activity requires lipids to activate a switch at the first transmission interface.

PubMed

Loo, Tip W; Clarke, David M

2016-04-01

P-glycoprotein (P-gp) is an ABC (ATP-Binding Cassette) drug pump. A common feature of ABC proteins is that they are organized into two wings. Each wing contains a transmembrane domain (TMD) and a nucleotide-binding domain (NBD). Drug substrates and ATP bind at the interface between the TMDs and NBDs, respectively. Drug transport involves ATP-dependent conformational changes between inward- (open, NBDs far apart) and outward-facing (closed, NBDs close together) conformations. P-gps crystallized in the presence of detergent show an open structure. Human P-gp is inactive in detergent but basal ATPase activity is restored upon addition of lipids. The lipids might cause closure of the wings to bring the NBDs close together to allow ATP hydrolysis. We show however, that cross-linking the wings together did not activate ATPase activity when lipids were absent suggesting that lipids may induce other structural changes required for ATPase activity. We then tested the effect of lipids on disulfide cross-linking of mutants at the first transmission interface between intracellular loop 4 (TMD2) and NBD1. Mutants L443C/S909C and L443C/R905C but not G471C/S909C and V472C/S909C were cross-linked with oxidant when in membranes. The mutants were then purified and cross-linked with or without lipids. Mutants G471C/S909C and V472C/S909C cross-linked only in the absence of lipids whereas mutants L443C/S909C and L443C/R905C were cross-linked only in the presence of lipids. The results suggest that lipids activate a switch at the first transmission interface and that the structure of P-gp is different in detergents and lipids. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

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

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.

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.

A Novel P1B-type Mn2+-transporting ATPase Is Required for Secreted Protein Metallation in Mycobacteria*

PubMed Central

Padilla-Benavides, Teresita; Long, Jarukit E.; Raimunda, Daniel; Sassetti, Christopher M.; Argüello, José M.

2013-01-01

Transition metals are central for bacterial virulence and host defense. P1B-ATPases are responsible for cytoplasmic metal efflux and play roles either in limiting cytosolic metal concentrations or in the maturation of secreted metalloproteins. The P1B-ATPase, CtpC, is required for Mycobacterium tuberculosis survival in a mouse model (Sassetti, C. M., and Rubin, E. J. (2003) Genetic requirements for mycobacterial survival during infection. Proc. Natl. Acad. Sci. U.S.A. 100, 12989–12994). CtpC prevents Zn2+ toxicity, suggesting a role in Zn2+ export from the cytosol (Botella, H., Peyron, P., Levillain, F., Poincloux, R., Poquet, Y., Brandli, I., Wang, C., Tailleux, L., Tilleul, S., Charriere, G. M., Waddell, S. J., Foti, M., Lugo-Villarino, G., Gao, Q., Maridonneau-Parini, I., Butcher, P. D., Castagnoli, P. R., Gicquel, B., de Chastellièr, C., and Neyrolles, O. (2011) Mycobacterial P1-type ATPases mediate resistance to zinc poisoning in human macrophages. Cell Host Microbe 10, 248–259). However, key metal-coordinating residues and the overall structure of CtpC are distinct from Zn2+-ATPases. We found that isolated CtpC has metal-dependent ATPase activity with a strong preference for Mn2+ over Zn2+. In vivo, CtpC is unable to complement Escherichia coli lacking a functional Zn2+-ATPase. Deletion of M. tuberculosis or Mycobacterium smegmatis ctpC leads to cytosolic Mn2+ accumulation but no alterations in other metals levels. Whereas ctpC-deficient M. tuberculosis is sensitive to extracellular Zn2+, the M. smegmatis mutant is not. Both ctpC mutants are sensitive to oxidative stress, which might explain the Zn2+-sensitive phenotype of the M. tuberculosis ctpC mutant. CtpC is a high affinity/slow turnover ATPase, suggesting a role in protein metallation. Consistent with this hypothesis, mutation of CtpC leads to a decrease of Mn2+ bound to secreted proteins and of the activity of secreted Fe/Mn-superoxide dismutase, particularly in M. smegmatis. Alterations in the

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

Redox Activation of the Universally Conserved ATPase YchF by Thioredoxin 1.

PubMed

Hannemann, Liya; Suppanz, Ida; Ba, Qiaorui; MacInnes, Katherine; Drepper, Friedel; Warscheid, Bettina; Koch, Hans-Georg

2016-01-20

YchF/Ola1 are unconventional members of the universally conserved GTPase family because they preferentially hydrolyze ATP rather than GTP. These ATPases have been associated with various cellular processes and pathologies, including DNA repair, tumorigenesis, and apoptosis. In particular, a possible role in regulating the oxidative stress response has been suggested for both bacterial and human YchF/Ola1. In this study, we analyzed how YchF responds to oxidative stress and how it potentially regulates the antioxidant response. Our data identify a redox-regulated monomer-dimer equilibrium of YchF as a key event in the functional cycle of YchF. Upon oxidative stress, the oxidation of a conserved and surface-exposed cysteine residue promotes YchF dimerization, which is accompanied by inhibition of the ATPase activity. No dimers were observed in a YchF mutant lacking this cysteine. In vitro, the YchF dimer is dissociated by thioredoxin 1 (TrxA) and this stimulates the ATPase activity. The physiological significance of the YchF-thioredoxin 1 interaction was demonstrated by in vivo cross-linking, which validated this interaction in living cells. This approach also revealed that both the ATPase domain and the helical domain of YchF are in contact with TrxA. YchF/Ola1 are the first redox-regulated members of the universally conserved GTPase family and are inactivated by oxidation of a conserved cysteine residue within the nucleotide-binding motif. Our data provide novel insights into the regulation of the so far ill-defined YchF/Ola1 family of proteins and stipulate their role as negative regulators of the oxidative stress response.

[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.

Resveratrol modulates ATPase activity of liposome-reconstituted ABCG1.

PubMed

de Athayde Moncorvo Collado, Alejandro; Corbalán, Natalia; Homolya, László; Morero, Roberto; Minahk, Carlos

2013-08-02

ABCG1 is a half-sized transporter with an unquestionable importance in cholesterol homeostasis. So far, its expression and thus its activity was suggested to be regulated at transcriptional level by LXR and PPAR agonists including polyphenols. However, it is unknown whether there are other mechanisms of up-regulation of ABCG1 activity. In the present work resveratrol was shown to induce a nearly twofold increase in ATPase activity of reconstituted ABCG1. Evidence is presented for the first time suggesting that resveratrol is able to activate ABCG1 activity by an alternative mechanism that involves an indirect interaction. Copyright © 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

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

A plasma membrane-type Ca[sup 2+]-ATPase of 120 kilodaltons on the endoplasmic reticulum from carrot (Daucus carota) cells

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

Chen, F.H.; Ratterman, D.M.; Sze, H.

1993-06-01

Cytosolic Ca[sup 2+] levels are regulated in part by Ca[sup 2+]-pumping ATPases that export Ca[sup 2+] from the cytoplasm; The types and properties of Ca[sup 2+] pumps in plants are not well understood. The kinetic properties of a 120-kD phosphoenzyme (PE) intermediate formed during the reaction cycle of a Ca[sup 2+]-ATPase from suspension-cultured carrot (Daucus carota) cells are characterized. Only one Ca[sup 2+]-dependent phosphoprotein was formed when carrot membrane vesicles were incubated with [[gamma]-[sup 32]P]ATP. Formation of this 120-kD phosphoprotein was inhibited by vanadate, enhanced by La[sup 3+], and decreased by hydroxylamine, confirming its identification as an intermediate of amore » phosphorylated-type Ca[sup 2+]-translocating ATPase. The 120-kD Ca[sup 2+]-ATPase was most abundant in endoplasmic reticulum-enriched fractions, in which the Ca[sup 2+]-ATPase was estimated to be 0.1% of membrane protein. Direct quantitation of Ca[sup 2+]-dependent phosphoprotein was used to examine the kinetics of PE formation. PE formation exhibited a K[sub m] for Ca[sup 2+] of 1 to 2 [mu]m and a K[sub m] for ATP of 67 nm. Relative affinities of substrates, determined by competition experiments, were 0.075 [mu]m for ATP, 1 [mu]m for ADP, 100 [mu]m for ITP, and 250 [mu]m for GTP. Thapsigargin and cyclopiazonic acid, specific inhibitors of animal sarcoplasmic/endoplasmic reticulum Ca[sup 2+]-ATPase, had no effect on PE formation; erythrosin B inhibited with 50% inhibition at <0.1 [mu]m. Calmodulin (1 [mu]m) stimulated PE formation by 25%. The results indicate that the carrot 120-kD Ca[sup 2+]-ATPase is similar but not identical to animal plasma membrane-type Ca[sup 2+]-ATPase and yet is located on endomembranes, such as the endoplasmic reticulum. This type of Ca[sup 2+] pump may reside on the cortical endoplasmic reticulum, thought to play a major role in anchoring the cytoskeleton and in facilitating secretion. 34 refs., 9 figs., 3 tabs.« less

Trypsin digestion for determining orientation of ATPase in Halobacterium saccharovorum membrane vesicles

NASA Technical Reports Server (NTRS)

Kristjansson, H.; Hochstein, L. I.

1986-01-01

Membranes prepared by low pressure disruption of cells exhibited no ATPase activity in the absence of Triton X-100, although 43% of the total menadione reductase activity was detected. Trypsin digestion reduced menadione reductase activity by 45% whereas ATPase activity was not affected. Disruption of the membrane fraction at higher pressure solubilized about 45% of the ATPase activity. The soluble activity was still enhanced by Triton X-100, suggesting that the detergent, besides disrupting membrane vesicles, also activated the ATPase. The discrepancy in localization of menadione reductase and ATPase activities raised questions regarding the reliability of using a single marker enzyme as an indicator of vesicle orientation.

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.

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

CGP-37157 inhibits the sarcoplasmic reticulum Ca²+ ATPase and activates ryanodine receptor channels in striated muscle.

PubMed

Neumann, Jake T; Diaz-Sylvester, Paula L; Fleischer, Sidney; Copello, Julio A

2011-01-01

7-Chloro-5-(2-chlorophenyl)-1,5-dihydro-4,1-benzothiazepin-2(3H)-one [CGP-37157 (CGP)], a benzothiazepine derivative of clonazepam, is commonly used as a blocker of the mitochondrial Na+/Ca²+ exchanger. However, evidence suggests that CGP could also affect other targets, such as L-type Ca²+ channels and plasmalemma Na+/Ca²+ exchanger. Here, we tested the possibility of a direct modulation of ryanodine receptor channels (RyRs) and/or sarco/endoplasmic reticulum Ca²+-stimulated ATPase (SERCA) by CGP. In the presence of ruthenium red (inhibitor of RyRs), CGP decreased SERCA-mediated Ca²+ uptake of cardiac and skeletal sarcoplasmic reticulum (SR) microsomes (IC₅₀ values of 6.6 and 9.9 μM, respectively). The CGP effects on SERCA activity correlated with a decreased V(max) of ATPase activity of SERCA-enriched skeletal SR fractions. CGP (≥ 5 μM) also increased RyR-mediated Ca²+ leak from skeletal SR microsomes. Planar bilayer studies confirmed that both cardiac and skeletal RyRs are directly activated by CGP (EC(50) values of 9.4 and 12.0 μM, respectively). In summary, we found that CGP inhibits SERCA and activates RyR channels. Hence, the action of CGP on cellular Ca²+ homeostasis reported in the literature of cardiac, skeletal muscle, and other nonmuscle systems requires further analysis to take into account the contribution of all CGP-sensitive Ca²+ transporters.

CGP-37157 Inhibits the Sarcoplasmic Reticulum Ca2+ ATPase and Activates Ryanodine Receptor Channels in Striated Muscle

PubMed Central

Neumann, Jake T.; Diaz-Sylvester, Paula L.; Fleischer, Sidney

2011-01-01

7-Chloro-5-(2-chlorophenyl)-1,5-dihydro-4,1-benzothiazepin-2(3H)-one [CGP-37157 (CGP)], a benzothiazepine derivative of clonazepam, is commonly used as a blocker of the mitochondrial Na+/Ca2+ exchanger. However, evidence suggests that CGP could also affect other targets, such as L-type Ca2+ channels and plasmalemma Na+/Ca2+ exchanger. Here, we tested the possibility of a direct modulation of ryanodine receptor channels (RyRs) and/or sarco/endoplasmic reticulum Ca2+-stimulated ATPase (SERCA) by CGP. In the presence of ruthenium red (inhibitor of RyRs), CGP decreased SERCA-mediated Ca2+ uptake of cardiac and skeletal sarcoplasmic reticulum (SR) microsomes (IC50 values of 6.6 and 9.9 μM, respectively). The CGP effects on SERCA activity correlated with a decreased Vmax of ATPase activity of SERCA-enriched skeletal SR fractions. CGP (≥5 μM) also increased RyR-mediated Ca2+ leak from skeletal SR microsomes. Planar bilayer studies confirmed that both cardiac and skeletal RyRs are directly activated by CGP (EC50 values of 9.4 and 12.0 μM, respectively). In summary, we found that CGP inhibits SERCA and activates RyR channels. Hence, the action of CGP on cellular Ca2+ homeostasis reported in the literature of cardiac, skeletal muscle, and other nonmuscle systems requires further analysis to take into account the contribution of all CGP-sensitive Ca2+ transporters. PMID:20923851

Different thermostabilities of sarcoplasmic reticulum (Ca2+ + Mg2+)-ATPases from rabbit and trout muscles.

