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Sample records for glutamate dehydrogenase activity

  1. Aminotransferase and glutamate dehydrogenase activities in lactobacilli and streptococci.

    PubMed

    Peralta, Guillermo Hugo; Bergamini, Carina Viviana; Hynes, Erica Rut

    2016-01-01

    Aminotransferases and glutamate dehydrogenase are two main types of enzymes involved in the initial steps of amino acid catabolism, which plays a key role in the cheese flavor development. In the present work, glutamate dehydrogenase and aminotransferase activities were screened in twenty one strains of lactic acid bacteria of dairy interest, either cheese-isolated or commercial starters, including fifteen mesophilic lactobacilli, four thermophilic lactobacilli, and two streptococci. The strains of Streptococcus thermophilus showed the highest glutamate dehydrogenase activity, which was significantly elevated compared with the lactobacilli. Aspartate aminotransferase prevailed in most strains tested, while the levels and specificity of other aminotransferases were highly strain- and species-dependent. The knowledge of enzymatic profiles of these starter and cheese-isolated cultures is helpful in proposing appropriate combinations of strains for improved or increased cheese flavor. PMID:27266631

  2. Aminotransferase and glutamate dehydrogenase activities in lactobacilli and streptococci.

    PubMed

    Peralta, Guillermo Hugo; Bergamini, Carina Viviana; Hynes, Erica Rut

    2016-01-01

    Aminotransferases and glutamate dehydrogenase are two main types of enzymes involved in the initial steps of amino acid catabolism, which plays a key role in the cheese flavor development. In the present work, glutamate dehydrogenase and aminotransferase activities were screened in twenty one strains of lactic acid bacteria of dairy interest, either cheese-isolated or commercial starters, including fifteen mesophilic lactobacilli, four thermophilic lactobacilli, and two streptococci. The strains of Streptococcus thermophilus showed the highest glutamate dehydrogenase activity, which was significantly elevated compared with the lactobacilli. Aspartate aminotransferase prevailed in most strains tested, while the levels and specificity of other aminotransferases were highly strain- and species-dependent. The knowledge of enzymatic profiles of these starter and cheese-isolated cultures is helpful in proposing appropriate combinations of strains for improved or increased cheese flavor.

  3. Effects of aluminum on activity of krebs cycle enzymes and glutamate dehydrogenase in rat brain homogenate.

    PubMed

    Zatta, P; Lain, E; Cagnolini, C

    2000-05-01

    Aluminum is a neurotoxic agent for animals and humans that has been implicated as an etiological factor in several neurodegenerative diseases and as a destabilizer of cell membranes. Due to its high reactivity, Al3+ is able to interfere with several biological functions, including enzymatic activities in key metabolic pathways. In this paper we report that, among the enzymes that constitute the Krebs cycle, only two are activated by aluminum: alpha-ketoglutarate dehydrogenase and succinate dehydrogenase. In contrast, aconitase, shows decreased activity in the presence of the metal ion. Al3+ also inhibits glutamate dehydrogenase, an allosteric enzyme that is closely linked to the Krebs cycle. A possible correlation between aluminum, the Krebs cycle and aging processes is discussed.

  4. Modulation of activity of Bacillus subtilis regulatory proteins GltC and TnrA by glutamate dehydrogenase.

    PubMed

    Belitsky, Boris R; Sonenshein, Abraham L

    2004-06-01

    The Bacillus subtilis gltAB operon, encoding glutamate synthase, requires a specific positive regulator, GltC, for its expression and is repressed by the global regulatory protein TnrA. The factor that controls TnrA activity, a complex of glutamine synthetase and a feedback inhibitor, such as glutamine, is known, but the signal for modulation of GltC activity has remained elusive. GltC-dependent gltAB expression was drastically reduced when cells were grown in media containing arginine or ornithine or proline, all of which are inducers and substrates of the Roc catabolic pathway. Analysis of gltAB expression in mutants with various defects in the Roc pathway indicated that rocG-encoded glutamate dehydrogenase was required for such repression, suggesting that the substrates or products of this enzyme are the real effectors of GltC. Given that RocG is an enzyme of glutamate catabolism, the main regulatory role of GltC may be prevention of a futile cycle of glutamate synthesis and degradation in the presence of arginine-related amino acids or proline. In addition, high activity of glutamate dehydrogenase was incompatible with activity of TnrA.

  5. Mechanism of hyperinsulinism in short-chain 3-hydroxyacyl-CoA dehydrogenase deficiency involves activation of glutamate dehydrogenase.

    PubMed

    Li, Changhong; Chen, Pan; Palladino, Andrew; Narayan, Srinivas; Russell, Laurie K; Sayed, Samir; Xiong, Guoxiang; Chen, Jie; Stokes, David; Butt, Yasmeen M; Jones, Patricia M; Collins, Heather W; Cohen, Noam A; Cohen, Akiva S; Nissim, Itzhak; Smith, Thomas J; Strauss, Arnold W; Matschinsky, Franz M; Bennett, Michael J; Stanley, Charles A

    2010-10-01

    The mechanism of insulin dysregulation in children with hyperinsulinism associated with inactivating mutations of short-chain 3-hydroxyacyl-CoA dehydrogenase (SCHAD) was examined in mice with a knock-out of the hadh gene (hadh(-/-)). The hadh(-/-) mice had reduced levels of plasma glucose and elevated plasma insulin levels, similar to children with SCHAD deficiency. hadh(-/-) mice were hypersensitive to oral amino acid with decrease of glucose level and elevation of insulin. Hypersensitivity to oral amino acid in hadh(-/-) mice can be explained by abnormal insulin responses to a physiological mixture of amino acids and increased sensitivity to leucine stimulation in isolated perifused islets. Measurement of cytosolic calcium showed normal basal levels and abnormal responses to amino acids in hadh(-/-) islets. Leucine, glutamine, and alanine are responsible for amino acid hypersensitivity in islets. hadh(-/-) islets have lower intracellular glutamate and aspartate levels, and this decrease can be prevented by high glucose. hadh(-/-) islets also have increased [U-(14)C]glutamine oxidation. In contrast, hadh(-/-) mice have similar glucose tolerance and insulin sensitivity compared with controls. Perifused hadh(-/-) islets showed no differences from controls in response to glucose-stimulated insulin secretion, even with addition of either a medium-chain fatty acid (octanoate) or a long-chain fatty acid (palmitate). Pull-down experiments with SCHAD, anti-SCHAD, or anti-GDH antibodies showed protein-protein interactions between SCHAD and GDH. GDH enzyme kinetics of hadh(-/-) islets showed an increase in GDH affinity for its substrate, α-ketoglutarate. These studies indicate that SCHAD deficiency causes hyperinsulinism by activation of GDH via loss of inhibitory regulation of GDH by SCHAD.

  6. Regulation of Glutamate Dehydrogenase Activity in Relation to Carbon Limitation and Protein Catabolism in Carrot Cell Suspension Cultures 1

    PubMed Central

    Robinson, Sharon A.; Stewart, George R.; Phillips, Richard

    1992-01-01

    Glutamate dehydrogenase (GDH) specific activity and function have been studied in cell suspension cultures of carrot (Daucus carota L. cv Chantenay) in response to carbon and nitrogen supply in the culture medium. The specific activity of GDH was derepressed in sucrose-starved cells concomitant with protein catabolism, ammonium excretion, and the accumulation of metabolically active amino acids. The addition of sucrose led to a rapid decrease in GDH specific activity, an uptake of ammonium from the medium, and a decrease in amino acid levels. The extent of GDH derepression was correlated positively with cellular glutamate concentration. These findings strengthen the view that the function of GDH is the catabolism of glutamate, which under conditions of carbon stress provides carbon skeletons for tricarboxylic acid cycle activity. PMID:16668745

  7. Characterization of the glutamate dehydrogenase activity of Gigantocotyle explanatum and Gastrothylax crumenifer (Trematoda: Digenea).

    PubMed

    Abidi, S M A; Khan, P; Saifullah, M K

    2009-12-01

    Glutamate dehydrogenase (GLDH) (EC 1.4.1.3) is a ubiquitous enzyme, which is present at the protein and carbohydrate metabolism crossroads. The enzyme activity was investigated in biliary and rumen amphistomes, Gigantocotyle explanatum and Gastrothylax crumenifer, respectively, infecting the Indian water buffalo Bubalus bubalis. The enzyme activity was consistently higher in G. explanatum as compared to G. crumenifer, where NAD(H) was utilized as coenzyme and the pH optima was recorded at 8. The K(m) and V(max) values for α-ketoglutarate were 2.1 mM and 9.09 units in G. explanatum, whereas 3.03 mM and 1.90 units in G. crumenifer, respectively. Among the allosteric modulator nucleotides, AMP, ADP, ATP, GMP, CMP and UMP, only AMP enhanced GLDH activity in G. crumenifer while ADP was stimulatory in G. explanatum. The amino acid leucine stimulated the GLDH activity in both the amphistomes while alanine was stimulatory only in G. crumenifer. Pronounced interspecific differences in response to different metabolic inhibitors like diethyldithiocarbamate, semicarbazide hydrochloride and mercurial ions were also observed. The osmotic stress alters the enzyme activity, particularly in hypertonic saline the GLDH activity increased significantly (p < 0.01) in G. explanatum, while insignificant effects were observed in rumen dwelling G. crumenifer. Histoenzymology revealed region/tissue specific distribution of GLDH with prominent staining in tissues like vitellaria, lymph system and tegument/subtegument, thus showing specific distribution of GLDH indicating differential metabolic state. Such intergeneric differences in GLDH activity could also be a consequence of occupying different microenvironments within the same host.

  8. Evidence for synergistic control of glutamate biosynthesis by glutamate dehydrogenases and glutamate in Bacillus subtilis.