PubMed

de Toledo, F G; Albuquerque, M C; Goulart, B H; Chini, E N

1995-05-01

Trout and rabbit (Ca2+ + Mg2+)-ATPases from sarcoplasmic reticulum were compared for differences in thermal inactivation and susceptibility to trypsin digestion. The trout ATPase is more heat-sensitive than the rabbit ATPase and is stabilized by Ca2+, Na+, K+ and nucleotides. Solubilization of both ATPases shows that the two ATPases have different protein-intrinsic inactivation kinetics. When digested by trypsin, the two ATPases display different cleavage patterns. The present results indicate that the trout and rabbit ATPases have dissimilarities in protein structure that may explain the differences in thermal inactivation kinetics.

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.

Redox Activation of the Universally Conserved ATPase YchF by Thioredoxin 1

PubMed Central

Hannemann, Liya; Suppanz, Ida; Ba, Qiaorui; MacInnes, Katherine; Drepper, Friedel; Warscheid, Bettina

2016-01-01

Abstract Aims: YchF/Ola1 are unconventional members of the universally conserved GTPase family because they preferentially hydrolyze ATP rather than GTP. These ATPases have been associated with various cellular processes and pathologies, including DNA repair, tumorigenesis, and apoptosis. In particular, a possible role in regulating the oxidative stress response has been suggested for both bacterial and human YchF/Ola1. In this study, we analyzed how YchF responds to oxidative stress and how it potentially regulates the antioxidant response. Results: Our data identify a redox-regulated monomer–dimer equilibrium of YchF as a key event in the functional cycle of YchF. Upon oxidative stress, the oxidation of a conserved and surface-exposed cysteine residue promotes YchF dimerization, which is accompanied by inhibition of the ATPase activity. No dimers were observed in a YchF mutant lacking this cysteine. In vitro, the YchF dimer is dissociated by thioredoxin 1 (TrxA) and this stimulates the ATPase activity. The physiological significance of the YchF-thioredoxin 1 interaction was demonstrated by in vivo cross-linking, which validated this interaction in living cells. This approach also revealed that both the ATPase domain and the helical domain of YchF are in contact with TrxA. Innovation: YchF/Ola1 are the first redox-regulated members of the universally conserved GTPase family and are inactivated by oxidation of a conserved cysteine residue within the nucleotide-binding motif. Conclusion: Our data provide novel insights into the regulation of the so far ill-defined YchF/Ola1 family of proteins and stipulate their role as negative regulators of the oxidative stress response. Antioxid. Redox Signal. 24, 141–156. PMID:26160547

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.

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.

Comparative effects of aluminum and ouabain on synaptosomal choline uptake, acetylcholine release and (Na+/K+)ATPase.

PubMed

Silva, Virgília S; Nunes, M Alexandra; Cordeiro, J Miguel; Calejo, Ana I; Santos, Sofia; Neves, Paulo; Sykes, António; Morgado, Fernando; Dunant, Yves; Gonçalves, Paula P

2007-07-17

Closing the gap between adverse health effects of aluminum and its mechanisms of action still represents a huge challenge. Cholinergic dysfunction has been implicated in neuronal injury induced by aluminum. Previously reported data also indicate that in vivo and in vitro exposure to aluminum inhibits the mammalian (Na(+)/K(+))ATPase, an ubiquitous plasma membrane pump. This study was undertaken with the specific aim of determining whether in vitro exposure to AlCl(3) and ouabain, the foremost utilized selective inhibitor of (Na(+)/K(+))ATPase, induce similar functional modifications of cholinergic presynaptic nerve terminals, by comparing their effects on choline uptake, acetylcholine release and (Na(+)/K(+))ATPase activity, on subcellular fractions enriched in synaptic nerve endings isolated from rat brain, cuttlefish optic lobe and torpedo electric organ. Results obtained show that choline uptake by rat synaptosomes was inhibited by submillimolar AlCl(3), whereas the amount of choline taken up by synaptosomes isolated from cuttlefish and torpedo remained unchanged. Conversely, choline uptake was reduced by ouabain to a large extent in all synaptosomal preparations analyzed. In contrast to ouabain, which modified the K(+) depolarization evoked release of acetylcholine by rat, cuttlefish and torpedo synaptosomal fractions, AlCl(3) induced reduction of stimulated acetylcholine release was only observed when rat synaptosomes were challenged. Finally, it was observed that the aluminum effect on cuttlefish and torpedo synaptosomal (Na(+)/K(+))ATPase activity was slight when compared to its inhibitory action on mammalian (Na(+)/K(+))ATPase. In conclusion, inhibition of (Na(+)/K(+))ATPase by AlCl(3) and ouabain jeopardized the high-affinity (Na(+)-dependent, hemicholinium-3 sensitive) uptake of choline and the Ca(2+)-dependent, K(+) depolarization evoked release of acetylcholine by rat, cuttlefish and torpedo synaptosomal fractions. The effects of submillimolar AlCl(3

Combined effects of EGFR tyrosine kinase inhibitors and vATPase inhibitors in NSCLC cells

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

Jin, Hyeon-Ok; Hong, Sung-Eun; Kim, Chang Soon

2015-08-15

Despite excellent initial clinical responses of non-small cell lung cancer (NSCLC) patients to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs), many patients eventually develop resistance. According to a recent report, vacuolar H + ATPase (vATPase) is overexpressed and is associated with chemotherapy drug resistance in NSCLC. We investigated the combined effects of EGFR TKIs and vATPase inhibitors and their underlying mechanisms in the regulation of NSCLC cell death. We found that combined treatment with EGFR TKIs (erlotinib, gefitinib, or lapatinib) and vATPase inhibitors (bafilomycin A1 or concanamycin A) enhanced synergistic cell death compared to treatments with each drugmore » alone. Treatment with bafilomycin A1 or concanamycin A led to the induction of Bnip3 expression in an Hif-1α dependent manner. Knock-down of Hif-1α or Bnip3 by siRNA further enhanced cell death induced by bafilomycin A1, suggesting that Hif-1α/Bnip3 induction promoted resistance to cell death induced by the vATPase inhibitors. EGFR TKIs suppressed Hif-1α and Bnip3 expression induced by the vATPase inhibitors, suggesting that they enhanced the sensitivity of the cells to these inhibitors by decreasing Hif-1α/Bnip3 expression. Taken together, we conclude that EGFR TKIs enhance the sensitivity of NSCLC cells to vATPase inhibitors by decreasing Hif-1α/Bnip3 expression. We suggest that combined treatment with EGFR TKIs and vATPase inhibitors is potentially effective for the treatment of NSCLC. - Highlights: • Co-treatment with EGFR TKIs and vATPase inhibitors induces synergistic cell death • EGFR TKIs enhance cell sensitivity to vATPase inhibitors via Hif-1α downregulation • Co-treatment of these inhibitors is potentially effective for the treatment of NSCLC.« less

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

Changes in antioxidant status, protein concentration, acetylcholinesterase, (Na+,K+)-, and Mg2+ -ATPase activities in the brain of hyper- and hypothyroid adult rats.

PubMed

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

2005-06-01

It is a common knowledge that metabolic reactions increase in hyperthyroidism and decrease in hypothyroidism. The aim of this work was to investigate how the metabolic reactions could affect the total antioxidant status (TAS), protein concentration (PC) and the activities of acetylcholinesterase (AChE), (Na+,K+)-ATPase and Mg2+ -ATPase in the brain of hyper- and hypothyroid adult male rats. Hyperthyroidism was induced in rats by subcutaneous administration of thyroxine (25 microg/l00 g body weight) once daily for 14 days, while hypothyroidism was induced by oral administration of propylthiouracil (0.05%) for 21 days. TAS, PC, and enzyme activities were evaluated spectrophotometrically in the homogenated brain of each animal. TAS, PC, and Mg2+ -ATPase activity were found unaffected in hyperthyroidism, while AChE and Na+,K+ -ATPase activities were reduced by 25% (p < 0.01). In contrast, TAS, (Na+,K+)-ATPase and Mg2+-ATPase activities were found to be increased (approx. 23-30%, p < 0.001) in the hypothyroid brain, while AChE activity and PC were shown to be inhibited (approx. 23-30%, p < 0.001). These changes on brain enzyme activities may reflect the different metabolic effects of hyper- and hypothyroidism. Such changes of the enzyme activities may differentially modulate the brain intracellular Mg2+, neural excitability, as well as the uptake and release of biogenic amines.

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

The Na+/K+-ATPase and the amyloid-beta peptide aβ1-40 control the cellular distribution, abundance and activity of TRPC6 channels.

PubMed

Chauvet, Sylvain; Boonen, Marielle; Chevallet, Mireille; Jarvis, Louis; Abebe, Addis; Benharouga, Mohamed; Faller, Peter; Jadot, Michel; Bouron, Alexandre

2015-11-01

The Na(+)/K(+)-ATPase interacts with the non-selective cation channels TRPC6 but the functional consequences of this association are unknown. Experiments performed with HEK cells over-expressing TRPC6 channels showed that inhibiting the activity of the Na(+)/K(+)-ATPase with ouabain reduced the amount of TRPC6 proteins and depressed Ca(2+) entry through TRPC6. This effect, not mimicked by membrane depolarization with KCl, was abolished by sucrose and bafilomycin-A, and was partially sensitive to the intracellular Ca(2+) chelator BAPTA/AM. Biotinylation and subcellular fractionation experiments showed that ouabain caused a multifaceted redistribution of TRPC6 to the plasma membrane and to an endo/lysosomal compartment where they were degraded. The amyloid beta peptide Aβ(1-40), another inhibitor of the Na(+)/K(+)-ATPase, but not the shorter peptide Aβ1-16, reduced TRPC6 protein levels and depressed TRPC6-mediated responses. In cortical neurons from embryonic mice, ouabain, veratridine (an opener of voltage-gated Na(+) channel), and Aβ(1-40) reduced TRPC6-mediated Ca(2+) responses whereas Aβ(1-16) was ineffective. Furthermore, when Aβ(1-40) was co-added together with zinc acetate it could no longer control TRPC6 activity. Altogether, this work shows the existence of a functional coupling between the Na(+)/K(+)-ATPase and TRPC6. It also suggests that the abundance, distribution and activity of TRPC6 can be regulated by cardiotonic steroids like ouabain and the naturally occurring peptide Aβ(1-40) which underlines the pathophysiological significance of these processes. Copyright © 2015 Elsevier B.V. All rights reserved.

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

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.

Antenna Effect on the Organic Spacer-Modified Eu-Doped Layered Gadolinium Hydroxide for the Detection of Vanadate Ions over a Wide pH Range.

PubMed

Jeong, Heejin; Lee, Byung-Il; Byeon, Song-Ho

2016-05-04

The excitation of the adsorbed vanadate group led to the red emission arising from the efficient energy transfer to Eu-doped layered gadolinium hydroxide (LGdH:Eu). This light-harvesting antenna effect allowed LGdH:Eu to detect selectively a vanadate in aqueous solution at different pHs. Because vanadate exists in various forms by extensive oligomerization and protonation reactions in aqueous solution depending on pH, it is important to detect a vanadate regardless of its form over a wide pH range. In particular, spacer molecules with long alkyl chains greatly facilitated access of a vanadate antenna into the interlayer surface of LGdH:Eu. The concomitant increase in adsorption capacity of LGdH:Eu achieved a strong antenna effect of vanadate on the red emission from Eu(3+). When a suspension containing LGdH:Eu nanosheets (1.0 g/L) was used, the vanadate concentration down to 1 × 10(-5) M could even be visually monitored, and the detection limit based on the (5)D0 → (7)F2 emission intensity could reach 4.5 × 10(-8) M.

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

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.

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

Electrical Conductivity Mechanism in Unconventional Lead Vanadate Glasses

NASA Astrophysics Data System (ADS)

Abdel-Wahab, F.; Merazga, A.; Montaser, A. A.