    PubMed

    Stannek, Lorena; Thiele, Martin J; Ischebeck, Till; Gunka, Katrin; Hammer, Elke; Völker, Uwe; Commichau, Fabian M

    2015-09-01

    In the Gram-positive bacterium, Bacillus subtilis glutamate is synthesized by the glutamine synthetase and the glutamate synthase (GOGAT). During growth with carbon sources that exert carbon catabolite repression, the rocG glutamate dehydrogenase (GDH) gene is repressed and the transcription factor GltC activates the expression of the GOGAT encoding gltAB genes. In the presence of amino acids of the glutamate family, the GDH RocG is synthesized and the enzyme prevents GltC from binding to DNA. The dual control of glutamate biosynthesis allows the efficient utilization of the available nutrients. Here we provide genetic and biochemical evidence that, like RocG, also the paralogous GDH GudB can inhibit the transcription factor GltC, thereby controlling glutamate biosynthesis. Contradictory previous observations show that high level of GDH activity does not result in permanent inhibition of GltC. By controlling the intracellular levels of glutamate through feeding with exogenous arginine, we observed that the GDH-dependent control of GltC and thus expression of the gltAB genes inversely correlates with the glutamate pool. These results suggest that the B. subtilis GDHs RocG and GudB in fact act as glutamate sensors. In conclusion, the GDH-mediated control of glutamate biosynthesis seems to depend on the intracellular glutamate concentration. PMID:25711804

  9. The maximum activities of hexokinase, phosphorylase, phosphofructokinase, glycerol phosphate dehydrogenases, lactate dehydrogenase, octopine dehydrogenase, phosphoenolpyruvate carboxykinase, nucleoside diphosphatekinase, glutamate-oxaloacetate transaminase and arginine kinase in relation to carbohydrate utilization in muscles from marine invertebrates.

    PubMed Central

    Zammit, V A; Newsholme, E A

    1976-01-01

    Comparison of the activities of hexokinase, phosphorylase and phosphofructokinase in muscles from marine invertebrates indicates that they can be divided into three groups. First, the activities of the three enzymes are low in coelenterate muscles, catch muscles of molluscs and muscles of echinoderms; this indicates a low rate of carbohydrate (and energy) utilization by these muscles. Secondly, high activities of phosphorylase and phosphofructokinase relative to those of hexokinase are found in, for example, lobster abdominal and scallop snap muscles; this indicates that these muscles depend largely on anaerobic degradation of glycogen for energy production. Thirdly, high activities of hexokinase are found in the radular muscles of prosobranch molluscs and the fin muscles of squids; this indicates a high capacity for glucose utilization, which is consistent with the high activities of enzymes of the tricarboxylic acid cycle in these muscles [Alp, Newsholme & Zammit (1976) Biochem. J. 154, 689-700]. 2. The activities of lactate dehydrogenase, octopine dehydrogenase, phosphoenolpyruvate carboxykinase, cytosolic and mitochondrial glycerol 3-phosphate dehydrogenase and glutamate-oxaloacetate transaminase were measured in order to provide a qualitative indication of the importance of different processes for oxidation of glycolytically formed NADH. The muscles are divided into four groups: those that have a high activity of lactate dehydrogenase relative to the activities of phosphofructokinase (e.g. crustacean muscles); those that have high activities of octopine dehydrogenase but low activities of lactate dehydrogenase (e.g. scallop snap muscle); those that have moderate activities of both lactate dehydrogenase and octopine dehydrogenase (radular muscles of prosobranchs), and those that have low activities of both lactate dehydrogenase and octopine dehydrogenase, but which possess activities of phosphoenolpyruvate carboxykinase (oyster adductor muscles). It is

  10. Adaptation of methods for glutamate dehydrogenase and alcohol dehydrogenase activities to a centrifugal analyser: assessment of their clinical use in anoxic states of the liver.

    PubMed Central

    Shephard, M D; Penberthy, L A; Berry, M N

    1987-01-01

    Sensitive, precise, and rapid methods for the measurement of alcohol dehydrogenase (ADH) and glutamate dehydrogenase (GDH) were developed on the Cobas Bio centrifugal analyser. The optimal pH for ADH in caucasians was 9.8. Non-linearity of ADH enzyme activity was observed when samples were diluted in saline; linearity was restored when inactivated serum was used as diluent. ADH was shown to be a sensitive index of liver anoxia due to cardiorespiratory disturbance (clinical sensitivity 90%) and generalised anoxia. GDH exhibited sensitivity equal to that of alanine aminotransferase (ALT) but was inferior to gamma-glutamyltransferase (GGT) in the detection of specific liver disease. Both ADH and GDH were sensitive indicators of alcoholic liver disease. PMID:2890662

  11. Green Tea Polyphenols Control Dysregulated Glutamate Dehydrogenase in Transgenic Mice by Hijacking the ADP Activation Site

    SciTech Connect

    Li, Changhong; Li, Ming; Chen, Pan; Narayan, Srinivas; Matschinsky, Franz M.; Bennett, Michael J.; Stanley, Charles A.; Smith, Thomas J.

    2012-05-09

    Glutamate dehydrogenase (GDH) catalyzes the oxidative deamination of L-glutamate and, in animals, is extensively regulated by a number of metabolites. Gain of function mutations in GDH that abrogate GTP inhibition cause the hyperinsulinism/hyperammonemia syndrome (HHS), resulting in increased pancreatic {beta}-cell responsiveness to leucine and susceptibility to hypoglycemia following high protein meals. We have previously shown that two of the polyphenols from green tea (epigallocatechin gallate (EGCG) and epicatechin gallate (ECG)) inhibit GDH in vitro and that EGCG blocks GDH-mediated insulin secretion in wild type rat islets. Using structural and site-directed mutagenesis studies, we demonstrate that ECG binds to the same site as the allosteric regulator, ADP. Perifusion assays using pancreatic islets from transgenic mice expressing a human HHS form of GDH demonstrate that the hyperresponse to glutamine caused by dysregulated GDH is blocked by the addition of EGCG. As observed in HHS patients, these transgenic mice are hypersensitive to amino acid feeding, and this is abrogated by oral administration of EGCG prior to challenge. Finally, the low basal blood glucose level in the HHS mouse model is improved upon chronic administration of EGCG. These results suggest that this common natural product or some derivative thereof may prove useful in controlling this genetic disorder. Of broader clinical implication is that other groups have shown that restriction of glutamine catabolism via these GDH inhibitors can be useful in treating various tumors. This HHS transgenic mouse model offers a highly useful means to test these agents in vivo.

  12. Green tea polyphenols control dysregulated glutamate dehydrogenase in transgenic mice by hijacking the ADP activation site.

    PubMed

    Li, Changhong; Li, Ming; Chen, Pan; Narayan, Srinivas; Matschinsky, Franz M; Bennett, Michael J; Stanley, Charles A; Smith, Thomas J

    2011-09-30

    Glutamate dehydrogenase (GDH) catalyzes the oxidative deamination of L-glutamate and, in animals, is extensively regulated by a number of metabolites. Gain of function mutations in GDH that abrogate GTP inhibition cause the hyperinsulinism/hyperammonemia syndrome (HHS), resulting in increased pancreatic β-cell responsiveness to leucine and susceptibility to hypoglycemia following high protein meals. We have previously shown that two of the polyphenols from green tea (epigallocatechin gallate (EGCG) and epicatechin gallate (ECG)) inhibit GDH in vitro and that EGCG blocks GDH-mediated insulin secretion in wild type rat islets. Using structural and site-directed mutagenesis studies, we demonstrate that ECG binds to the same site as the allosteric regulator, ADP. Perifusion assays using pancreatic islets from transgenic mice expressing a human HHS form of GDH demonstrate that the hyperresponse to glutamine caused by dysregulated GDH is blocked by the addition of EGCG. As observed in HHS patients, these transgenic mice are hypersensitive to amino acid feeding, and this is abrogated by oral administration of EGCG prior to challenge. Finally, the low basal blood glucose level in the HHS mouse model is improved upon chronic administration of EGCG. These results suggest that this common natural product or some derivative thereof may prove useful in controlling this genetic disorder. Of broader clinical implication is that other groups have shown that restriction of glutamine catabolism via these GDH inhibitors can be useful in treating various tumors. This HHS transgenic mouse model offers a highly useful means to test these agents in vivo.

  13. [Features of glutamate dehydrogenase in fetal and adult rumen tissue].

    PubMed

    Kalachniuk, H I; Fomenko, I S; Kalachniuk, L H; Kavai, Sh; Marounek, M; Savka, O H

    2001-01-01

    Glutamate dehydrogenase (GDH) from rumen mucosa of cow fetus, liver and two forms from mucosa (bacterial and tissue) of the adult animal were partly purified and characterized. The activity of the bacterial glutamate dehydrogenase was shown to depend on qualities of a biomass of microbes, adhered on surface of rumen mucosa. All enzymes from tissues (GDHTRF, TRC, TLC), revealed the hypersensibility to increase in the concentration medium of Zn2+, guanosine triphosphate (GTP), acting here in a role of negative modulators, and also adenosine monophosphate (AMP) and leucine, which acted as activators. However, in the same concentrations these effectors do not influence the activity of the bacterial glutamate dehydrogenase. And if all tissues enzymes are highly specific to coenzyme NADH, the bacterial ones almost in 3 times is more active at NADPH use. PMID:11642036

  14. Side-chain interactions in the regulatory domain of human glutamate dehydrogenase determine basal activity and regulation.