2017-03-01

Lead vanadate glasses of the system (V2O5)_{1-x}(PbO)x with x = 0.4, 0.45, 0.5, 0.55, 0.6 have been prepared by the press-quenching technique. The dc (σ (0)) and ac (σ (ω )) electrical conductivities were measured in the temperature range from 150 to 420 K and the frequency range from 102 to 106 Hz. The electrical properties are shown to be sensitive to composition. The experimental results have been analyzed within the framework of different models. The dc conductivity is found to be proportional to Tp with the exponent p ranging from 8.2 to 9.8, suggesting that the transport is determined by a multi-phonon process of weak electron-lattice coupling. The ac conductivity is explained by the percolation path approximation (PPA). In this model, σ (ω ) is closely related to the σ (0) and fitting the experimental data produces a dielectric relaxation time τ in good agreement with the expected value in both magnitude and temperature dependence.

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.

Effect of an ADP analog on isometric force and ATPase activity of active muscle fibers.

PubMed

Karatzaferi, Christina; Myburgh, Kathryn H; Chinn, Marc K; Franks-Skiba, Kathleen; Cooke, Roger

2003-04-01

The role played by ADP in modulating cross-bridge function has been difficult to study, because it is hard to buffer ADP concentration in skinned muscle preparations. To solve this, we used an analog of ADP, spin-labeled ADP (SL-ADP). SL-ADP binds tightly to myosin but is a very poor substrate for creatine kinase or pyruvate kinase. Thus ATP can be regenerated, allowing well-defined concentrations of both ATP and SL-ADP. We measured isometric ATPase rate and isometric tension as a function of both [SL-ADP], 0.1-2 mM, and [ATP], 0.05-0.5 mM, in skinned rabbit psoas muscle, simulating fresh or fatigued states. Saturating levels of SL-ADP increased isometric tension (by P'), the absolute value of P' being nearly constant, approximately 0.04 N/mm(2), in variable ATP levels, pH 7. Tension decreased (50-60%) at pH 6, but upon addition of SL-ADP, P' was still approximately 0.04 N/mm(2). The ATPase was inhibited competitively by SL-ADP with an inhibition constant, K(i), of approximately 240 and 280 microM at pH 7 and 6, respectively. Isometric force and ATPase activity could both be fit by a simple model of cross-bridge kinetics.

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

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.

New possibilities of neodymium-doped vanadate crystals as active media for diode-pumped lasers

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

Vlasov, V I; Garnov, S V; Zavartsev, Yu D

The spectral and lasing parameters of Nd:GdVO{sub 4}, Nd:YVO{sub 4}, and Nd:Gd{sub 0.7}Y{sub 0.3}VO{sub 4} vanadate crystals cut along the c axis are studied. Lasing is obtained for the first time in a nonselective resonator at the {sup 4}F{sub 3/2}-{sup 4}I{sub 11/2} transition at 1065.5 nm in a Nd:GdVO{sub 4} crystal. Tuning is realised in the range from 1062.3 to 1066.1 nm and two-frequency lasing is obtained. (special issue devoted to the 25th anniversary of the a.m. prokhorov general physics institute)

Comparison of the vanadate oxidase method with the diazo method for serum bilirubin determination in dog, monkey, and rat.

PubMed

Ameri, Mehrdad; Schnaars, Henry; Sibley, John; Honor, David

2011-01-01

The most widely used method for bilirubin concentration determination is the diazo method, which measures the color of azobilirubin. The vanadate oxidase method is based on oxidation of bilirubin to biliverdin by vanadate. The objective of this study was to compare total and direct bilirubin concentration ([Bt] and [Bd], respectively) determined by the diazo and vanadate oxidase methods in pooled serum samples from dogs, monkeys, and rats spiked with panels of different concentrations of bilirubin standards. Pooled serum samples from 40 dogs, 40 monkeys, and 60 rats were spiked with either ditaurine conjugates of bilirubin or a standard reference material. The results obtained from both assays were compared using Deming regression analysis. The intra- and interassay precision, expressed as a percentage of the coefficient of variation (%CV), was determined for [Bt] and [Bd], and the mean percentage of recovery was calculated. The vanadate oxidase method displayed an excellent correlation (r  =  0.99-1.00) with the diazo method. Using Deming regression, there were minimal negative or positive constant and proportional biases for [Bt] and [Bd]. The precision studies revealed that the vanadate oxidase method has comparable between-run and within-run CVs to those of the diazo method. The recovery study demonstrated that the diazo method more closely approximates the expected values of [Bt]. In conclusion, the vanadate oxidase method is a simple and rapid method that can be employed as an alternative to the diazo method when interfering substances are present in the serum samples of dog, monkey, and rat.

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.

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

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.

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

Relationship of the Membrane ATPase from Halobacterium saccharovorum to Vacuolar ATPases

NASA Technical Reports Server (NTRS)

Stan-Lotter, Helga; Bowman, Emma J.; Hochstein, Lawrence I.

1991-01-01

Polyclonal antiserum against subunit A (67 kDa) of the vacuolar ATPase from Neurospora crassa reacted with subunit I (87 kDa) from a membrane ATPase of the extremely halophilic archaebacterium Halobacterium saccharovorum. The halobacterial ATPase was inhibited by nitrate and N-ethylmaleimide; the extent of the latter inhibition was diminished in the presence of adenosine di- or triphosphates. 4-Chloro-7-nitrobenzofurazan in- hibited the hatobacterial ATPase also in a nucleotide- protectable manner; the bulk of inhibitor was associated with subunit II (60 kDa). The data suggested that this halobacterial ATPase may have conserved structural features from both the vacuotar and the F-type ATPases.

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

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

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

Control of Gastric H,K-ATPase Activity by Cations, Voltage and Intracellular pH Analyzed by Voltage Clamp Fluorometry in Xenopus Oocytes

PubMed Central

Dürr, Katharina L.; Tavraz, Neslihan N.; Friedrich, Thomas

2012-01-01

Whereas electrogenic partial reactions of the Na,K-ATPase have been studied in depth, much less is known about the influence of the membrane potential on the electroneutrally operating gastric H,K-ATPase. In this work, we investigated site-specifically fluorescence-labeled H,K-ATPase expressed in Xenopus oocytes by voltage clamp fluorometry to monitor the voltage-dependent distribution between E1P and E2P states and measured Rb+ uptake under various ionic and pH conditions. The steady-state E1P/E2P distribution, as indicated by the voltage-dependent fluorescence amplitudes and the Rb+ uptake activity were highly sensitive to small changes in intracellular pH, whereas even large extracellular pH changes affected neither the E1P/E2P distribution nor transport activity. Notably, intracellular acidification by approximately 0.5 pH units shifted V0.5, the voltage, at which the E1P/E2P ratio is 50∶50, by −100 mV. This was paralleled by an approximately two-fold acceleration of the forward rate constant of the E1P→E2P transition and a similar increase in the rate of steady-state cation transport. The temperature dependence of Rb+ uptake yielded an activation energy of ∼90 kJ/mol, suggesting that ion transport is rate-limited by a major conformational transition. The pronounced sensitivity towards intracellular pH suggests that proton uptake from the cytoplasmic side controls the level of phosphoenzyme entering the E1P→E2P conformational transition, thus limiting ion transport of the gastric H,K-ATPase. These findings highlight the significance of cellular mechanisms contributing to increased proton availability in the cytoplasm of gastric parietal cells. Furthermore, we show that extracellular Na+ profoundly alters the voltage-dependent E1P/E2P distribution indicating that Na+ ions can act as surrogates for protons regarding the E2P→E1P transition. The complexity of the intra- and extracellular cation effects can be rationalized by a kinetic model suggesting

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

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.

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.

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.

Deficient erythrocyte NaK-ATPase activity in different affective states in bipolar affective disorder and normalization by lithium therapy.

PubMed

Hokin-Neaverson, M; Jefferson, J W

1989-01-01

This report expands on previously presented evidence for a trait-dependent deficiency of erythrocyte sodium pump activity in bipolar affective disorder. Several parameters of erythrocyte NaK-ATPase activity in different affective states and the effects of lithium therapy were examined. In lithium-free bipolar affective disorder patients, the mean percent differences from the individual sex- and age-matched controls for erythrocyte sodium pump activity were: manic + hypomanic group, -21.5%, n = 16, p less than 0.02; depressed group, -12.4%, n = 14, p less than 0.02; euthymic group, -6.9%, n = 18, p less than 0.10. Ouabain-sensitive potassium ion uptake was less than the controls in the manic group (-25.4%, n = 3, p less than 0.02), and in the combined affectively ill group, manic + depressed (-23.4%, n = 7, p less than 0.02). Cell ouabain binding was less than the controls in the manic group (-19.0%, n = 6, p less than 0.05). NaK-ATPase activity in washed erythrocyte membranes (ghosts) was significantly lower than the controls in the manic group (-14.5%, n = 4, p less than 0.02), and the mean value for the whole group (manic + depressed + euthymic) was lower than the controls (-11.8%, n = 18, p less than 0.05). Values for ouabain binding in ghosts from the bipolar subjects were not significantly different from the matched controls.(ABSTRACT TRUNCATED AT 250 WORDS)

The Influence of Fatty Acid Methyl Esters (FAMEs) in the Biochemistry and the Na(+)/K(+)-ATPase Activity of Culex quinquefasciatus Larvae.

PubMed

Silva, Lilian N D; Ribeiro-Neto, José A; Valadares, Jéssica M M; Costa, Mariana M; Lima, Luciana A R S; Grillo, Luciano A M; Cortes, Vanessa F; Santos, Herica L; Alves, Stênio N; Barbosa, Leandro A

2016-08-01

Culex quinquefasciatus is the main vector of lymphatic filariasis and combating this insect is of great importance to public health. There are reports of insects that are resistant to the products currently used to control this vector, and therefore, the search for new products has increased. In the present study, we have evaluated the effects of fatty acid methyl esters (FAMEs) that showed larvicidal activity against C. quinquefasciatus, on glucose, total protein, and triacylglycerol contents and Na(+)/K(+)-ATPase activity in mosquito larvae. The exposure of the fourth instar larvae to the compounds caused a decrease in the total protein content and an increase in the activity of the Na(+)/K(+)-ATPase. Furthermore, the direct effect of FAMEs on cell membrane was assessed on purified pig kidney Na(+)/K(+)-ATPase membranes, erythrocyte ghost membranes, and larvae membrane preparation. No modifications on total phospholipids and cholesterol content were found after FAMEs 20 min treatment on larvae membrane preparation, but only 360 µg/mL FAME 2 was able to decrease total phospholipid of erythrocyte ghost membrane. Moreover, only 60 and 360 µg/mL FAME 3 caused an activation of purified Na(+)/K(+)-ATPase, that was an opposite effect of FAMEs treatment in larvae membrane preparation, and caused an inhibition of the pump activity. These data together suggest that maybe FAMEs can modulate the Na(+)/K(+)-ATPase on intact larvae for such mechanisms and not for a direct effect, one time that the direct effect of FAMEs in membrane preparation decreased the activity of Na(+)/K(+)-ATPase. The biochemical changes caused by the compounds were significant and may negatively influence the development and survival of C. quinquefasciatus larvae.

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.

Salt stress reduces kernel number of corn by inhibiting plasma membrane H+-ATPase activity.

PubMed

Jung, Stephan; Hütsch, Birgit W; Schubert, Sven

2017-04-01

Salt stress affects yield formation of corn (Zea mays L.) at various physiological levels resulting in an overall grain yield decrease. In this study we investigated how salt stress affects kernel development of two corn cultivars (cvs. Pioneer 3906 and Fabregas) at and shortly after pollination. In an earlier study, we found an accumulation of hexoses in the kernel tissue. Therefore, it was hypothesized that hexose uptake into developing endosperm and embryo might be inhibited. Hexoses are transported into the developing endosperm by carriers localized in the plasma membrane (PM). The transport is driven by the pH gradient which is built up by the PM H + -ATPase. It was investigated whether the PM H + -ATPase activity in developing corn kernels was inhibited by salt stress, which would cause a lower pH gradient resulting in impaired hexose import and finally in kernel abortion. Corn grown under control and salt stress conditions was harvested 0 and 2 days after pollination (DAP). Under salt stress sucrose and hexose concentrations in kernel tissue were higher 0 and 2 DAP. Kernel PM H + -ATPase activity was not affected at 0 DAP, but it was reduced at 2 DAP. This is in agreement with the finding, that kernel growth and thus kernel setting was not affected in the salt stress treatment at pollination, but it was reduced 2 days later. It is concluded that inhibition of PM H + -ATPase under salt stress impaired the energization of hexose transporters into the cells, resulting in lower kernel growth and finally in kernel abortion. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

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.

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

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.

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

Palmitate-Induced Vacuolar-Type H+-ATPase Inhibition Feeds Forward Into Insulin Resistance and Contractile Dysfunction.