    PubMed

    Mastorodemos, Vasileios; Kanavouras, Konstantinos; Sundaram, Shobana; Providaki, Maria; Petraki, Zoe; Kokkinidis, Michael; Zaganas, Ioannis; Logothetis, Diomedes E; Plaitakis, Andreas

    2015-04-01

    Glutamate Dehydrogenase (GDH) is central to the metabolism of glutamate, a major excitatory transmitter in mammalian central nervous system (CNS). hGDH1 is activated by ADP and L-leucine and powerfully inhibited by GTP. Besides this housekeeping hGDH1, duplication led to an hGDH2 isoform that is expressed in the human brain dissociating its function from GTP control. The novel enzyme has reduced basal activity (4-6% of capacity) while remaining remarkably responsive to ADP/L-leucine activation. While the molecular basis of this evolutionary adaptation remains unclear, substitution of Ser for Arg443 in hGDH1 is shown to diminish basal activity (< 2% of capacity) and abrogate L-leucine activation. To explore whether the Arg443Ser mutation disrupts hydrogen bonding between Arg443 and Ser409 of adjacent monomers in the regulatory domain ('antenna'), we replaced Ser409 by Arg or Asp in hGDH1. The Ser409Arg-1 change essentially replicated the Arg443Ser-1 mutation effects. Molecular dynamics simulation predicted that Ser409 and Arg443 of neighboring monomers come in close proximity in the open conformation and that introduction of Ser443-1 or Arg409-1 causes them to separate with the swap mutation (Arg409/Ser443) reinstating their proximity. A swapped Ser409Arg/Arg443Ser-1 mutant protein, obtained in recombinant form, regained most of the wild-type hGDH1 properties. Also, when Ser443 was replaced by Arg443 in hGDH2 (as occurs in hGDH1), the Ser443Arg-2 mutant acquired most of the hGDH1 properties. Hence, side-chain interactions between 409 and 443 positions in the 'antenna' region of hGDHs are crucial for basal catalytic activity, allosteric regulation, and relative resistance to thermal inactivation. PMID:25620628

  15. Chicken liver glutamate dehydrogenase (GDH) demonstrates a histone H3 specific protease (H3ase) activity in vitro.

    PubMed

    Purohit, Jogeswar S; Tomar, Raghuvir S; Panigrahi, Anil K; Pandey, Shashibhal M; Singh, Divya; Chaturvedi, Madan M

    2013-11-01

    Site-specific proteolysis of the N or C-terminus of histone tails has emerged as a novel form of irreversible post-translational modifications assigned to histones. Though there are many reports describing histone specific proteolysis, there are very few studies on purification of a histone specific protease. Here, we demonstrate a histone H3 specific protease (H3ase) activity in chicken liver nuclear extract. H3ase was purified to homogeneity and identified as glutamate dehydrogenase (GDH) by sequencing. A series of biochemical experiments further confirmed that the H3ase activity was due to GDH. The H3ase clipped histone H3 products were sequenced by N-terminal sequencing and the precise clipping sites of H3ase were mapped. H3ase activity was only specific to chicken liver as it was not demonstrated in other tissues like heart, muscle and brain of chicken. We assign a novel serine like protease activity to GDH which is specific to histone H3. PMID:23856561

  16. Theoretical analysis of the glutamate dehydrogenase kinetics under physiological conditions.

    PubMed

    Popova, S V; Reich, J G

    1983-01-01

    A kinetic model of the glutamate dehydrogenase reaction has been formulated for the reversible reaction including all seven reactants (substrates and cofactors NAD(H) and NADP(H)). The model parameters have been evaluated from published initial-rate data. Analysis of the model at cofactor concentration near to that in the intact mitochondrion has shown that the competition for active sites between cofactors and substrates simultaneously present in mitochondria diminishes the steady-state rate of the reaction by a factor of 10 to 100 as compared to the maximal reaction rate. The model predicts near-equilibrium of the reaction substrates with NAD+/NADH cofactor pair and off-equilibrium with NADP+/NADPH. Substrate cycling with futile transfer of hydrogen from NADP+-system to NAD+-system has been found to account under in vivo conditions for no more than 2% of the maximal glutamate dehydrogenase activity in the mitochondria.

  17. Dysfunctional TCA-Cycle Metabolism in Glutamate Dehydrogenase Deficient Astrocytes.

    PubMed

    Nissen, Jakob D; Pajęcka, Kamilla; Stridh, Malin H; Skytt, Dorte M; Waagepetersen, Helle S

    2015-12-01

    Astrocytes take up glutamate in the synaptic area subsequent to glutamatergic transmission by the aid of high affinity glutamate transporters. Glutamate is converted to glutamine or metabolized to support intermediary metabolism and energy production. Glutamate dehydrogenase (GDH) and aspartate aminotransferase (AAT) catalyze the reversible reaction between glutamate and α-ketoglutarate, which is the initial step for glutamate to enter TCA cycle metabolism. In contrast to GDH, AAT requires a concomitant interconversion of oxaloacetate and aspartate. We have investigated the role of GDH in astrocyte glutamate and glucose metabolism employing siRNA mediated knock down (KD) of GDH in cultured astrocytes using stable and radioactive isotopes for metabolic mapping. An increased level of aspartate was observed upon exposure to [U-(13) C]glutamate in astrocytes exhibiting reduced GDH activity. (13) C Labeling of aspartate and TCA cycle intermediates confirmed that the increased amount of aspartate is associated with elevated TCA cycle flux from α-ketoglutarate to oxaloacetate, i.e. truncated TCA cycle. (13) C Glucose metabolism was elevated in GDH deficient astrocytes as observed by increased de novo synthesis of aspartate via pyruvate carboxylation. In the absence of glucose, lactate production from glutamate via malic enzyme was lower in GDH deficient astrocytes. In conclusions, our studies reveal that metabolism via GDH serves an important anaplerotic role by adding net carbon to the TCA cycle. A reduction in GDH activity seems to cause the astrocytes to up-regulate activity in pathways involved in maintaining the amount of TCA cycle intermediates such as pyruvate carboxylation as well as utilization of alternate substrates such as branched chain amino acids.

  18. A membrane-bound form of glutamate dehydrogenase possesses an ATP-dependent high-affinity microtubule-binding activity.

    PubMed Central

    Rajas, F; Rousset, B

    1993-01-01

    We previously identified a 50 kDa membrane protein which bound to in vitro assembled microtubules [Mithieux and Rousset (1989) J. Biol. Chem. 264, 4664-4668]. This protein exhibited the expected properties for mediating the ATP-dependent association of vesicles with microtubules [Mithieux, Audebet and Rousset (1988) Biochim. Biophys. Acta 969, 121-130]. The 50 kDa membrane protein (MP50), initially extracted in very low amount from isolated pig thyroid lysosomes/endosomes, has now been purified from membrane preparations of crude vesicle fractions from pig liver and brain. MP50 was isolated from detergent-solubilized membrane protein by affinity chromatography on immobilized ATP; 3-5 mg of MP50 was obtained from 100 g of liver tissue. Phase partitioning in Triton X-114 indicated that MP50 is a peripheral membrane protein. Radioiodinated liver MP50 bound to microtubules assembled in vitro. The binding was inhibited by ATP (Ki = 0.76 mM) and displaced by unlabelled liver or brain MP50. Equilibrium binding studies yielded KD values of 1.8 x 10(-7) M. By N-terminal amino acid sequence analysis, MP50 was identified as glutamate dehydrogenase (GDH), by comparison of V8 protease peptide maps of MP50 with purified liver GDH. Liver MP50 exhibited a low GDH activity; 4-5 units/mg compared with 18 and 34 units/mg for purified bovine and rat liver GDH respectively. Bovine and rat liver GDH yielded six spots from pI 5.7 to 7.2 when analysed by two-dimensional electrophoresis; in contrast, MP50 gave one main spot (corresponding to spot 2 of liver GDH) with a pI of approx. 6.5. Soluble liver GDH from commercial sources exhibited a very low or no microtubule-binding activity. In conclusion, we have found a membrane-bound form of GDH capable of specific and nucleotide-sensitive interaction with microtubules. Our data suggest that GDH isoproteins, the number of which has been undervalued up to now, could have cellular functions other than that of an enzyme. Images Figure 1 Figure 3

  19. Untangling the glutamate dehydrogenase allosteric nightmare.

    PubMed

    Smith, Thomas J; Stanley, Charles A

    2008-11-01

    Glutamate dehydrogenase (GDH) is found in all living organisms, but only animal GDH is regulated by a large repertoire of metabolites. More than 50 years of research to better understand the mechanism and role of this allosteric network has been frustrated by its sheer complexity. However, recent studies have begun to tease out how and why this complex behavior evolved. Much of GDH regulation probably occurs by controlling a complex ballet of motion necessary for catalytic turnover and has evolved concomitantly with a long antenna-like feature of the structure of the enzyme. Ciliates, the 'missing link' in GDH evolution, might have created the antenna to accommodate changing organelle functions and was refined in humans to, at least in part, link amino acid catabolism with insulin secretion.

  20. A novel glutamate dehydrogenase from bovine brain: purification and characterization.

    PubMed

    Lee, J; Kim, S W; Cho, S W

    1995-08-01

    A soluble form of novel glutamate dehydrogenase has been purified from bovine brain. The preparation was homogeneous on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and composed of six identical subunits having a subunit size of 57,500 Da. The biochemical properties of glutamate dehydrogenase such as N-terminal amino acids sequences, kinetic parameters, amino acids analysis, and optimum pH were examined in both reductive amination of alpha-ketoglutarate and oxidative deamination of glutamate. N-terminal amino acid sequences of the bovine brain enzyme showed the significant differences in the first 5 amino acids compared to other glutamate dehydrogenases from various sources. These results indicate that glutamate dehydrogenase isolated from bovine brain is a novel polypeptide.

  1. Glutamate dehydrogenase from pumpkin cotyledons: characterization and isoenzymes.

    PubMed

    Chou, K H; Splittstoesser, W E

    1972-04-01

    Glutamate dehydrogenase from pumpkin (Cucurbita moschata Pior. cultivar Dickinson Field) cotyledons was found in both soluble and particulate fractions with the bulk of the activity in the soluble fraction. Both enzymes used NAD(H) and NADP(H) but NAD(H) was favored. The enzymes were classified as glutamate-NAD oxidoreductase, deaminating (EC 1.4.1.3). Both enzymes were heat stable, had a pH optimum for reductive amination of 8.0, and were inhibited by high concentrations of NH(4) (+) or alpha-ketoglutarate. The soluble enzyme was more sensitive to NH(4) (+) inhibition and was activated by metal ions after ammonium sulfate fractionation while the solubilized particulate enzyme was not. Inhibition by ethylenediaminetetraacetate was restored by several divalent ions and inhibition by p-hydroxymercuribenzoate was reversed by glutathione. Particulate glutamate dehydrogenase showed a greater activity with NADP. The molecular weights of the enzymes are 250,000. Separation of the enzymes by disc gel electrophoresis showed that during germination the soluble isoenzymes increased from 1 to 7 in number, while only one particulate isoenzyme was found at any time. This particulate isoenzyme was identical with one of the soluble isoenzymes. A number of methods indicated that the soluble isoenzymes were not simply removed from the particulate fraction and that true isoenzymes were found.

  2. Crystal structure of a chimaeric bacterial glutamate dehydrogenase.

    PubMed

    Oliveira, Tânia; Sharkey, Michael A; Engel, Paul C; Khan, Amir R

    2016-06-01

    Glutamate dehydrogenases (EC 1.4.1.2-4) catalyse the oxidative deamination of L-glutamate to α-ketoglutarate using NAD(P)(+) as a cofactor. The bacterial enzymes are hexameric, arranged with 32 symmetry, and each polypeptide consists of an N-terminal substrate-binding segment (domain I) followed by a C-terminal cofactor-binding segment (domain II). The catalytic reaction takes place in the cleft formed at the junction of the two domains. Distinct signature sequences in the nucleotide-binding domain have been linked to the binding of NAD(+) versus NADP(+), but they are not unambiguous predictors of cofactor preference. In the absence of substrate, the two domains move apart as rigid bodies, as shown by the apo structure of glutamate dehydrogenase from Clostridium symbiosum. Here, the crystal structure of a chimaeric clostridial/Escherichia coli enzyme has been determined in the apo state. The enzyme is fully functional and reveals possible determinants of interdomain flexibility at a hinge region following the pivot helix. The enzyme retains the preference for NADP(+) cofactor from the parent E. coli domain II, although there are subtle differences in catalytic activity. PMID:27303899

  3. Correlation of loss of activity of human aldehyde dehydrogenase with reaction of bromoacetophenone with glutamic acid-268 and cysteine-302 residues. Partial-sites reactivity of aldehyde dehydrogenase.

    PubMed Central

    Abriola, D P; MacKerell, A D; Pietruszko, R

    1990-01-01

    Bromoacetophenone (2-bromo-1-phenylethanone) has been characterized as an affinity reagent for human aldehyde dehydrogenase (EC 1.2.1.3) [MacKerell, MacWright & Pietruszko (1986) Biochemistry 25, 5182-5189], and has been shown to react specifically with the Glu-268 residue [Abriola, Fields, Stein, MacKerell & Pietruszko (1987) Biochemistry 26, 5679-5684] with an apparent inactivation stoichiometry of two molecules of bromoacetophenone per molecule of enzyme. The specificity of bromoacetophenone for reaction with Glu-268, however, is not absolute, owing to the extreme reactivity of this reagent. When bromo[14C]acetophenone was used to label the human cytoplasmic E1 isoenzyme radioactively and tryptic fragmentation was carried out, peptides besides that containing Glu-268 were found to have reacted with reagent. These peptides were purified by h.p.l.c. and analysed by sequencing and scintillation counting to quantify radioactive label in the material from each cycle of sequencing. Reaction of bromoacetophenone with the aldehyde dehydrogenase molecule during enzyme activity loss occurs with two residues, Glu-268 and Cys-302. The activity loss, however, appears to be proportional to incorporation of label at Glu-268. The large part of incorporation of label at Cys-302 occurs after the activity loss is essentially complete. With both Glu-268 and Cys-302, however, the incorporation of label stops after one molecule of bromoacetophenone has reacted with each residue. Reaction with other residues continues after activity loss is complete. PMID:1968743

  4. Plasma aspartate aminotransferase (AST), glutamate dehydrogenase (GLDH) and gamma-glutamyl transpeptidase (GGT) activities in water buffaloes with experimental subclinical fasciolosis.

    PubMed

    Yang, Q; Mao, W H; Ferre, I; Bayón, J E; Mao, X Z; González-Gallego, J

    1998-07-31

    The effect of chronic Fasciola hepatica infection on the activity of plasma aspartate aminotransferase (AST), glutamate dehydrogenase (GLDH) and gamma-glutamyl transpeptidase (GGT) was investigated in water buffaloes dosed daily with 60 F. hepatica metacercariae over 20 days. Experimental fluke infection caused no clinical signs but provoked an increase in plasma level of IgG directed against F. hepatica from 4 weeks after infection. There was a significant increase in plasma AST from 6 weeks post-infection. Maximal values were reached at 14 weeks and remained significantly elevated by 23 weeks. Plasma GLDH was significantly elevated from 6 to 21 weeks post-infection. Significant increases in plasma GGT occurred from 8 to 26 weeks post-infection, reaching maximal values at 15 weeks. This study shows that plasma enzyme activities may be useful in studies of fluke-induced liver damage in water buffaloes.

  5. The Isoenzyme 7 of Tobacco NAD(H)-Dependent Glutamate Dehydrogenase Exhibits High Deaminating and Low Aminating Activities in Vivo1[OA

    PubMed Central

    Skopelitis, Damianos S.; Paranychianakis, Nikolaos V.; Kouvarakis, Antonios; Spyros, Apostolis; Stephanou, Euripides G.; Roubelakis-Angelakis, Kalliopi A.

    2007-01-01

    Following the discovery of glutamine synthetase/glutamate (Glu) synthase, the physiological roles of Glu dehydrogenase (GDH) in nitrogen metabolism in plants remain obscure and is the subject of considerable controversy. Recently, transgenics were used to overexpress the gene encoding for the β-subunit polypeptide of GDH, resulting in the GDH-isoenzyme 1 deaminating in vivo Glu. In this work, we present transgenic tobacco (Nicotiana tabacum) plants overexpressing the plant gdh gene encoding for the α-subunit polypeptide of GDH. The levels of transcript correlated well with the levels of total GDH protein, the α-subunit polypeptide, and the abundance of GDH-anionic isoenzymes. Assays of transgenic plant extracts revealed high in vitro aminating and low deaminating activities. However, gas chromatography/mass spectrometry analysis of the metabolic fate of 15NH4 or [15N]Glu revealed that GDH-isoenzyme 7 mostly deaminates Glu and also exhibits low ammonium assimilating activity. These and previous results firmly establish the direction of the reactions catalyzed by the anionic and cationic isoenzymes of GDH in vivo under normal growth conditions and reveal a paradox between the in vitro and in vivo enzyme activities. PMID:17932305

  6. Glutamate dehydrogenase: structure, allosteric regulation, and role in insulin homeostasis.

    PubMed

    Li, Ming; Li, Changhong; Allen, Aron; Stanley, Charles A; Smith, Thomas J

    2014-01-01

    Glutamate dehydrogenase (GDH) is a homohexameric enzyme that catalyzes the reversible oxidative deamination of L-glutamate to 2-oxoglutarate. Only in the animal kingdom is this enzyme heavily allosterically regulated by a wide array of metabolites. The major activators are ADP and leucine and inhibitors include GTP, palmitoyl CoA, and ATP. Spontaneous mutations in the GTP inhibitory site that lead to the hyperinsulinism/hyperammonemia (HHS) syndrome have shed light as to why mammalian GDH is so tightly regulated. Patients with HHS exhibit hypersecretion of insulin upon consumption of protein and concomitantly extremely high levels of ammonium in the serum. The atomic structures of four new inhibitors complexed with GDH complexes have identified three different allosteric binding sites. Using a transgenic mouse model expressing the human HHS form of GDH, at least three of these compounds blocked the dysregulated form of GDH in pancreatic tissue. EGCG from green tea prevented the hyper-response to amino acids in whole animals and improved basal serum glucose levels. The atomic structure of the ECG-GDH complex and mutagenesis studies is directing structure-based drug design using these polyphenols as a base scaffold. In addition, all of these allosteric inhibitors are elucidating the atomic mechanisms of allostery in this complex enzyme.

  7. The structure and allosteric regulation of mammalian glutamate dehydrogenase.

    PubMed

    Li, Ming; Li, Changhong; Allen, Aron; Stanley, Charles A; Smith, Thomas J

    2012-03-15

    Glutamate dehydrogenase (GDH) is a homohexameric enzyme that catalyzes the reversible oxidative deamination of l-glutamate to 2-oxoglutarate. Only in the animal kingdom is this enzyme heavily allosterically regulated by a wide array of metabolites. The major activators are ADP and leucine, while the most important inhibitors include GTP, palmitoyl CoA, and ATP. Recently, spontaneous mutations in the GTP inhibitory site that lead to the hyperinsulinism/hyperammonemia (HHS) syndrome have shed light as to why mammalian GDH is so tightly regulated. Patients with HHS exhibit hypersecretion of insulin upon consumption of protein and concomitantly extremely high levels of ammonium in the serum. The atomic structures of four new inhibitors complexed with GDH complexes have identified three different allosteric binding sites. Using a transgenic mouse model expressing the human HHS form of GDH, at least three of these compounds were found to block the dysregulated form of GDH in pancreatic tissue. EGCG from green tea prevented the hyper-response to amino acids in whole animals and improved basal serum glucose levels. The atomic structure of the ECG-GDH complex and mutagenesis studies is directing structure-based drug design using these polyphenols as a base scaffold. In addition, all of these allosteric inhibitors are elucidating the atomic mechanisms of allostery in this complex enzyme.