PubMed

Liu, Yilin; Steinbusch, Laura K M; Nabben, Miranda; Kapsokalyvas, Dimitris; van Zandvoort, Marc; Schönleitner, Patrick; Antoons, Gudrun; Simons, Peter J; Coumans, Will A; Geomini, Amber; Chanda, Dipanjan; Glatz, Jan F C; Neumann, Dietbert; Luiken, Joost J F P

2017-06-01

Dietary fat overconsumption leads to myocardial lipid accumulation through mechanisms that are incompletely resolved. Previously, we identified increased translocation of the fatty acid transporter CD36 from its endosomal storage compartment to the sarcolemma as the primary mechanism of excessive myocellular lipid import. Here, we show that increased CD36 translocation is caused by alkalinization of endosomes resulting from inhibition of proton pumping activity of vacuolar-type H + -ATPase (v-ATPase). Endosomal alkalinization was observed in hearts from rats fed a lard-based high-fat diet and in rodent and human cardiomyocytes upon palmitate overexposure, and appeared as an early lipid-induced event preceding the onset of insulin resistance. Either genetic or pharmacological inhibition of v-ATPase in cardiomyocytes exposed to low palmitate concentrations reduced insulin sensitivity and cardiomyocyte contractility, which was rescued by CD36 silencing. The mechanism of palmitate-induced v-ATPase inhibition involved its dissociation into two parts: the cytosolic V 1 and the integral membrane V 0 subcomplex. Interestingly, oleate also inhibits v-ATPase function, yielding triacylglycerol accumulation but not insulin resistance. In conclusion, lipid oversupply increases CD36-mediated lipid uptake that directly impairs v-ATPase function. This feeds forward to enhanced CD36 translocation and further increased lipid uptake. In the case of palmitate, its accelerated uptake ultimately precipitates into cardiac insulin resistance and contractile dysfunction. © 2017 by the American Diabetes Association.

The insulin-like effect of vanadate on lipolysis in rat adipocytes is not accompanied by an insulin-like effect on tyrosine phosphorylation.

PubMed

Mooney, R A; Bordwell, K L; Luhowskyj, S; Casnellie, J E

1989-01-01

Tyrosine phosphorylation of the insulin receptor and other intracellular proteins in rat adipocytes was examined using an immunoblot technique with antiphosphotyrosine antibody. Insulin at 10(-7) M increased the tyrosine phosphorylation of the 95K subunit of the insulin receptor (15-fold) and proteins of 180K (7-fold) and 60K (23-fold). Increases in insulin-dependent phosphorylation of the three proteins were detectable at 10(-10) M insulin and attained steady state within 30 sec of insulin (10(-7) M) addition. Small effects of insulin (less than 30% increases) were observed on proteins of 120K and 53K. In contrast to insulin, the effects of vanadate on tyrosine phosphorylation were small and nonspecific. Vanadate increased tyrosine phosphorylation of the 95K insulin receptor beta-subunit and the 120K and 60K proteins similarly, with increases of 1.5- to 3-fold at 1 mM and 2-fold or less at 200 and 50 microM. Vanadate-dependent tyrosine phosphorylation of the 180K protein increased to a maximum of only 30% at 200 microM. Tyrosine phosphorylation of the 53K protein was somewhat larger, approaching 4-fold at 1 mM vanadate. The concentration of insulin and vanadate that inhibited isoproterenol-dependent lipolysis were not comparable to those that increased tyrosine phosphorylation. Vanadate at 1 mM was more potent as an antilipolytic agent than 10(-9) M insulin (93% vs. 81%), yet increased tyrosine phosphorylation of the 95K insulin receptor beta-subunit only as effectively as 10(-10) M insulin (which inhibited lipolysis only 42%). The dissimilar responses were even more pronounced when antilipolysis was compared to tyrosine phosphorylation of the 180K and 60K proteins. For example, insulin at 10(-9) M increased tyrosine phosphorylation of the 180K protein 2.9-fold, while 1 mM vanadate had a negligible effect (10% increase). Thus, vanadate exerts an insulin-like effect on lipolysis, yet its effects on tyrosine phosphorylation differ from those of insulin.

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

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.

Characterization of the temperature-sensitive reaction of F1-ATPase by using single-molecule manipulation

PubMed Central

Watanabe, Rikiya; Noji, Hiroyuki

2014-01-01

F1-ATPase (F1) is a rotary motor protein that couples ATP hydrolysis to mechanical rotation with high efficiency. In our recent study, we observed a highly temperature-sensitive (TS) step in the reaction catalyzed by a thermophilic F1 that was characterized by a rate constant remarkably sensitive to temperature and had a Q10 factor of 6–19. Since reactions with high Q10 values are considered to involve large conformational changes, we speculated that the TS reaction plays a key role in the rotation of F1. To clarify the role of the TS reaction, in this study, we conducted a stall and release experiment using magnetic tweezers, and assessed the torque generated during the TS reaction. The results indicate that the TS reaction generates the same amount of rotational torque as does ATP binding, but more than that generated during ATP hydrolysis. Thus, we confirmed that the TS reaction contributes significantly to the rotation of F1. PMID:24825532

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.

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.

Optical study of active ion transport in lipid vesicles containing reconstituted Na,K-ATPase.

PubMed

Apell, H J; Marcus, M M; Anner, B M; Oetliker, H; Läuger, P

1985-01-01

A fluorescence method is described for the measurement of ATP-driven ion fluxes in lipid vesicles containing purified Na,K-ATPase. The membrane voltage of enzyme containing vesicles was measured by using a voltage-sensitive indocyanine dye. By addition of valinomycin the vesicle membrane is made selectively permeable to K+ so that the membrane voltage approaches the Nernst potential for K+. With constant external K+ concentration, the time course of internal K+ concentration can be continuously measured as change of the fluorescence signal after activation of the pump. The optical method has a higher time resolution than tracer-flux experiments and allows an accurate determination of initial flux rates. From the temperature dependence of active K+ transport its activation energy was determined to be 115 kJ/mol. ATP-stimulated electrogenic pumping can be measured as fast fluorescence change when the membrane conductance is low (i.e., at low or zero valinomycin concentration). In accordance with expectation, the amplitude of the fast signal change increases with decreasing passive ion permeability of the vesicle membrane. The resolution of the charge movement is so high that a few pump turnovers can be easily detected.

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.

Simultaneous speciation analysis of chromate, molybdate, tungstate and vanadate in welding fume alkaline extracts by HPLC-ICP-MS.

PubMed

Ščančar, Janez; Berlinger, Balázs; Thomassen, Yngvar; Milačič, Radmila

2015-09-01

A novel analytical procedure was developed for the simultaneous speciation analysis of chromate, molybdate, tungstate and vanadate by anion-exchange high performance liquid chromatography hyphenated to inductively coupled plasma mass spectrometry (HPLC-ICP-MS). Linear gradient elution from 100% water to 100% 0.7 M NaCl was applied for chromatographic separation of metal species. In standard aqueous solution at neutral pH molybdate, tungstate and vanadate exist in several aqueous species, while chromate is present as a single CrO4(2-) species. Consequently, only chromate can be separated from this solution in a sharp chromatographic peak. For obtaining sharp chromatographic peaks for molybdate, tungstate and vanadate, the pH of aqueous standard solutions was raised to 12. At highly alkaline conditions single CrO4(2-), MoO4(2-) and WO4(2-) are present and were eluted in sharp chromatographic peaks, while VO4(3-) species, which predominates at pH 12 was eluted in slightly broaden peak. In a mixture of aqueous standard solutions (pH 12) chromate, molybdate, tungstate and vanadate were eluted at retention times from 380 to 420 s, 320 to 370 s, 300 to 350 s and 240 to 360 s, respectively. Eluted species were simultaneously detected on-line by ICP-MS recording m/z 52, 95, 182 and 51. The developed procedure was successfully applied to the analysis of leachable concentrations of chromate, molybdate, tungstate and vanadate in alkaline extracts (2% NaOH+3% Na2CO3) of manual metal arc (MMA) welding fumes loaded on filters. Good repeatability and reproducibility of measurement (RSD±3.0%) for the investigated species were obtained in both aqueous standard solutions (pH 12) and in alkaline extracts of welding fumes. Low limits of detection (LODs) were found for chromate (0.02 ng Cr mL(-1)), molybdate (0.1 ng Mo mL(-1)), tungstate (0.1 ng W mL(-1)) and vanadate (0.2 ng V mL(-1)). The accuracy of analytical procedure for the determination of chromate was checked by analysis of

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

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

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

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.

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.

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.

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.

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.

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

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.

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

Subcellular localization of calcium and Ca-ATPase activity during nuclear maturation in Bufo arenarum oocytes.

PubMed

Ramos, Inés; Cisint, Susana B; Crespo, Claudia A; Medina, Marcela F; Fernández, Silvia N

2009-08-01

The localization of calcium and Ca-ATPase activity in Bufo arenarum oocytes was investigated by ultracytochemical techniques during progesterone-induced nuclear maturation, under in vitro conditions. No Ca2+ deposits were detected in either control oocytes or progesterone-treated ones for 1-2 h. At the time when nuclear migration started, electron dense deposits of Ca2+ were visible in vesicles, endoplasmic reticulum cisternae and in the space between the annulate lamellae membranes. Furthermore, Ca-ATPase activity was also detected in these membrane structures. As maturation progressed, the cation deposits were observed in the cytomembrane structures, which underwent an important reorganization and redistribution. Thus, they moved from the subcortex and became located predominantly in the oocyte cortex area when nuclear maturation ended. Ca2+ stores were observed in vesicles surrounding or between the cortical granules, which are aligned close to the plasma membrane. The positive Ca-ATPase reaction in these membrane structures could indicate that the calcium deposit is an ATP-dependent process. Our results suggest that during oocyte maturation calcium would be stored in membrane structures where it remains available for release at the time of fertilization. Data obtained under our experimental conditions indicate that calcium from the extracellular medium would be important for the oocyte maturation process.

Altered erythrocyte sodium-lithium counter-transport and Na+/K(+)-ATPase activity in cystic fibrosis.

PubMed

Luczay, A; Vásárhelyi, B; Dobos, M; Holics, K; Ujhelyi, R; Tulassay, T

1997-03-01

Patients with cystic fibrosis (CF) exhibit normal concentrations of sodium and chloride in spite of the disturbance of Cl- and Na+ transport in epithelial cells. To characterize compensatory mechanisms in the regulation of sodium homeostasis, erythrocytes of 13 CF patients were analysed for sodium-lithium counter-transport (SLC), Na+/K(+)-ATPase activity and intracellular sodium content. Values were compared to those of healthy controls. Patients with CF had normal serum sodium and chloride concentrations and renal excretions of these ions were within the physiological range. Intracellular sodium concentration was similar in the CF and the control group (6.8 +/- 2.2 vs 5.7 +/- 1.0 mmol/l RBCs). Red blood cells' SLC and Na+/ K(+)-ATPase activity were elevated in CF patients (381 +/- 106 mumol/h/l RBCs vs 281 +/- 64; p < 0.01) and (445 +/- 129 mumol ATP mg prot/h vs 322 +/- 84, p < 0.01). Our study demonstrates that transmembrane cation transport systems are highly activated in CF. The increased sodium transport may be part of a compensatory mechanism of sodium homeostasis in children with CF.

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

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.

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.

Honeycomb-like S = 5/2 Spin–Lattices in Manganese(II) Vanadates

DOE PAGES

Sanjeewa, Liurukara. D.; McGuire, Michael A.; McMillen, Colin D.; ...

2016-08-26

We synthesized new complex manganese vanadate materials as high-quality single crystals in multi-millimeter lengths using a high-temperature, high-pressure hydrothermal method. We grew one compound, Mn 5(VO 4) 2(OH) 4, from Mn 2O 3 and V 2O 5 in 3 M CsOH at 580 °C and 1.5 kbar. Changing the mineralizer to 1 M CsOH/3MCsCl leads to the formation of another product, Mn 6O(VO 4) 2(OH). Both compounds were structurally characterized by single-crystal X-ray diffraction (Mn 5(VO 4) 2(OH) 4: C2/m, Z = 2, a = 9.6568(9) Å, b = 9.5627(9) Å, c = 5.4139(6) Å, β = 98.529(8)°; Mn 6O(VOmore » 4) 2(OH): P21/m, Z = 2, a = 8.9363(12) Å, b = 6.4678(8) Å, c = 10.4478(13) Å, β = 99.798(3)°), revealing interesting low-dimensional transition-metal features. Mn 5(VO 4) 2(OH) 4 possesses complex honeycomb-type Mn–O layers, built from edge-sharing [MnO 6] octahedra in the bc plane, with bridging vanadate groups connecting these layers along the a-axis. Mn 6O(VO 4) 2(OH) presents a more complicated structure with both octahedral [MnO 6] and trigonal bipyramidal [MnO 5] units. A different pattern of planar honeycomb sheets are formed by edge-shared [MnO6] octahedra, and these sublattices are connected through edge-shared dimers of [MnO 5] trigonal bipyramids to form corrugated sheets. Vanadate groups again condense the sheets into a three-dimensional framework. Infrared and Raman spectroscopies indicated the presence of OH groups and displayed characteristic Raman scattering due to vanadate groups. Furthermore, temperature-dependent magnetic studies indicated Curie–Weiss behavior above 100 K with significant anti-ferromagnetic coupling for both compounds, with further complex magnetic behavior at lower temperatures. The data indicate canted anti-ferromagnetic order below 57 K in Mn 5(VO 4) 2(OH) 4 and below 45 K in Mn 6O(VO 4) 2(OH). Members of another class of compounds, K 2M 3(VO 4) 2(OH) 2 (M = Mn, Co), also containing a honeycomb-type sublattice, were also synthesized to

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.