  8. Delineation of an in vivo inhibitor for Aspergillus glutamate dehydrogenase.

    PubMed

    Choudhury, Rajarshi; Noor, Shahid; Varadarajalu, Lakshmi Prabha; Punekar, Narayan S

    2008-01-01

    NADP-glutamate dehydrogenase (NADP-GDH) along with glutamine synthetase plays a pivotal role in ammonium assimilation. Specific inhibitors were valuable in defining the importance of glutamine synthetase in nitrogen metabolism. Selective in vivo inhibition of NADP-GDH has so far been an elusive desideratum. Isophthalate, a potent in vitro inhibitor of Aspergillus niger NADP-GDH [Noor S, Punekar NS. Allosteric NADP-glutamate dehydrogenase from aspergilli: purification, characterization and implications for metabolic regulation at the carbon-nitrogen interface. Microbiology 2005;151:1409-19], was evaluated for its efficacy in vivo. Dimethyl ester of isophthalate (DMIP), but not isophthalate, inhibited A. niger growth on agar as well as in liquid culture. This was ascribed to the inability of isophthalate to enter fungal mycelia. Subsequent to DMIP addition however, intracellular isophthalate could be demonstrated. Apart from NAD-GDH, no other enzyme including NAD-glutamate synthase was inhibited by isophthalate. A cross-over at NADP-GDH step of metabolism was observed as a direct consequence of isophthalate (formed in vivo from DMIP) inhibiting this enzyme. Addition of ammonium to DMIP-treated A. niger mycelia resulted in intensive vacuolation, retraction of cytoplasm and autolysis. Taken together, these results implicate glutamate dehydrogenase and NADP-GDH in particular, as a key target of in vivo isophthalate inhibition during ammonium assimilation. PMID:22578865

  9. Use of immobilized glutamate dehydrogenase to synthesize /sup 13/N-labeled L-amino acids

    SciTech Connect

    Cooper, A.J.L.; Gelbard, A.S.

    1981-02-01

    By utilizing glutamate dehydrogenase immobilized onto CNBr-activated Sepharose it is possible to synthesize six L-/sup 13/N-amino acids in high radiochemical yield (5-140 mCi) and in high (> 99%) radiochemical purity. These /sup 13/N-amino acid solutions are potentially suitable for whole body and organ imaging in large animals and man.

  10. Glutamates 78 and 122 in the active site of saccharopine dehydrogenase contribute to reactant binding and modulate the basicity of the acid-base catalysts.

    PubMed

    Ekanayake, Devi K; Andi, Babak; Bobyk, Kostyantyn D; West, Ann H; Cook, Paul F

    2010-07-01

    Saccharopine dehydrogenase catalyzes the NAD-dependent oxidative deamination of saccharopine to give l-lysine and alpha-ketoglutarate. There are a number of conserved hydrophilic, ionizable residues in the active site, all of which must be important to the overall reaction. In an attempt to determine the contribution to binding and rate enhancement of each of the residues in the active site, mutations at each residue are being made, and double mutants are being made to estimate the interrelationship between residues. Here, we report the effects of mutations of active site glutamate residues, Glu(78) and Glu(122), on reactant binding and catalysis. Site-directed mutagenesis was used to generate E78Q, E122Q, E78Q/E122Q, E78A, E122A, and E78A/E122A mutant enzymes. Mutation of these residues increases the positive charge of the active site and is expected to affect the pK(a) values of the catalytic groups. Each mutant enzyme was completely characterized with respect to its kinetic and chemical mechanism. The kinetic mechanism remains the same as that of wild type enzymes for all of the mutant enzymes, with the exception of E78A, which exhibits binding of alpha-ketoglutarate to E and E.NADH. Large changes in V/K(Lys), but not V, suggest that Glu(78) and Glu(122) contribute binding energy for lysine. Shifts of more than a pH unit to higher and lower pH of the pK(a) values observed in the V/K(Lys) pH-rate profile of the mutant enzymes suggests that the presence of Glu(78) and Glu(122) modulates the basicity of the catalytic groups.

  11. Bacterial cell-surface displaying of thermo-tolerant glutamate dehydrogenase and its application in L-glutamate assay.

    PubMed

    Song, Jianxia; Liang, Bo; Han, Dongfei; Tang, Xiangjiang; Lang, Qiaolin; Feng, Ruirui; Han, Lihui; Liu, Aihua

    2015-03-01

    In this paper, glutamate dehydrogenase (Gldh) is reported to efficiently display on Escherichia coli cell surface by using N-terminal region of ice the nucleation protein as an anchoring motif. The presence of Gldh was confirmed by SDS-PAGE and enzyme activity assay. Gldh was detected mainly in the outer membrane fraction, suggesting that the Gldh was displayed on the bacterial cell surface. The optimal temperature and pH for the bacteria cell-surface displayed Gldh (bacteria-Gldh) were 70°C and 9.0, respectively. Additionally, the fusion protein retained almost 100% of its initial enzymatic activity after 1 month incubation at 4°C. Transition metal ions could inhibit the enzyme activity to different extents, while common anions had little adverse effect on enzyme activity. Importantly, the displayed Gldh is most specific to l-glutamate reported so far. The bacterial Gldh was enabled to catalyze oxidization of l-glutamate with NADP(+) as cofactor, and the resultant NADPH can be detected spectrometrically at 340nm. The bacterial-Gldh based l-glutamate assay was established, where the absorbance at 340nm increased linearly with the increasing l-glutamate concentration within the range of 10-400μM. Further, the proposed approach was successfully applied to measure l-glutamate in real samples. PMID:25659635

  12. Demonstration of glutamate dehydrogenase isozymes in beef heart mitochondria.

    PubMed

    McDaniel, H; Bosing-Schneider, R; Jenkins, R; Rasched, I; Sund, H

    1986-01-15

    Glutamate dehydrogenase (GDH) has been purified from beef heart mitochondria and compared with crystalline beef liver GDH. The specific activity of heart GDH was 127 units and of liver GDH 80 units. Heart GDH subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis had a protein corresponding to liver GDH and a smaller molecular weight protein. On agarose gel electrophoresis heart GDH activity was resolved into two fractions (with or without protease inhibitors) while liver had only one fraction. One of the heart fractions moved with liver GDH on electrophoresis. Thermal stability studies showed heart and liver GDH activity differed. Mouse antibodies to liver GDH precipitated both liver and heart GDH on double immunodiffusion. Mouse antibodies to liver GDH identified on nitrocellulose paper the polypeptide band of liver and heart GDH that were the same molecular weight but did not cross-react with the smaller molecular weight polypeptide present in heart GDH. Trypsin digestion of the two major protein bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis of purified GDH from beef heart mitochondria did not show any overlapping peptides. We conclude beef heart GDH activity is composed of two isozymes. One is the same as beef liver GDH, and the other is a smaller molecular weight protein. We propose the terms GDH-LM for the liver GDH isozyme and GDH-HM for the smaller molecular weight isozyme present in heart mitochondria but not liver.

  13. Glutamate dehydrogenases: the why and how of coenzyme specificity.

    PubMed

    Engel, Paul C

    2014-01-01

    NAD(+) and NADP(+), chemically similar and with almost identical standard oxidation-reduction potentials, nevertheless have distinct roles, NAD(+) serving catabolism and ATP generation whereas NADPH is the biosynthetic reductant. Separating these roles requires strict specificity for one or the other coenzyme for most dehydrogenases. In many organisms this holds also for glutamate dehydrogenases (GDH), NAD(+)-dependent for glutamate oxidation, NADP(+)-dependent for fixing ammonia. In higher animals, however, GDH has dual specificity. It has been suggested that GDH in mitochondria reacts only with NADP(H), the NAD(+) reaction being an in vitro artefact. However, contrary evidence suggests mitochondrial GDH not only reacts with NAD(+) but maintains equilibrium using the same pool as accessed by β-hydroxybutyrate dehydrogenase. Another complication is the presence of an energy-linked dehydrogenase driving NADP(+) reduction by NADH, maintaining the coenzyme pools at different oxidation-reduction potentials. Its coexistence with GDH makes possible a futile cycle, control of which is not yet properly explained. Structural studies show NAD(+)-dependent, NADP(+)-dependent and dual-specificity GDHs are closely related and a few site-directed mutations can reverse specificity. Specificity for NAD(+) or for NADP(+) has probably emerged repeatedly during evolution, using different structural solutions on different occasions. In various GDHs the P7 position in the coenzyme-binding domain plays a key role. However, whereas in other dehydrogenases an acidic P7 residue usually hydrogen bonds to the 2'- and 3'-hydroxyls, dictating NAD(+) specificity, among GDHs, depending on detailed conformation of surrounding residues, an acidic P7 may permit binding of NAD(+) only, NADP(+) only, or in higher animals both.

  14. Butachlor impact on protein, free amino acid and glutamine contents, and on activity levels of aminotransferases, glutamate dehydrogenase and glutamine synthetase in the fresh water snail, Pila globosa (Swainson).

    PubMed

    Rajyalakshmi, T; Srinivas, T; Swamy, K V; Mohan, P M

    1996-08-01

    Biochemical changes followed in the freshwater snail Pila globosa (Swainson) during exposure to sublethal concentrations of the herbicide butachlor (26.6 ppm) in the ambient medium, at 3,6,12,24 and 48 h intervals, were marked by a significant decrease in total and soluble proteins, and an increase in free amino acids in foot and hepatopancreas up to 12 h before gradually recovering. Aminotransferase activities and glutamine content decreased during the early periods of exposure, while glutamate dehydrogenase activity increased. After an initial elevation, glutamate synthetase activity decreased at later intervals. Maximum effect of butachlor on the enzymes was seen after 12 h exposure. The extent of increase or decrease in different parameters examined varied between the two tissues studied. These changes are discussed in relation to the toxic stress of butachlor.

  15. Isocitrate dehydrogenases and oxoglutarate dehydrogenase activities of baker's yeast grown in a variety of hypoxic conditions.

    PubMed

    Machado, A; Nuñez de Castro, I; Mayor, F

    1975-02-28

    The activities of isocitrate dehydrogenase (NAD), isocitrate dehydrogenase (NADP) and oxoglutarate dehydrogenase have been investigated in Saccharomyces cerevisiae grown in a variety of aerobic and hypoxic conditions, the latter including oxygen deprivation, high glucose concentration, addition of inhibitors of mitochondrial protein synthesis, respiratory inhibition by azide, and impaired respiration mutants. All hypoxic conditions led to a marked decrease of oxoglutarate dehydrogenase and significant decreases of the two isocitrate dehydrogenases. According to its kinetic properties, the NAD-isocitrate dehydrogenase will not be operative in hypoxia "in vivo". From these and other related facts it is concluded that hypoxic conditions in yeast generally lead to a splitting of the tricarboxylic acid cycle and that glutamate synthesis in these conditions takes place through the coupling of the NADP-linked isocitrate and glutamate dehydrogenases.

  16. Amperometric L-glutamate biosensor based on bacterial cell-surface displayed glutamate dehydrogenase.

    PubMed

    Liang, Bo; Zhang, Shu; Lang, Qiaolin; Song, Jianxia; Han, Lihui; Liu, Aihua

    2015-07-16

    A novel L-glutamate biosensor was fabricated using bacteria surface-displayed glutamate dehydrogenase (Gldh-bacteria). Here the cofactor NADP(+)-specific dependent Gldh was expressed on the surface of Escherichia coli using N-terminal region of ice nucleation protein (INP) as the anchoring motif. The cell fractionation assay and SDS-PAGE analysis indicated that the majority of INP-Gldh fusion proteins were located on the surface of cells. The biosensor was fabricated by successively casting polyethyleneimine (PEI)-dispersed multi-walled carbon nanotubes (MWNTs), Gldh-bacteria and Nafion onto the glassy carbon electrode (Nafion/Gldh-bacteria/PEI-MWNTs/GCE). The MWNTs could not only significantly lower the oxidation overpotential towards NAPDH, which was the product of NADP(+) involving in the oxidation of glutamate by Gldh, but also enhanced the current response. Under the optimized experimental conditions, the current-time curve of the Nafion/Gldh-bacteria/PEI-MWNTs/GCE was performed at +0.52 V (vs. SCE) by amperometry varying glutamate concentration. The current response was linear with glutamate concentration in two ranges (10 μM-1 mM and 2-10 mM). The low limit of detection was estimated to be 2 μM glutamate (S/N=3). Moreover, the proposed biosensor is stable, specific, reproducible and simple, which can be applied to real samples detection.

  17. Amperometric L-glutamate biosensor based on bacterial cell-surface displayed glutamate dehydrogenase.

    PubMed

    Liang, Bo; Zhang, Shu; Lang, Qiaolin; Song, Jianxia; Han, Lihui; Liu, Aihua

    2015-07-16

    A novel L-glutamate biosensor was fabricated using bacteria surface-displayed glutamate dehydrogenase (Gldh-bacteria). Here the cofactor NADP(+)-specific dependent Gldh was expressed on the surface of Escherichia coli using N-terminal region of ice nucleation protein (INP) as the anchoring motif. The cell fractionation assay and SDS-PAGE analysis indicated that the majority of INP-Gldh fusion proteins were located on the surface of cells. The biosensor was fabricated by successively casting polyethyleneimine (PEI)-dispersed multi-walled carbon nanotubes (MWNTs), Gldh-bacteria and Nafion onto the glassy carbon electrode (Nafion/Gldh-bacteria/PEI-MWNTs/GCE). The MWNTs could not only significantly lower the oxidation overpotential towards NAPDH, which was the product of NADP(+) involving in the oxidation of glutamate by Gldh, but also enhanced the current response. Under the optimized experimental conditions, the current-time curve of the Nafion/Gldh-bacteria/PEI-MWNTs/GCE was performed at +0.52 V (vs. SCE) by amperometry varying glutamate concentration. The current response was linear with glutamate concentration in two ranges (10 μM-1 mM and 2-10 mM). The low limit of detection was estimated to be 2 μM glutamate (S/N=3). Moreover, the proposed biosensor is stable, specific, reproducible and simple, which can be applied to real samples detection. PMID:26073813

  18. Evolution of glutamate dehydrogenase regulation of insulin homeostasis is an example of molecular exaptation.

    PubMed

    Allen, Aron; Kwagh, Jae; Fang, Jie; Stanley, Charles A; Smith, Thomas J

    2004-11-16

    Glutamate dehydrogenase (GDH) is found in all organisms and catalyzes the oxidative deamination of glutamate to 2-oxoglutarate. While this enzyme does not exhibit allosteric regulation in plants, bacteria, or fungi, its activity is tightly controlled by a number of compounds in mammals. We have previously shown that this regulation plays an important role in insulin homeostasis in humans and evolved concomitantly with a 48-residue "antenna" structure. As shown here, the antenna and some of the allosteric regulation first appears in the Ciliates. This primitive regulation is mediated by fatty acids and likely reflects the gradual movement of fatty acid oxidation from the peroxisomes to the mitochondria as the Ciliates evolved away from plants, fungi, and other protists. Mutagenesis studies where the antenna is deleted support this contention by demonstrating that the antenna is essential for fatty acid regulation. When the antenna from the Ciliates is spliced onto human GDH, it was found to fully communicate all aspects of mammalian regulation. Therefore, we propose that glutamate dehydrogenase regulation of insulin secretion is a example of exaptation at the molecular level where the antenna and associated fatty acid regulation was created to accommodate the changes in organelle function in the Ciliates and then later used to link amino acid catabolism and/or regulation of intracellular glutamate/glutamine levels in the pancreatic beta cells with insulin homeostasis in mammals.

  19. Cellular and subcellular localization of hexokinase, glutamate dehydrogenase, and alanine aminotransferase in the honeybee drone retina.

    PubMed

    Veuthey, A L; Tsacopoulos, M; Millan de Ruiz, L; Perrottet, P

    1994-05-01

    Subcellular localization of hexokinase in the honeybee drone retina was examined following fractionation of cell homogenate using differential centrifugation. Nearly all hexokinase activity was found in the cytosolic fraction, following a similar distribution as the cytosolic enzymatic marker, phosphoglycerate kinase. The distribution of enzymatic markers of mitochondria (succinate dehydrogenase, rotenone-insensitive cytochrome c reductase, and adenylate kinase) indicated that the outer mitochondrial membrane was partly damaged, but their distributions were different from that of hexokinase. The activity of hexokinase in purified suspensions of cells was fivefold higher in glial cells than in photoreceptors. This result is consistent with the hypothesis based on quantitative 2-deoxy[3H]glucose autoradiography that only glial cells phosphorylate significant amounts of glucose to glucose-6-phosphate. The activities of alanine aminotransferase and to a lesser extent of glutamate dehydrogenase were higher in the cytosolic than in the mitochondrial fraction. This important cytosolic activity of glutamate dehydrogenase was consistent with the higher activity found in mitochondria-poor glial cells. In conclusion, this distribution of enzymes is consistent with the model of metabolic interactions between glial and photoreceptor cells in the intact bee retina. PMID:8158142

  20. Cellular and subcellular localization of hexokinase, glutamate dehydrogenase, and alanine aminotransferase in the honeybee drone retina.

    PubMed

    Veuthey, A L; Tsacopoulos, M; Millan de Ruiz, L; Perrottet, P

    1994-05-01

    Subcellular localization of hexokinase in the honeybee drone retina was examined following fractionation of cell homogenate using differential centrifugation. Nearly all hexokinase activity was found in the cytosolic fraction, following a similar distribution as the cytosolic enzymatic marker, phosphoglycerate kinase. The distribution of enzymatic markers of mitochondria (succinate dehydrogenase, rotenone-insensitive cytochrome c reductase, and adenylate kinase) indicated that the outer mitochondrial membrane was partly damaged, but their distributions were different from that of hexokinase. The activity of hexokinase in purified suspensions of cells was fivefold higher in glial cells than in photoreceptors. This result is consistent with the hypothesis based on quantitative 2-deoxy[3H]glucose autoradiography that only glial cells phosphorylate significant amounts of glucose to glucose-6-phosphate. The activities of alanine aminotransferase and to a lesser extent of glutamate dehydrogenase were higher in the cytosolic than in the mitochondrial fraction. This important cytosolic activity of glutamate dehydrogenase was consistent with the higher activity found in mitochondria-poor glial cells. In conclusion, this distribution of enzymes is consistent with the model of metabolic interactions between glial and photoreceptor cells in the intact bee retina.

  1. The structure and allosteric regulation of glutamate dehydrogenase.

    PubMed

    Li, Ming; Li, Changhong; Allen, Aron; Stanley, Charles A; Smith, Thomas J

    2011-09-01

    Glutamate dehydrogenase (GDH) has been extensively studied for more than 50 years. Of particular interest is the fact that, while considered by most to be a 'housekeeping' enzyme, the animal form of GDH is heavily regulated by a wide array of allosteric effectors and exhibits extensive inter-subunit communication. While the chemical mechanism for GDH has remained unchanged through epochs of evolution, it was not clear how or why animals needed to evolve such a finely tuned form of this enzyme. As reviewed here, recent studies have begun to elucidate these issues. Allosteric regulation first appears in the Ciliates and may have arisen to accommodate evolutionary changes in organelle function. The occurrence of allosteric regulation appears to be coincident with the formation of an 'antenna' like feature rising off the tops of the subunits that may be necessary to facilitate regulation. In animals, this regulation further evolved as GDH became integrated into a number of other regulatory pathways. In particular, mutations in GDH that abrogate GTP inhibition result in dangerously high serum levels of insulin and ammonium. Therefore, allosteric regulation of GDH plays an important role in insulin homeostasis. Finally, several compounds have been identified that block GDH-mediated insulin secretion that may be to not only find use in treating these insulin disorders but to kill tumors that require glutamine metabolism for cellular energy.

  2. Metabolic control of cell division in α-proteobacteria by a NAD-dependent glutamate dehydrogenase.

    PubMed

    Beaufay, François; De Bolle, Xavier; Hallez, Régis

    2016-01-01

    Prior to initiate energy-consuming processes, such as DNA replication or cell division, cells need to evaluate their metabolic status. We have recently identified and characterized a new connection between metabolism and cell division in the α-proteobacterium Caulobacter crescentus. We showed that an NAD-dependent glutamate dehydrogenase (GdhZ) coordinates growth with cell division according to its enzymatic activity. Here we report the conserved role of GdhZ in controlling cell division in another α-proteobacterium, the facultative intracellular pathogen Brucella abortus. We also discuss the importance of amino acids as a main carbon source for α-proteobacteria.

  3. Glutamate 636 of the Escherichia coli pyruvate dehydrogenase-E1 participates in active center communication and behaves as an engineered acetolactate synthase with unusual stereoselectivity.