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.

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.

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.

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

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

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

Myosin Activator Omecamtiv Mecarbil Increases Myocardial Oxygen Consumption and Impairs Cardiac Efficiency Mediated by Resting Myosin ATPase Activity.

PubMed

Bakkehaug, Jens Petter; Kildal, Anders Benjamin; Engstad, Erik Torgersen; Boardman, Neoma; Næsheim, Torvind; Rønning, Leif; Aasum, Ellen; Larsen, Terje Steinar; Myrmel, Truls; How, Ole-Jakob

2015-07-01

Omecamtiv mecarbil (OM) is a novel inotropic agent that prolongs systolic ejection time and increases ejection fraction through myosin ATPase activation. We hypothesized that a potentially favorable energetic effect of unloading the left ventricle, and thus reduction of wall stress, could be counteracted by the prolonged contraction time and ATP-consumption. Postischemic left ventricular dysfunction was created by repetitive left coronary occlusions in 7 pigs (7 healthy pigs also included). In both groups, systolic ejection time and ejection fraction increased after OM (0.75 mg/kg loading for 10 minutes, followed by 0.5 mg/kg/min continuous infusion). Cardiac efficiency was assessed by relating myocardial oxygen consumption to the cardiac work indices, stroke work, and pressure-volume area. To circumvent potential neurohumoral reflexes, cardiac efficiency was additionally assessed in ex vivo mouse hearts and isolated myocardial mitochondria. OM impaired cardiac efficiency; there was a 31% and 23% increase in unloaded myocardial oxygen consumption in healthy and postischemic pigs, respectively. Also, the oxygen cost of the contractile function was increased by 63% and 46% in healthy and postischemic pigs, respectively. The increased unloaded myocardial oxygen consumption was confirmed in OM-treated mouse hearts and explained by an increased basal metabolic rate. Adding the myosin ATPase inhibitor, 2,3-butanedione monoxide abolished all surplus myocardial oxygen consumption in the OM-treated hearts. Omecamtiv mecarbil, in a clinically relevant model, led to a significant myocardial oxygen wastage related to both the contractile and noncontractile function. This was mediated by that OM induces a continuous activation in resting myosin ATPase. © 2015 American Heart Association, Inc.

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.

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.

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.

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

Neuronal-specific endoplasmic reticulum Mg(2+)/Ca(2+) ATPase Ca(2+) sequestration in mixed primary hippocampal culture homogenates.

PubMed

Parsons, J Travis; Sun, David A; DeLorenzo, Robert J; Churn, Severn B

2004-07-01

Endoplasmic reticulum Mg(2+)/Ca(2+) ATPase Ca(2+) sequestration is crucial for maintenance of neuronal Ca(2+) homeostasis. The use of cell culture in conjunction with modern Ca(2+) imaging techniques has been invaluable in elucidating these mechanisms. While imaging protocols evaluate endoplasmic reticulum Ca(2+) loads, measurement of Mg(2+)/Ca(2+) ATPase activity is indirect, comparing cytosolic Ca(2+) levels in the presence or absence of the Mg(2+)/Ca(2+) ATPase inhibitor thapsigargin. Direct measurement of Mg(2+)/Ca(2+) ATPase by isolation of microsomes is impossible due to the minuscule amounts of protein yielded from cultures used for imaging. In the current study, endoplasmic reticulum Mg(2+)/Ca(2+) ATPase Ca(2+) sequestration was measured in mixed homogenates of neurons and glia from primary hippocampal cultures. It was demonstrated that Ca(2+) uptake was mediated by the endoplasmic reticulum Mg(2+)/Ca(2+) ATPase due to its dependence on ATP and Mg(2+), enhancement by oxalate, and inhibition by thapsigargin. It was also shown that neuronal Ca(2+) uptake, mediated by the type 2 sarco(endo)plasmic reticulum Ca(2+) ATPase isoform, could be distinguished from glial Ca(2+) uptake in homogenates composed of neurons and glia. Finally, it was revealed that Ca(2+) uptake was sensitive to incubation on ice, extremely labile in the absence of protease inhibitors, and significantly more stable under storage conditions at -80 degrees C.

Two halide-containing cesium manganese vanadates: synthesis, characterization, and magnetic properties

DOE PAGES

Smith Pellizzeri, Tiffany M.; McGuire, Michael A.; McMillen, Colin D.; ...

2018-01-24

In this study, two new halide-containing cesium manganese vanadates have been synthesized by a high-temperature (580 °C) hydrothermal synthetic method from aqueous brine solutions. One compound, Cs 3Mn(VO 3) 4Cl, (1) was prepared using a mixed cesium hydroxide/chloride mineralizer, and crystallizes in the polar noncentrosymmetric space group Cmm2, with a = 16.7820(8) Å, b = 8.4765(4) Å, c = 5.7867(3) Å. This structure is built from sinusoidal zig-zag (VO 3) n chains that run along the b-axis and are coordinated to Mn 2+ containing (MnO 4Cl) square-pyramidal units that are linked together to form layers. The cesium cations reside betweenmore » the layers, but also coordinate to the chloride ion, forming a cesium chloride chain that also propagates along the b-axis. The other compound, Cs 2Mn(VO 3) 3F, (2) crystallizes in space group Pbca with a = 7.4286(2) Å, b = 15.0175(5) Å, c = 19.6957(7) Å, and was prepared using a cesium fluoride mineralizer. The structure is comprised of corner sharing octahedral Mn 2+ chains, with trans fluoride ligands acting as bridging units, whose ends are capped by (VO 3) n vanadate chains to form slabs. The cesium atoms reside between the manganese vanadate layers, and also play an integral part in the structure, forming a cesium fluoride chain that runs along the b-axis. Both compounds were characterized by single-crystal X-ray diffraction, powder X-ray diffraction, and single-crystal Raman spectroscopy. Additionally, the magnetic properties of 2 were investigated. Lastly, above 50 K, it displays behavior typical of a low dimensional system with antiferromagnetic interactions, as to be expected for linear chains of manganese(II) within the crystal structure.« less

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

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.

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.

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

A proton pump ATPase with testis-specific E1-subunit isoform required for acrosome acidification.

PubMed

Sun-Wada, Ge-Hong; Imai-Senga, Yoko; Yamamoto, Akitsugu; Murata, Yoshiko; Hirata, Tomoyuki; Wada, Yoh; Futai, Masamitsu

2002-05-17

The vacuolar-type H(+)-ATPases (V-ATPases) are a family of multimeric proton pumps involved in a wide variety of physiological processes. We have identified two novel mouse genes, Atp6e1 and Atp6e2, encoding testis-specific (E1) and ubiquitous (E2) V-ATPase subunit E isoforms, respectively. The E1 transcript appears about 3 weeks after birth, corresponding to the start of meiosis, and is expressed specifically in round spermatids in seminiferous tubules. Immunohistochemistry with isoform-specific antibodies revealed that the V-ATPase with E1 and a2 isoforms is located specifically in developing acrosomes of spermatids and acrosomes in mature sperm. In contrast, the E2 isoform was expressed in all tissues examined and present in the perinuclear compartments of spermatocytes. The E1 isoform exhibits 70% identity with the E2, and both isoforms functionally complemented a null mutation of the yeast counterpart VMA4, indicating that they are bona fide V-ATPase subunits. The chimeric enzymes showed slightly lower K(m)(ATP) than yeast V-ATPase. Consistent with the temperature-sensitive growth of Deltavma4-expressing E1 isoform, vacuolar membrane vesicles exhibited temperature-sensitive coupling between ATP hydrolysis and proton transport. These results suggest that E1 isoform is essential for energy coupling involved in acidification of acrosome.

Global analysis of gene expression in mineralizing fish vertebra-derived cell lines: new insights into anti-mineralogenic effect of vanadate

PubMed Central

2011-01-01

Background Fish has been deemed suitable to study the complex mechanisms of vertebrate skeletogenesis and gilthead seabream (Sparus aurata), a marine teleost with acellular bone, has been successfully used in recent years to study the function and regulation of bone and cartilage related genes during development and in adult animals. Tools recently developed for gilthead seabream, e.g. mineralogenic cell lines and a 4 × 44K Agilent oligo-array, were used to identify molecular determinants of in vitro mineralization and genes involved in anti-mineralogenic action of vanadate. Results Global analysis of gene expression identified 4,223 and 4,147 genes differentially expressed (fold change - FC > 1.5) during in vitro mineralization of VSa13 (pre-chondrocyte) and VSa16 (pre-osteoblast) cells, respectively. Comparative analysis indicated that nearly 45% of these genes are common to both cell lines and gene ontology (GO) classification is also similar for both cell types. Up-regulated genes (FC > 10) were mainly associated with transport, matrix/membrane, metabolism and signaling, while down-regulated genes were mainly associated with metabolism, calcium binding, transport and signaling. Analysis of gene expression in proliferative and mineralizing cells exposed to vanadate revealed 1,779 and 1,136 differentially expressed genes, respectively. Of these genes, 67 exhibited reverse patterns of expression upon vanadate treatment during proliferation or mineralization. Conclusions Comparative analysis of expression data from fish and data available in the literature for mammalian cell systems (bone-derived cells undergoing differentiation) indicate that the same type of genes, and in some cases the same orthologs, are involved in mechanisms of in vitro mineralization, suggesting their conservation throughout vertebrate evolution and across cell types. Array technology also allowed identification of genes differentially expressed upon exposure of fish cell lines to vanadate

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).

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.

Effect of Bi2O3 on structural, optical, and other physical properties of semiconducting zinc vanadate glasses

NASA Astrophysics Data System (ADS)

Punia, R.; Kundu, R. S.; Hooda, J.; Dhankhar, S.; Dahiya, Sajjan; Kishore, N.

2011-08-01

Zinc bismuth vanadate glasses with compositions 50V2O5-xBi2O3-(50-x) ZnO have been prepared using a conventional melt-quenching method and the solubility limit of Bi2O3 in zinc vanadate glass system has been investigated using x-ray diffraction. Density has been measured using Archimedes' principle; molar volume (Vm) and crystalline volumes (Vc) have also been estimated. With an increase in Bi2O3 content, there is an increase in density and molar volume of the glass samples. The glass transition temperature (Tg) and Hurby coefficient (Kgl) have been determined using differential scanning calorimetry (DSC) and are observed to increase with increase in Bi2O3 content (i.e., x), up to x = 15, thereby indicating the structural modifications and increased thermal stability of zinc vanadate glasses on addition of Bi2O3. FTIR spectra have been recorded and the analysis of FTIR shows that the structure depends upon the Bi2O3 content in the glass compositions. On addition of Bi2O3 into the zinc vanadate system, the structure of V2O5 changes from VO4 tetrahedral to VO5 trigonal bi-pyramid configuration. The optical parameters have been calculated by using spectroscopic ellipsometry for bulk oxide glasses (perhaps used first time for bulk glasses) and optical bandgap energy is found to increase with increase in Bi2O3 content.

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.

Physical and electrochemical properties of alkaline earth doped, rare earth vanadates

NASA Astrophysics Data System (ADS)

Adijanto, Lawrence; Balaji Padmanabhan, Venu; Holmes, Kevin J.; Gorte, Raymond J.; Vohs, John M.

2012-06-01

The effect of partial substitution of alkaline earth (AE) ions, Sr2+ and Ca2+, for the rare earth (RE) ions, La3+, Ce3+, Pr3+, and Sm3+, on the physical properties of REVO4 compounds were investigated. The use of the Pechini method to synthesize the vanadates allowed for high levels of AE substitution to be obtained. Coulometric titration was used to measure redox isotherms for these materials and showed that the addition of the AE ions increased both reducibility and electronic conductivity under typical solid oxide fuel cell (SOFC) anode conditions, through the formation of compounds with mixed vanadium valence. In spite of their high electronic conductivity, REVO4-yttira stabilized zirconia (YSZ) composite anodes exhibited only modest performance when used in SOFCs operating with H2 fuel at 973 K due to their low catalytic activity. High performance was obtained, however, after the addition of a small amount of catalytically active Pd to the anode.