    PubMed

    Nemeria, Natalia; Tittmann, Kai; Joseph, Ebenezer; Zhou, Leon; Vazquez-Coll, Michelle B; Arjunan, Palaniappa; Hübner, Gerhard; Furey, William; Jordan, Frank

    2005-06-01

    The residue Glu636 is located near the thiamine diphosphate (ThDP) binding site of the Escherichia coli pyruvate dehydrogenase complex E1 subunit (PDHc-E1), and to probe its function two variants, E636A and E636Q were created with specific activities of 2.5 and 26% compared with parental PDHc-E1. According to both fluorescence binding and kinetic assays, the E636A variant behaved according to half-of-the-sites mechanism with respect to ThDP. In contrast, with the E636Q variant a K(d,ThDP) = 4.34 microM and K(m,ThDP) = 11 microM were obtained with behavior more reminiscent of the parental enzyme. The CD spectra of both variants gave evidence for formation of the 1',4'-iminopyrimidine tautomer on binding of phosphonolactylthiamine diphosphate, a stable analog of the substrate-ThDP covalent complex. Rapid formation of optically active (R)-acetolactate by both variants, but not by the parental enzyme, was observed by CD and NMR spectroscopy. The acetolactate configuration produced by the Glu636 variants is opposite that produced by the enzyme acetolactate synthase and the Asp28-substituted variants of yeast pyruvate decarboxylase, suggesting that the active centers of the two sets of enzymes exhibit different facial selectivity (re or si) vis à vis pyruvate. The tryptic peptide map (mass spectral analysis) revealed that the Glu636 substitution changed the mobility of a loop comprising amino acid residues from the ThDP binding fold. Apparently, the residue Glu636 has important functions both in active center communication and in protecting the active center from undesirable "carboligase" side reactions.

  4. Purification and properties of NADP-dependent glutamate dehydrogenase from Ruminococcus flavefaciens FD-1.

    PubMed Central

    Duncan, P A; White, B A; Mackie, R I

    1992-01-01

    Glutamate dehydrogenase (GDH) (L-glutamate:NADP+ oxidoreductase, deaminating, EC 1.4.1.4) from the cellulolytic ruminal bacterium Ruminococcus flavefaciens has been purified and characterized. The native enzyme and subunit are 280 and 48 kDa, respectively, suggesting that the native enzyme is a hexamer. The enzyme requires 0.5 M KCl for optimal activity and has a pH optimum of 6.9 to 7.0. The Kms for ammonia, alpha-ketoglutarate, and glutamate are 19, 0.41, and 62 mM, respectively. The sigmoidal NADPH saturation curve revealed positive cooperativity for the binding of this coenzyme. The first residue in the N-terminal amino acid sequence from R. flavefaciens GDH was alanine, suggesting that the protein may be modified posttranslationally. Comparison of the N-terminal sequence with those of Escherichia coli, Salmonella typhimurium, and Clostridium symbiosum revealed only 39% amino acid homologies. The GDH from R. flavefaciens was unique in that its specific activity was highest during ammonia-limited growth but was not affected by ammonia shock treatment (20 mM). Images PMID:1335719

  5. Modular coenzyme specificity: a domain-swopped chimera of glutamate dehydrogenase.

    PubMed

    Sharkey, Michael A; Engel, Paul C

    2009-11-01

    Domain-swopped chimeras of the glutamate dehydrogenases from Clostridium symbiosum (CsGDH) (NAD(+)-specific) and Escherichia coli (EcGDH) (NADP(+)-specific) have been produced, with the aim of testing the localization of determinants of coenzyme specificity. An active chimera consisting of the substrate-binding domain (Domain I) of CsGDH and the coenzyme-binding domain (Domain II) of EcGDH has been purified to homogeneity, and a thorough kinetic analysis has been carried out. Results indicate that selectivity for the phosphorylated coenzyme does indeed reside solely in Domain II; the chimera utilizes NAD(+) at 0.8% of the rate observed with NADP(+), similar to the 0.5% ratio for EcGDH. Positive cooperativity toward L-glutamate, characteristic of CsGDH, has been retained with Domain I. An unforeseen feature of this chimera, however, is that, although glutamate cooperativity occurs only at higher pH values in the parent CsGDH, the chimeric protein shows it over the full pH range explored. Also surprising is that the chimera is capable of catalysing severalfold higher reaction rates (V(max)) in both directions than either of the parent enzymes from which it is constructed.

  6. Characterization of a pyridine nucleotide-nonspecific glutamate dehydrogenase from Bacteroides thetaiotaomicron.

    PubMed Central

    Glass, T L; Hylemon, P B

    1980-01-01

    An oxidized nicotinamide adenine dinucleotide phosphate/oxidized nicotinamide adenine dinucleotide (NADP+/NAD+) nonspecific L-glutamate dehydrogenase from Bacteroides thetaiotaomicron was purified 40-fold (NADP+ or NAD+ activity) over crude cell extract by heat treatment, (NH4)2SO2 fractionation, diethylaminoethyl-cellulose, Bio-Gel A 1.5m, and hydroxylapatite chromatography. Both NADP+- and NAD+-dependent activities coeluted from all chromatographic treatments. Moreover, a constant ratio of NADP+/NAD+ specific activities was demonstrated at each purification step. Both activities also comigrated in 6% nondenaturing polyacrylamide gels. Affinity chromatography of the 40-fold-purified enzyme using Procion RED HE-3B gave a preparation containing both NADP+- and NAD+-linked activities which showed a single protein band of 48,5000 molecular weight after sodium dodecyl sulfate-polyacrylamide gradient gel electrophoresis. The dual pyridine nucleotide nature of the enzyme was most readily apparent in the oxidative direction. Reductively, the enzyme was 30-fold more active with reduced NADP than with reduced NAD. Nonlinear concave 1/V versus 1/S plots were observed for reduced NADP and NH4Cl. Salts (0.1 M) stimulated the NADP+-linked reaction, inhibited the NAD+-linked reaction, and had little effect on the reduced NADP-dependent reaction. The stimulatory effect of salts (NADP+) was nonspecific, regardless of the anion or cation, whereas the degree of NAD+-linked inhibition decreased in the order to I- greater than Br- greater than Cl- greater than F-. Both NADP+ and NAD+ glutamate dehydrogenase activities were also detected in cell extracts from representative strains of other bacteroides deoxyribonucleic acid homology groups. Images PMID:7364728

  7. Mediator-less highly sensitive voltammetric detection of glutamate using glutamate dehydrogenase/vertically aligned CNTs grown on silicon substrate.

    PubMed

    Gholizadeh, Azam; Shahrokhian, Saeed; zad, Azam Iraji; Mohajerzadeh, Shamsoddin; Vosoughi, Manouchehr; Darbari, Sara; Sanaee, Zeinab

    2012-01-15

    A sensitive glutamate biosensor is prepared based on glutamate dehydrogenase/vertically aligned carbon nanotubes (GLDH, VACNTs). Vertically aligned carbon nanotubes were grown on a silicon substrate by direct current plasma enhanced chemical vapor deposition (DC-PECVD) method. The electrochemical behavior of the synthesized VACNTs was investigated by cyclic voltammetry and electrochemical impedance spectroscopic methods. Glutamate dehydrogenase covalently attached on tip of VACNTs. The electrochemical performance of the electrode for detection of glutamate was investigated by cyclic and differential pulse voltammetry. Differential pulse voltammetric determinations of glutamate are performed in mediator-less condition and also, in the presence of 1 and 5 μM thionine as electron mediator. The linear calibration curve of the concentration of glutamate versus peak current is investigated in a wide range of 0.1-500 μM. The mediator-less biosensor has a low detection limit of 57 nM and two linear ranges of 0.1-20 μM with a sensitivity of 0.976 mA mM(-1) cm(-2) and 20-300 μM with a sensitivity of 0.182 mA mM(-1) cm(-2). In the presence of 1 μM thionine as an electron mediator, the prepared biosensor shows a low detection limit of 68 nM and two linear ranges of 0.1-20 with a calibration sensitivity of 1.17 mA mM(-1) cm(-2) and 20-500 μM with a sensitivity of 0.153 mA mM(-1) cm(-2). The effects of the other biological compounds on the voltammetric behavior of the prepared biosensor and its response stability are investigated. The results are demonstrated that the GLDH/VACNTs electrode even without electron mediator is a suitable basic electrode for detection of glutamate. PMID:22040749

  8. A NAD-dependent glutamate dehydrogenase coordinates metabolism with cell division in Caulobacter crescentus.

    PubMed

    Beaufay, François; Coppine, Jérôme; Mayard, Aurélie; Laloux, Géraldine; De Bolle, Xavier; Hallez, Régis

    2015-07-01

    Coupling cell cycle with nutrient availability is a crucial process for all living cells. But how bacteria control cell division according to metabolic supplies remains poorly understood. Here, we describe a molecular mechanism that coordinates central metabolism with cell division in the α-proteobacterium Caulobacter crescentus. This mechanism involves the NAD-dependent glutamate dehydrogenase GdhZ and the oxidoreductase-like KidO. While enzymatically active GdhZ directly interferes with FtsZ polymerization by stimulating its GTPase activity, KidO bound to NADH destabilizes lateral interactions between FtsZ protofilaments. Both GdhZ and KidO share the same regulatory network to concomitantly stimulate the rapid disassembly of the Z-ring, necessary for the subsequent release of progeny cells. Thus, this mechanism illustrates how proteins initially dedicated to metabolism coordinate cell cycle progression with nutrient availability.

  9. A NAD-dependent glutamate dehydrogenase coordinates metabolism with cell division in Caulobacter crescentus

    PubMed Central

    Beaufay, François; Coppine, Jérôme; Mayard, Aurélie; Laloux, Géraldine; De Bolle, Xavier; Hallez, Régis

    2015-01-01

    Coupling cell cycle with nutrient availability is a crucial process for all living cells. But how bacteria control cell division according to metabolic supplies remains poorly understood. Here, we describe a molecular mechanism that coordinates central metabolism with cell division in the α-proteobacterium Caulobacter crescentus. This mechanism involves the NAD-dependent glutamate dehydrogenase GdhZ and the oxidoreductase-like KidO. While enzymatically active GdhZ directly interferes with FtsZ polymerization by stimulating its GTPase activity, KidO bound to NADH destabilizes lateral interactions between FtsZ protofilaments. Both GdhZ and KidO share the same regulatory network to concomitantly stimulate the rapid disassembly of the Z-ring, necessary for the subsequent release of progeny cells. Thus, this mechanism illustrates how proteins initially dedicated to metabolism coordinate cell cycle progression with nutrient availability. PMID:25953831

  10. Structure of NADP+-dependent glutamate dehydrogenase from Escherichia coli: Reflections on the basis of coenzyme specificity in the family of glutamate dehydrogenases

    PubMed Central

    Sharkey, Michael A.; Oliveira, Tânia F.; Engel, Paul C.; Khan, Amir R.