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.

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.

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.

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.

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

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.

Response of Ca2+-ATPase to clinorotaion of pea seedlings. O. M. Nedukha and E. L. Kordyum

NASA Astrophysics Data System (ADS)

Nedukha, Olena

2016-07-01

The present study was aimed to reveal of response of Ca2+-ATPase activity of cortex cells in distal elongation zone of Pisum sativum root to slow clinorotation. Pea seedlings were grown on a horizontal clinostat (2 rpm) and in the stationary control for 6 days. The electron-cytochemical method was used to examine the effects of imitated microgravity on the distribution of Ca2+-ATPase in outer layers of root cortex. The quantitative analysis of the density of cytochemical reaction products was measured using the Image J program. Electron microscopy showed the presence of electron-dense lead phosphate precipitated grains, the enzymatic activity reaction products on the plasma membrane, membranes of vesicular structures, endoplasmic reticulum (ER) and on organelles envelope in both of samples of the stationary control and clinorotated seedlings. We revealed the sensitivity of Ca2+-ATPase to clinorotation. The quantitative analysis of the area and density of enzymatic activity reaction products revealed that clinorotation led to the decrease of 3.4 times the density of reaction products on the plasma membrane and the increase of reaction products density on endomembranes and organelles membranes, in particular: in 2.2 times on mitochondria membranes; in 1.3 times - on membranes of ER; in 2.5 times - on tonoplast; by an order of magnitude greater - on contacting membranes of organelles with plasma membrane in comparison with such in cells of control samples. The data analysis can indicate an intensification of calcium pump on endomembranes, on envelopes of cytoplasmic organelles and nucleus. The obtained data suggest that the redistribution of Ca2+-ATPase activity in cells can be mediated by the activation of certain isoforms of enzyme or/and by an activation of Ca2+/H+ antiporter in plasma membrane that helps to maintain optimal calcium balance in plant cells under imitated microgravity.

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.

Single-Molecule Analysis of the Rotation of F1-ATPase under High Hydrostatic Pressure

PubMed Central

Okuno, Daichi; Nishiyama, Masayoshi; Noji, Hiroyuki

2013-01-01

F1-ATPase is the water-soluble part of ATP synthase and is an ATP-driven rotary molecular motor that rotates the rotary shaft against the surrounding stator ring, hydrolyzing ATP. Although the mechanochemical coupling mechanism of F1-ATPase has been well studied, the molecular details of individual reaction steps remain unclear. In this study, we conducted a single-molecule rotation assay of F1 from thermophilic bacteria under various pressures from 0.1 to 140 MPa. Even at 140 MPa, F1 actively rotated with regular 120° steps in a counterclockwise direction, showing high conformational stability and retention of native properties. Rotational torque was also not affected. However, high hydrostatic pressure induced a distinct intervening pause at the ATP-binding angles during continuous rotation. The pause was observed under both ATP-limiting and ATP-saturating conditions, suggesting that F1 has two pressure-sensitive reactions, one of which is evidently ATP binding. The rotation assay using a mutant F1(βE190D) suggested that the other pressure-sensitive reaction occurs at the same angle at which ATP binding occurs. The activation volumes were determined from the pressure dependence of the rate constants to be +100 Å3 and +88 Å3 for ATP binding and the other pressure-sensitive reaction, respectively. These results are discussed in relation to recent single-molecule studies of F1 and pressure-induced protein unfolding. PMID:24094404

Two ATPases

PubMed Central

Senior, Alan E.

2012-01-01

In this article, I reflect on research on two ATPases. The first is F1F0-ATPase, also known as ATP synthase. It is the terminal enzyme in oxidative phosphorylation and famous as a nanomotor. Early work on mitochondrial enzyme involved purification in large amount, followed by deduction of subunit composition and stoichiometry and determination of molecular sizes of holoenzyme and individual subunits. Later work on Escherichia coli enzyme utilized mutagenesis and optical probes to reveal the molecular mechanism of ATP hydrolysis and detailed facets of catalysis. The second ATPase is P-glycoprotein, which confers multidrug resistance, notably to anticancer drugs, in mammalian cells. Purification of the protein in large quantity allowed detailed characterization of catalysis, formulation of an alternating sites mechanism, and recently, advances in structural characterization. PMID:22822068

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

Non-Watson–Crick interactions between PNA and DNA inhibit the ATPase activity of bacteriophage T4 Dda helicase

PubMed Central

Tackett, Alan J.; Corey, David R.; Raney, Kevin D.

2002-01-01

Peptide nucleic acid (PNA) is a DNA mimic in which the nucleobases are linked by an N-(2-aminoethyl) glycine backbone. Here we report that PNA can interact with single-stranded DNA (ssDNA) in a non-sequence-specific fashion. We observed that a 15mer PNA inhibited the ssDNA-stimulated ATPase activity of a bacteriophage T4 helicase, Dda. Surprisingly, when a fluorescein-labeled 15mer PNA was used in binding studies no interaction was observed between PNA and Dda. However, fluorescence polarization did reveal non-sequence-specific interactions between PNA and ssDNA. Thus, the inhibition of ATPase activity of Dda appears to result from depletion of the available ssDNA due to non-Watson–Crick binding of PNA to ssDNA. Inhibition of the ssDNA-stimulated ATPase activity was observed for several PNAs of varying length and sequence. To study the basis for this phenomenon, we examined self-aggregation by PNAs. The 15mer PNA readily self-aggregates to the point of precipitation. Since PNAs are hydrophobic, they aggregate more than DNA or RNA, making the study of this phenomenon essential for understanding the properties of PNA. Non-sequence-specific interactions between PNA and ssDNA were observed at moderate concentrations of PNA, suggesting that such interactions should be considered for antisense and antigene applications. PMID:11842106

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 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.

Torque-coupled thermodynamic model for FoF1 -ATPase

NASA Astrophysics Data System (ADS)

Ai, Guangkuo; Liu, Pengfei; Ge, Hao

2017-05-01

FoF1 -ATPase is a motor protein complex that utilizes transmembrane ion flow to drive the synthesis of adenosine triphosphate (ATP) from adenosine diphosphate (ADP) and phosphate (Pi). While many theoretical models have been proposed to account for its rotary activity, most of them focus on the Fo or F1 portions separately rather than the complex as a whole. Here, we propose a simple but new torque-coupled thermodynamic model of FoF1 -ATPase. Solving this model at steady state, we find that the monotonic variation of each portion's efficiency becomes much more robust over a wide range of parameters when the Fo and F1 portions are coupled together, as compared to cases when they are considered separately. Furthermore, the coupled model predicts the dependence of each portion's kinetic behavior on the parameters of the other. Specifically, the power and efficiency of the F1 portion are quite sensitive to the proton gradient across the membrane, while those of the Fo portion as well as the related Michaelis constants for proton concentrations respond insensitively to concentration changes in the reactants of ATP synthesis. The physiological proton gradient across the membrane in the Fo portion is also shown to be optimal for the Michaelis constants of ADP and phosphate in the F1 portion during ATP synthesis. Together, our coupled model is able to predict key dynamic and thermodynamic features of the FoF1 -ATPase in vivo semiquantitatively, and suggests that such coupling approach could be further applied to other biophysical systems.

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

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.

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.

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

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.

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.

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.

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.

Preliminary study of gill NA+,K+-ATPase activity in juvenile spring chinook salmon following electroshock or handling stress

USGS Publications Warehouse

VanderKooi, S.P.; Gale, William L.; Maule, A.G.

2000-01-01

We compared gill Na+,K+-ATPase in subyearling and yearling spring chinook salmon Oncorhynchus tshawytscha 3 h, 24 h, and 7 d after exposure to either a short pulsed DC electroshock (300 V, 50 Hz, 8-ms pulse duration) or an acute handling stress. Mean gill Na+,K+-ATPase values ranged from 7.5 to 11.8 ??mol inorganic phosphate (Pi) ?? (mg protein)-1 ?? h-1. No significant differences were detected, with the exception of electroshocked subyearlings 7 d after treatment. Increased activity was attributed to the presence of two influential values. No significant differences were detected after removal of these observations, so the increase was not considered biologically significant. Inclusion of the outliers did not alter our interpretation of the results given that the observed increase was slight compared with the magnitude of changes reported under experimental conditions and in migrating juvenile salmonids. The treatment groups underwent a typical stress response and had significantly elevated cortisol and glucose levels 3 h after treatment. Recovery to control levels occurred within 24 h for cortisol and from 24 h to 7 d for glucose. Our results lead to the conclusion that neither acute electroshock nor acute handling stress alters Na+,K+-ATPase activity in juvenile spring chinook salmon.

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.

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.

[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.

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.

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.

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

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

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

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.

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

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.

Low pH-Induced Changes of Antioxidant Enzyme and ATPase Activities in the Roots of Rice (Oryza sativa L.) Seedlings

PubMed Central

Zhang, Yi-Kai; Zhu, De-Feng; Zhang, Yu-Ping; Chen, Hui-Zhe; Xiang, Jing; Lin, Xian-Qing

2015-01-01

Soil acidification is the main problem in the current rice production. Here, the effects of low pH on the root growth, reactive oxygen species metabolism, plasma membrane functions, and the transcript levels of the related genes were investigated in rice seedlings (Oryza sativa L.) in a hydroponic system at pH 3.5, 4.5, and 5.5. There were two hybrid rice cultivars in this trial, including Yongyou 12 (YY12, a japonica hybrid) and Zhongzheyou 1 (ZZY1, an indica hybrid). Higher H+ activity markedly decreased root length, the proportion of fine roots, and dry matter production, but induced a significant accumulation of hydrogen peroxide (H2O2), and led to serious lipid peroxidation in the roots of the two varieties. The transcript levels of copper/zinc superoxide dismutase 1 (Cu/Zn SOD1), copper/zinc superoxide dismutase 2 (Cu/Zn SOD2), catalase A (CATA) and catalase B (CATB) genes in YY12 and ZZY1 roots were significantly down-regulated after low pH exposure for two weeks. Meanwhile, a significant decrease was observed in the expression of the P-type Ca2+-ATPases in roots at pH 3.5. The activities of antioxidant enzymes (SOD, CAT) and plasma membrane (PM) Ca2+-ATPase in the two varieties were dramatically inhibited by strong rhizosphere acidification. However, the expression levels of ascorbate peroxidase 1 (APX1) and PM H+-ATPase isoform 7 were up-regulated under H+ stress compared with the control. Significantly higher activities of APX and PM H+-ATPase could contribute to the adaptation of rice roots to low pH. PMID:25719552

Rotary ATPases

PubMed Central

Stewart, Alastair G.; Sobti, Meghna; Harvey, Richard P.; Stock, Daniela

2013-01-01

Rotary ATPases are molecular rotary motors involved in biological energy conversion. They either synthesize or hydrolyze the universal biological energy carrier adenosine triphosphate. Recent work has elucidated the general architecture and subunit compositions of all three sub-types of rotary ATPases. Composite models of the intact F-, V- and A-type ATPases have been constructed by fitting high-resolution X-ray structures of individual subunits or sub-complexes into low-resolution electron densities of the intact enzymes derived from electron cryo-microscopy. Electron cryo-tomography has provided new insights into the supra-molecular arrangement of eukaryotic ATP synthases within mitochondria and mass-spectrometry has started to identify specifically bound lipids presumed to be essential for function. Taken together these molecular snapshots show that nano-scale rotary engines have much in common with basic design principles of man made machines from the function of individual “machine elements” to the requirement of the right “fuel” and “oil” for different types of motors. PMID:23369889

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.

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.

Cysteine residues 244 and 458-459 within the catalytic subunit of Na,K-ATPase control the enzyme's hydrolytic and signaling function under hypoxic conditions.

PubMed

Petrushanko, Irina Yu; Mitkevich, Vladimir A; Lakunina, Valentina A; Anashkina, Anastasia A; Spirin, Pavel V; Rubtsov, Peter M; Prassolov, Vladimir S; Bogdanov, Nikolay B; Hänggi, Pascal; Fuller, William; Makarov, Alexander A; Bogdanova, Anna

2017-10-01

Our previous findings suggested that reversible thiol modifications of cysteine residues within the actuator (AD) and nucleotide binding domain (NBD) of the Na,K-ATPase may represent a powerful regulatory mechanism conveying redox- and oxygen-sensitivity of this multifunctional enzyme. S-glutathionylation of Cys244 in the AD and Cys 454-458-459 in the NBD inhibited the enzyme and protected cysteines' thiol groups from irreversible oxidation under hypoxic conditions. In this study mutagenesis approach was used to assess the role these cysteines play in regulation of the Na,K-ATPase hydrolytic and signaling functions. Several constructs of mouse α1 subunit of the Na,K-ATPase were produced in which Cys244, Cys 454-458-459 or Cys 244-454-458-459 were replaced by alanine. These constructs were expressed in human HEK293 cells. Non-transfected cells and those expressing murine α1 subunit were exposed to hypoxia or treated with oxidized glutathione (GSSG). Both conditions induced inhibition of the wild type Na,K-ATPase. Enzymes containing mutated mouse α1 lacking Cys244 or all four cysteines (Cys 244-454-458-459) were insensitive to hypoxia. Inhibitory effect of GSSG was observed for wild type murine Na,K-ATPase, but was less pronounced in Cys454-458-459Ala mutant and completely absent in the Cys244Ala and Cys 244-454-458-459Ala mutants. In cells, expressing wild type enzyme, ouabain induced activation of Src and Erk kinases under normoxic conditions, whereas under hypoxic conditions this effect was inversed. Cys454-458-459Ala substitution abolished Src kinase activation in response to ouabain treatment, uncoupled Src from Erk signaling, and interfered with O 2 -sensitivity of Na,K-ATPase signaling function. Moreover, modeling predicted that S-glutathionylation of Cys 458 and 459 should prevent inhibitory binding of Src to NBD. Our data indicate for the first time that cysteine residues within the AD and NBD influence hydrolytic as well as receptor function of the Na,K-ATPase

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.

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.

Thiourea modulates the expression and activity profile of mtATPase under salinity stress in seeds of Brassica juncea

PubMed Central

Srivastava, A. K.; Ramaswamy, N. K.; Mukopadhyaya, R.; Jincy, M. G. Chiramal; D'Souza, S. F.

2009-01-01

Background and Aims Large areas of the globe are becoming saline due to evapotranspiration and poor irrigation practices, and sustainability of agriculture is being seriously affected. Thiourea (TU) has been identified as an effective bioregulator imparting stress tolerance to crops. The molecular mechanisms involved in the TU-mediated response are considered in this study. Methods Differential display was performed in order to identify TU-modulated transcripts in Brassica juncea seeds exposed to various treatments (distilled water; 1 m NaCl; 1 m NaCl + 500 p.p.m. TU). The differential regulation of these transcripts was validated by quantitative real-time PCR. Key Results Thiourea treatment maintained the viability of seeds exposed to NaCl for 6 h. Expression analysis showed that the transcript level of alpha, beta, gamma, delta and epsilon subunits of mitochondrial ATPase (mtATPase) varied in seeds subjected to the different treatments for 1 h: expression level was significantly altered by 1 m NaCl relative to controls; however, in the NaCl + TU treatment it reverted back in an integrated manner. Similar results were obtained from time-kinetics studies of beta and delta subunits in roots of 8-d-old seedlings. These observations were also confirmed by the mtATPase activity from isolated mitochondria. The reversal in the expression and activity profile of mtATPase through the application of a bioregulator such as TU is a novel finding for any plant system. Conclusions The results suggest that TU treatment maintains the integrity and functioning of mitochondria in seeds as well as seedlings exposed to salinity. Thus, TU has the potential to be used as an effective bioregulator to impart salinity tolerance under field conditions, and might prove to be of high economic importance by opening new avenues for both basic and applied research. PMID:19033283

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

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.

Vacuolar ATPase in Phagosome-Lysosome Fusion

PubMed Central

Kissing, Sandra; Hermsen, Christina; Repnik, Urska; Nesset, Cecilie Kåsi; von Bargen, Kristine; Griffiths, Gareth; Ichihara, Atsuhiro; Lee, Beth S.; Schwake, Michael; De Brabander, Jef; Haas, Albert; Saftig, Paul

2015-01-01

The vacuolar H+-ATPase (v-ATPase) complex is instrumental in establishing and maintaining acidification of some cellular compartments, thereby ensuring their functionality. Recently it has been proposed that the transmembrane V0 sector of v-ATPase and its a-subunits promote membrane fusion in the endocytic and exocytic pathways independent of their acidification functions. Here, we tested if such a proton-pumping independent role of v-ATPase also applies to phagosome-lysosome fusion. Surprisingly, endo(lyso)somes in mouse embryonic fibroblasts lacking the V0 a3 subunit of the v-ATPase acidified normally, and endosome and lysosome marker proteins were recruited to phagosomes with similar kinetics in the presence or absence of the a3 subunit. Further experiments used macrophages with a knockdown of v-ATPase accessory protein 2 (ATP6AP2) expression, resulting in a strongly reduced level of the V0 sector of the v-ATPase. However, acidification appeared undisturbed, and fusion between latex bead-containing phagosomes and lysosomes, as analyzed by electron microscopy, was even slightly enhanced, as was killing of non-pathogenic bacteria by V0 mutant macrophages. Pharmacologically neutralized lysosome pH did not affect maturation of phagosomes in mouse embryonic cells or macrophages. Finally, locking the two large parts of the v-ATPase complex together by the drug saliphenylhalamide A did not inhibit in vitro and in cellulo fusion of phagosomes with lysosomes. Hence, our data do not suggest a fusion-promoting role of the v-ATPase in the formation of phagolysosomes. PMID:25903133

Vacuolar ATPase in phagosome-lysosome fusion.

PubMed

Kissing, Sandra; Hermsen, Christina; Repnik, Urska; Nesset, Cecilie Kåsi; von Bargen, Kristine; Griffiths, Gareth; Ichihara, Atsuhiro; Lee, Beth S; Schwake, Michael; De Brabander, Jef; Haas, Albert; Saftig, Paul

2015-05-29

The vacuolar H(+)-ATPase (v-ATPase) complex is instrumental in establishing and maintaining acidification of some cellular compartments, thereby ensuring their functionality. Recently it has been proposed that the transmembrane V0 sector of v-ATPase and its a-subunits promote membrane fusion in the endocytic and exocytic pathways independent of their acidification functions. Here, we tested if such a proton-pumping independent role of v-ATPase also applies to phagosome-lysosome fusion. Surprisingly, endo(lyso)somes in mouse embryonic fibroblasts lacking the V0 a3 subunit of the v-ATPase acidified normally, and endosome and lysosome marker proteins were recruited to phagosomes with similar kinetics in the presence or absence of the a3 subunit. Further experiments used macrophages with a knockdown of v-ATPase accessory protein 2 (ATP6AP2) expression, resulting in a strongly reduced level of the V0 sector of the v-ATPase. However, acidification appeared undisturbed, and fusion between latex bead-containing phagosomes and lysosomes, as analyzed by electron microscopy, was even slightly enhanced, as was killing of non-pathogenic bacteria by V0 mutant macrophages. Pharmacologically neutralized lysosome pH did not affect maturation of phagosomes in mouse embryonic cells or macrophages. Finally, locking the two large parts of the v-ATPase complex together by the drug saliphenylhalamide A did not inhibit in vitro and in cellulo fusion of phagosomes with lysosomes. Hence, our data do not suggest a fusion-promoting role of the v-ATPase in the formation of phagolysosomes. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

The emerging structure of vacuolar ATPases.

PubMed

Drory, Omri; Nelson, Nathan

2006-10-01

Bioenergetics and physiology of primary pumps have been revitalized by new insights into the mechanism of energizing biomembranes. Structural information is becoming available, and the three-dimensional structure of F-ATPase is being resolved. The growing understanding of the fundamental mechanism of energy coupling may revolutionize our view of biological processes. The F- and V-ATPases (vacuolar-type ATPase) exhibit a common mechanical design in which nucleotide-binding on the catalytic sector, through a cycle of conformation changes, drives the transmembrane passage of protons by turning a membrane-embedded rotor. This motor can run in forward or reverse directions, hydrolyzing ATP as it pumps protons uphill or creating ATP as protons flow downhill. In contrast to F-ATPases, whose primary function in eukaryotic cells is to form ATP at the expense of the proton-motive force (pmf), V-ATPases function exclusively as an ATP-dependent proton pump. The pmf generated by V-ATPases in organelles and membranes of eukaryotic cells is utilized as a driving force for numerous secondary transport processes. V- and F-ATPases have similar structure and mechanism of action, and several of their subunits evolved from common ancestors. Electron microscopy studies of V-ATPase revealed its general structure at low resolution. Recently, several structures of V-ATPase subunits, solved by X-ray crystallography with atomic resolution, were published. This, together with electron microscopy low-resolution maps of the whole complex, and biochemistry cross-linking experiments, allows construction of a structural model for a part of the complex that may be used as a working hypothesis for future research.

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

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.

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

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.

Structure and mechanism of Zn2+-transporting P-type ATPases

PubMed Central

Wang, Kaituo; Sitsel, Oleg; Meloni, Gabriele; Autzen, Henriette Elisabeth; Andersson, Magnus; Klymchuk, Tetyana; Nielsen, Anna Marie; Rees, Douglas C.; Nissen, Poul; Gourdon, Pontus

2014-01-01

Zinc is an essential micronutrient for all living organisms, required for signaling and proper function of a range of proteins involved in e.g. DNA-binding and enzymatic catalysis1. In prokaryotes and photosynthetic eukaryotes Zn2+-transporting P-type ATPases of class IB (ZntA) are crucial for cellular redistribution and detoxification of Zn2+ and related elements2,3. Here we present crystal structures representing the phosphoenzyme ground state (E2P) and a dephosphorylation intermediate (E2.Pi) of ZntA from Shigella sonnei, determined at 3.2 and 2.7 Å resolution, respectively. The structures reveal a similar fold as the Cu+-ATPases with an amphipathic helix at the membrane interface. A conserved electronegative funnel connects this region to the intramembranous high-affinity ion-binding site and may promote specific uptake of cellular Zn2+ ions. The E2P structure displays a wide extracellular release pathway reaching the invariant residues at the high-affinity site, including Cys392, Cys394 and Asp714. The pathway closes in the E2.Pi state where Asp714 interacts with the conserved Lys693, which possibly stimulates Zn2+ release as a built-in counter-ion, as also proposed for H+-ATPases. Indeed, transport studies in liposomes provide experimental support for ZntA activity without counter-transport. These findings suggest a mechanistic link between PIB-type Zn2+-ATPases and PIII-type H+-ATPases, and show at the same time structural features of the extracellular release pathway that resemble the PII-type ATPases such as the sarco(endo)plasmic reticulum Ca2+-ATPase4,5 (SERCA) and Na+,K+-ATPase6. PMID:25132545

Transcriptional regulators of Na,K-ATPase subunits

PubMed Central

Li, Zhiqin; Langhans, Sigrid A.

2015-01-01

The Na,K-ATPase classically serves as an ion pump creating an electrochemical gradient across the plasma membrane that is essential for transepithelial transport, nutrient uptake and membrane potential. In addition, Na,K-ATPase also functions as a receptor, a signal transducer and a cell adhesion molecule. With such diverse roles, it is understandable that the Na,K-ATPase subunits, the catalytic α-subunit, the β-subunit and the FXYD proteins, are controlled extensively during development and to accommodate physiological needs. The spatial and temporal expression of Na,K-ATPase is partially regulated at the transcriptional level. Numerous transcription factors, hormones, growth factors, lipids, and extracellular stimuli modulate the transcription of the Na,K-ATPase subunits. Moreover, epigenetic mechanisms also contribute to the regulation of Na,K-ATPase expression. With the ever growing knowledge about diseases associated with the malfunction of Na,K-ATPase, this review aims at summarizing the best-characterized transcription regulators that modulate Na,K-ATPase subunit levels. As abnormal expression of Na,K-ATPase subunits has been observed in many carcinoma, we will also discuss transcription factors that are associated with epithelial-mesenchymal transition, a crucial step in the progression of many tumors to malignant disease. PMID:26579519

Tight coupling of Na+/K+-ATPase with glycolysis demonstrated in permeabilized rat cardiomyocytes.

PubMed

Sepp, Mervi; Sokolova, Niina; Jugai, Svetlana; Mandel, Merle; Peterson, Pearu; Vendelin, Marko

2014-01-01

The effective integrated organization of processes in cardiac cells is achieved, in part, by the functional compartmentation of energy transfer processes. Earlier, using permeabilized cardiomyocytes, we demonstrated the existence of tight coupling between some of cardiomyocyte ATPases and glycolysis in rat. In this work, we studied contribution of two membrane ATPases and whether they are coupled to glycolysis--sarcoplasmic reticulum Ca2+ ATPase (SERCA) and plasmalemma Na+/K+-ATPase (NKA). While SERCA activity was minor in this preparation in the absence of calcium, major role of NKA was revealed accounting to ∼30% of the total ATPase activity which demonstrates that permeabilized cell preparation can be used to study this pump. To elucidate the contribution of NKA in the pool of ATPases, a series of kinetic measurements was performed in cells where NKA had been inhibited by 2 mM ouabain. In these cells, we recorded: ADP- and ATP-kinetics of respiration, competition for ADP between mitochondria and pyruvate kinase (PK), ADP-kinetics of endogenous PK, and ATP-kinetics of total ATPases. The experimental data was analyzed using a series of mathematical models with varying compartmentation levels. The results show that NKA is tightly coupled to glycolysis with undetectable flux of ATP between mitochondria and NKA. Such tight coupling of NKA to PK is in line with its increased importance in the pathological states of the heart when the substrate preference shifts to glucose.

MgATP hydrolysis destabilizes the interaction between subunit H and yeast V1-ATPase, highlighting H's role in V-ATPase regulation by reversible disassembly.

PubMed

Sharma, Stuti; Oot, Rebecca A; Wilkens, Stephan

2018-05-12

Vacuolar H+-ATPases (V-ATPases; V1Vo-ATPases) are rotary motor proton pumps that acidify intracellular compartments and in some tissues, the extracellular space. V-ATPase is regulated by reversible disassembly into autoinhibited V1-ATPase and Vo proton channel sectors. An important player in V-ATPase regulation is subunit H, which binds at the interface of V1 and Vo. H is required for MgATPase activity in holo V-ATPase, but also for stabilizing the MgADP inhibited state in membrane detached V1. However, how H fulfills these two functions is poorly understood. To characterize the H-V1 interaction and its role in reversible disassembly, we determined binding affinities of full length H and its N-terminal domain (HNT) for an isolated heterodimer of subunits E and G (EG), the N-terminal domain of subunit a (aNT), and V1 lacking subunit H (V1ΔH). Using isothermal titration calorimetry (ITC) and biolayer interferometry (BLI), we show that HNT binds EG with moderate affinity, that full length H binds aNT weakly, and that both H and HNT bind V1ΔH with high affinity. We also found that only one molecule of HNT binds V1ΔH with high affinity, suggesting conformational asymmetry of the three EG heterodimers in V1ΔH. Moreover, MgATP hydrolysis-driven conformational changes in V1 destabilized the interaction of H, or HNT, with V1ΔH, suggesting an interplay between MgADP inhibition and subunit H. Our observation that H binding is affected by MgATP hydrolysis in V1 points to H's role in the mechanism of reversible disassembly. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.

Diphenyl diselenide elicits antidepressant-like activity in rats exposed to monosodium glutamate: A contribution of serotonin uptake and Na(+), K(+)-ATPase activity.

PubMed

Quines, Caroline B; Rosa, Suzan G; Velasquez, Daniela; Da Rocha, Juliana T; Neto, José S S; Nogueira, Cristina W

2016-03-15

Depression is a disorder with symptoms manifested at the psychological, behavioral and physiological levels. Monosodium glutamate (MSG) is the most widely used additive in the food industry; however, some adverse effects induced by this additive have been demonstrated in experimental animals and humans, including functional and behavioral alterations. The aim of this study was to investigate the possible antidepressant-like effect of diphenyl diselenide (PhSe)2, an organoselenium compound with pharmacological properties already documented, in the depressive-like behavior induced by MSG in rats. Male and female newborn Wistar rats were divided in control and MSG groups, which received, respectively, a daily subcutaneous injection of saline (0.9%) or MSG (4g/kg/day) from the 1st to 5th postnatal day. At 60th day of life, animals received (PhSe)2 (10mg/kg, intragastrically) 25min before spontaneous locomotor and forced swimming tests (FST). The cerebral cortices of rats were removed to determine [(3)H] serotonin (5-HT) uptake and Na(+), K(+)-ATPase activity. A single administration of (PhSe)2 was effective against locomotor hyperactivity caused by MSG in rats. (PhSe)2 treatment protected against the increase in the immobility time and a decrease in the latency for the first episode of immobility in the FST induced by MSG. Furthermore, (PhSe)2 reduced the [(3)H] 5-HT uptake and restored Na(+), K(+)-ATPase activity altered by MSG. In the present study a single administration of (PhSe)2 elicited an antidepressant-like effect and decrease the synaptosomal [(3)H] 5-HT uptake and an increase in the Na(+), K(+)-ATPase activity in MSG-treated rats. Copyright © 2015 Elsevier B.V. All rights reserved.

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.

Selective inhibition of ATPase activity during contraction alters the activation of p38 MAP kinase isoforms in skeletal muscle

PubMed Central

Brault, Jeffrey J.; Pizzimenti, Natalie M.; Dentel, John N.; Wiseman, Robert W.

2013-01-01

Muscle contractions strongly activate p38 MAP kinases, but the precise contraction-associated sarcoplasmic event(s) (e.g. force production, energetic demands and/or calcium cycling) that activate these kinases are still unclear. We tested the hypothesis that during contraction the phosphorylation of p38 isoforms is sensitive to the increase in ATP demand relative to ATP supply. Energetic demands were inhibited using N-benzyl-p-toluene sulphonamide (BTS, type II actomyosin) and cyclopiazonic acid (CPA, SERCA). Extensor digitorum longus muscles from Swiss Webster mice were incubated in Ringer’s solution (37°C) with or without inhibitors and then stimulated at 10 Hz for 15 min. Muscles were immediately freeze-clamped for metabolite and western blot analysis. BTS and BTS+CPA treatment decreased force production by 85%, as measured by the tension time integral, while CPA alone potentiated force by 310%. In control muscles, contractions resulted in a 73% loss of ATP content and a concomitant 7-fold increase in IMP content, a measure of sustained energetic imbalance. BTS or CPA treatment lessened the loss of ATP, but BTS+CPA treatment completely eliminated the energetic imbalance since ATP and IMP levels were nearly equal to those of non-stimulated muscles. The independent inhibition of cytosolic ATPase activities had no effect on contraction-induced p38 MAPK phosphorylation, but combined treatment prevented the increase in phosphorylation of the γ isoform while the α/βisoforms unaffected. These observations suggest that an energetic signal may trigger phosphorylation of the p38γ isoform while other factors are involved in activating the α/β isoforms, and also may explain how contractions differentially activate signaling pathways. PMID:23296747

2,3-Dihydroxy-quinoxaline induces ATPase activity of Herpes Simplex Virus thymidine kinase.

PubMed

Zeifman, Alexey A; Novikov, Fedor N; Stroylov, Victor S; Stroganov, Oleg V; Chilov, Ghermes G; Skoblov, Alexander Y; Miroshnikov, Anatoly I; Skoblov, Yuri S

2014-01-31

2,3-Dihydroxy-quinoxaline, a small molecule that promotes ATPase catalytic activity of Herpes Simplex Virus thymidine kinase (HSV-TK), was identified by virtual screening. This compound competitively inhibited HSV-TK catalyzed phosphorylation of acyclovir with Ki=250 μM (95% CI: 106-405 μM) and dose-dependently increased the rate of the ATP hydrolysis with KM=112 μM (95% CI: 28-195 μM). The kinetic scheme consistent with this experimental data is proposed. Copyright © 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

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

Correlation between uncoupled ATP hydrolysis and heat production by the sarcoplasmic reticulum Ca2+-ATPase: coupling effect of fluoride.

PubMed

Reis, M; Farage, M; de Souza, A C; de Meis, L

2001-11-16

The sarcoplasmic reticulum Ca(2+)-ATPase transports Ca(2+) using the chemical energy derived from ATP hydrolysis. Part of the chemical energy is used to translocate Ca(2+) through the membrane (work) and part is dissipated as heat. The amount of heat produced during catalysis increases after formation of the Ca(2+) gradient across the vesicle membrane. In the absence of gradient (leaky vesicles) the amount of heat produced/mol of ATP cleaved is half of that measured in the presence of the gradient. After formation of the gradient, part of the ATPase activity is not coupled to Ca(2+) transport. We now show that NaF can impair the uncoupled ATPase activity with discrete effect on the ATPase activity coupled to Ca(2+) transport. For the control vesicles not treated with NaF, after formation of the gradient only 20% of the ATP cleaved is coupled to Ca(2+) transport, and the caloric yield of the total ATPase activity (coupled plus uncoupled) is 22.8 kcal released/mol of ATP cleaved. In contrast, the vesicles treated with NaF consume only the ATP needed to maintain the gradient, and the caloric yield of ATP hydrolysis is 3.1 kcal/mol of ATP. The slow ATPase activity measured in vesicles treated with NaF has the same Ca(2+) dependence as the control vesicles. This demonstrates unambiguously that the uncoupled activity is an actual pathway of the Ca(2+)-ATPase rather than a contaminating phosphatase. We conclude that when ATP hydrolysis occurs without coupled biological work most of the chemical energy is dissipated as heat. Thus, uncoupled ATPase activity appears to be the mechanistic feature underlying the ability of the Ca(2+)-ATPase to modulated heat production.

Stability and self-passivation of copper vanadate photoanodes under chemical, electrochemical, and photoelectrochemical operation

DOE PAGES

Zhou, Lan; Yan, Qimin; Yu, Jie; ...

2016-03-14

We discuss how deployment of solar fuels technology requires photoanodes and that long term stability, can be accomplished using light absorbers that self-passivate under operational conditions. We recently reported that several copper vanadates are promising photoanode materials, and their stability and self-passivation is demonstrated through a combination of Pourbaix calculations and combinatorial experimentation.

Identification of Residues in the Lipopolysaccharide ABC Transporter That Coordinate ATPase Activity with Extractor Function.

PubMed

Simpson, Brent W; Owens, Tristan W; Orabella, Matthew J; Davis, Rebecca M; May, Janine M; Trauger, Sunia A; Kahne, Daniel; Ruiz, Natividad

2016-10-18

The surface of most Gram-negative bacteria is covered with lipopolysaccharide (LPS), creating a permeability barrier against toxic molecules, including many antimicrobials. To assemble LPS on their surface, Gram-negative bacteria must extract newly synthesized LPS from the inner membrane, transport it across the aqueous periplasm, and translocate it across the outer membrane. The LptA to -G proteins assemble into a transenvelope complex that transports LPS from the inner membrane to the cell surface. The Lpt system powers LPS transport from the inner membrane by using a poorly characterized ATP-binding cassette system composed of the ATPase LptB and the transmembrane domains LptFG. Here, we characterize a cluster of residues in the groove region of LptB that is important for controlling LPS transport. We also provide the first functional characterization of LptFG and identify their coupling helices that interact with the LptB groove. Substitutions at conserved residues in these coupling helices compromise both the assembly and function of the LptB 2 FG complex. Defects in LPS transport conferred by alterations in the LptFG coupling helices can be rescued by changing a residue in LptB that is adjacent to functionally important residues in the groove region. This suppression is achieved by increasing the ATPase activity of the LptB 2 FG complex. Taken together, these data identify a specific binding site in LptB for the coupling helices of LptFG that is responsible for coupling of ATP hydrolysis by LptB with LptFG function to achieve LPS extraction. Lipopolysaccharide (LPS) is synthesized at the cytoplasmic membrane of Gram-negative bacteria and transported across several compartments to the cell surface, where it forms a barrier that protects these organisms from antibiotics. The LptB 2 FG proteins form an ATP-binding cassette (ABC) transporter that uses energy from ATP hydrolysis in the cytoplasm to facilitate extraction of LPS from the outer face of the

PfeT, a P1B4 -type ATPase, effluxes ferrous iron and protects Bacillus subtilis against iron intoxication.

PubMed

Guan, Guohua; Pinochet-Barros, Azul; Gaballa, Ahmed; Patel, Sarju J; Argüello, José M; Helmann, John D

2015-11-01

Iron is an essential element for nearly all cells and limited iron availability often restricts growth. However, excess iron can also be deleterious, particularly when cells expressing high affinity iron uptake systems transition to iron rich environments. Bacillus subtilis expresses numerous iron importers, but iron efflux has not been reported. Here, we describe the B. subtilis PfeT protein (formerly YkvW/ZosA) as a P1B4 -type ATPase in the PerR regulon that serves as an Fe(II) efflux pump and protects cells against iron intoxication. Iron and manganese homeostasis in B. subtilis are closely intertwined: a pfeT mutant is iron sensitive, and this sensitivity can be suppressed by low levels of Mn(II). Conversely, a pfeT mutant is more resistant to Mn(II) overload. In vitro, the PfeT ATPase is activated by both Fe(II) and Co(II), although only Fe(II) efflux is physiologically relevant in wild-type cells, and null mutants accumulate elevated levels of intracellular iron. Genetic studies indicate that PfeT together with the ferric uptake repressor (Fur) cooperate to prevent iron intoxication, with iron sequestration by the MrgA mini-ferritin playing a secondary role. Protection against iron toxicity may also be a key role for related P1B4 -type ATPases previously implicated in bacterial pathogenesis. © 2015 John Wiley & Sons Ltd.