    2013-01-01

    Summary Glutamate dehydrogenases (EC 1.4.1.2–4) catalyse the oxidative deamination of l-glutamate to α-ketoglutarate using NAD+ and/or NADP+ as a cofactor. Subunits of homo-hexameric bacterial enzymes comprise a substrate-binding Domain I followed by a nucleotide binding Domain II. The reaction occurs in a catalytic cleft between the two domains. Although conserved residues in the nucleotide-binding domains of various dehydrogenases have been linked to cofactor preferences, the structural basis for specificity in the glutamate dehydrogenase (GDH) family remains poorly understood. Here, the refined crystal structure of Escherichia coli GDH in the absence of reactants is described at 2.5Å resolution. Modelling of NADP+ in Domain II reveals the potential contribution of positively charged residues from a neighbouring α-helical hairpin to phosphate recognition. In addition, a serine residue that follows the P7 aspartate is presumed to form a hydrogen bond to the 2’-phosphate. Mutagenesis and kinetic analysis confirms the importance of these residues in NADP+ recognition. Surprisingly, one of the positively charged residues is conserved in all sequences of NAD+ dependent enzymes, but the conformations adopted by the corresponding regions in proteins whose structure has been solved preclude their contribution toward the co-ordination of the 2’-ribose phosphate of NADP+. These studies clarify the sequence/structure relationships in bacterial glutamate dehydrogenases, revealing that identical residues may specify different coenzyme preferences, depending on the structural context. Primary sequence alone is therefore not a reliable guide for predicting coenzyme specificity. We also consider how it is possible for a single sequence to accommodate both coenzymes in the dual specificity GDHs of animals. PMID:23879525

  11. Kinetic studies to determine the mechanism of regulation of bovine liver glutamate dehydrogenase by nucleotide effectors.

    PubMed

    Cook, P F

    1982-01-01

    A combination of kinetic and isotope effect studies in the presence and absence of the effectors ADP and GTP was used to elucidate the mechanism of regulation of bovine liver glutamate dehydrogenase. ADP at low concentrations of glutamate competes with TPN for free enzyme. GTP exhibits a similar effect at high concentrations (100 microM and above). When ADP binds at its allosteric site, it increases the off rates of both alpha-ketoglutarate and TPNH from their product complexes. This results in a decrease in V/K for both substrates, an increase in V, and an increase in the deuterium isotope effects for all three parameters so that they are all about 1.3. The rate of release of glutamate from E-TPNH-glutamate is also apparently enhanced since no substrate inhibition by glutamate is observed in the presence of ADP. The effect of GTP is in opposition to that of ADP in that GTP decreases the off rates for both TPN and glutamate from E-TPN-glutamate as well as the off rates for alpha-ketoglutarate and TPNH. This results in an increase in the V/K's for both substrates, a decrease in V, and a decrease in the deuterium isotope effects for all three parameters to a value of 1. Substrate inhibition by glutamate is also eliminated by GTP probably by preventing any significant accumulation of E-TPNH to which glutamate binds as an inhibitor.

  12. The structure of apo human glutamate dehydrogenase details subunit communication and allostery.

    PubMed

    Smith, Thomas J; Schmidt, Timothy; Fang, Jie; Wu, Jane; Siuzdak, Gary; Stanley, Charles A

    2002-05-01

    The structure of human glutamate dehydrogenase (GDH) has been determined in the absence of active site and regulatory ligands. Compared to the structures of bovine GDH that were complexed with coenzyme and substrate, the NAD binding domain is rotated away from the glutamate-binding domain. The electron density of this domain is more disordered the further it is from the pivot helix. Mass spectrometry results suggest that this is likely due to the apo form being more dynamic than the closed form. The antenna undergoes significant conformational changes as the catalytic cleft opens. The ascending helix in the antenna moves in a clockwise manner and the helix in the descending strand contracts in a manner akin to the relaxation of an extended spring. A number of spontaneous mutations in this antenna region cause the hyperinsulinism/hyperammonemia syndrome by decreasing GDH sensitivity to the inhibitor, GTP. Since these residues do not directly contact the bound GTP, the conformational changes in the antenna are apparently crucial to GTP inhibition. In the open conformation, the GTP binding site is distorted such that it can no longer bind GTP. In contrast, ADP binding benefits by the opening of the catalytic cleft since R463 on the pivot helix is pushed into contact distance with the beta-phosphate of ADP. These results support the previous proposal that purines regulate GDH activity by altering the dynamics of the NAD binding domain. Finally, a possible structural mechanism for negative cooperativity is presented.

  13. The simultaneous determination of NAD(H) and NADP(H) utilization by glutamate dehydrogenase.

    PubMed

    Treberg, Jason R; Brosnan, Margaret E; Brosnan, John T

    2010-11-01

    Glutamate dehydrogenase (GDH) from vertebrates is unusual among NAD(P)H-dependent dehydrogenases in that it can use either NAD(H) or NADP(H) as cofactor. In this study, we measure the rate of cofactor utilization by bovine GDH when both cofactors are present. Methods for both reaction directions were developed, and for the first time, to our knowledge, the GDH activity has been simultaneously studied in the presence of both NAD(H) and NADP(H). Our data indicate that NADP(H) has inhibitory effects on the rate of NAD(H) utilization by GDH, a characteristic of GDH not previously recognized. The response of GDH to allosteric activators in the presence of NAD(H) and NADP(H) suggests that ADP and leucine moderate much of the inhibitory effect of NADP(H) on the utilization of NAD(H). These results illustrate that simple assumptions of cofactor preference by mammalian GDH are incomplete without an appreciation of allosteric effects when both cofactors are simultaneously present.

  14. Interaction between glutamate dehydrogenase (GDH) and L-leucine catabolic enzymes: intersecting metabolic pathways.

    PubMed

    Hutson, Susan M; Islam, Mohammad Mainul; Zaganas, Ioannis

    2011-09-01

    Branched-chain amino acids (BCAAs) catabolism follows sequential reactions and their metabolites intersect with other metabolic pathways. The initial enzymes in BCAA metabolism, the mitochondrial branched-chain aminotransferase (BCATm), which deaminates the BCAAs to branched-chain α-keto acids (BCKAs); and the branched-chain α-keto acid dehydrogenase enzyme complex (BCKDC), which oxidatively decarboxylates the BCKAs, are organized in a supramolecular complex termed metabolon. Glutamate dehydrogenase (GDH1) is found in the metabolon in rat tissues. Bovine GDH1 binds to the pyridoxamine 5'-phosphate (PMP)-form of human BCATm (PMP-BCATm) but not to pyridoxal 5'-phosphate (PLP)-BCATm in vitro. This protein interaction facilitates reamination of the α-ketoglutarate (αKG) product of the GDH1 oxidative deamination reaction. Human GDH1 appears to act like bovine GDH1 but human GDH2 does not show the same enhancement of BCKDC enzyme activities. Another metabolic enzyme is also found in the metabolon is pyruvate carboxylase (PC). Kinetic results suggest that PC binds to the E1 decarboxylase of BCKDC but does not effect BCAA catabolism. The protein interaction of BCATm and GDH1 promotes regeneration of PLP-BCATm which then binds to BCKDC resulting in channeling of the BCKA products from BCATm first half reaction to E1 and promoting BCAA oxidation and net nitrogen transfer from BCAAs. The cycling of nitrogen through glutamate via the actions of BCATm and GDH1 releases free ammonia. Formation of ammonia may be important for astrocyte glutamine synthesis in the central nervous system. In peripheral tissue association of BCATm and GDH1 would promote BCAA oxidation at physiologically relevant BCAA concentrations. PMID:21621574

  15. Structural insights into domain movement and cofactor specificity of glutamate dehydrogenase from Corynebacterium glutamicum.

    PubMed

    Son, Hyeoncheol Francis; Kim, Il-Kwon; Kim, Kyung-Jin

    2015-04-10

    Glutamate dehydrogenase (GDH) is an enzyme involved in the synthesis of amino acids by converting glutamate to α-ketoglutarate, and vice versa. To investigate the molecular mechanism of GDH, we determined a crystal structure of the Corynebacterium glutamicum-derived GDH (CgGDH) in complex with its NADP cofactor and α-ketoglutarate substrate. CgGDH functions as a hexamer, and each CgGDH monomer comprises 2 separate domains; a Rossmann fold cofactor-binding domain and a substrate-binding domain. The structural comparison between the apo- and cofactor/substrate-binding forms revealed that the CgGDH enzyme undergoes a domain movement during catalysis. In the apo-form, CgGDH exists as an open state, and upon binding of the substrate and cofactor the protein undergoes a conformation change to a closed state. Our structural study also revealed that CgGDH has cofactor specificity for NADP, but not NAD, and this was confirmed by GDH activity measurements. Residues involved in the stabilization of the NADP cofactor and the α-ketoglutarate substrate were identified, and their roles in substrate/cofactor binding were confirmed by site-directed mutagenesis experiments.

  16. Using Cryo-EM to Map Small Ligands on Dynamic Metabolic Enzymes: Studies with Glutamate Dehydrogenase.

    PubMed

    Borgnia, Mario J; Banerjee, Soojay; Merk, Alan; Matthies, Doreen; Bartesaghi, Alberto; Rao, Prashant; Pierson, Jason; Earl, Lesley A; Falconieri, Veronica; Subramaniam, Sriram; Milne, Jacqueline L S

    2016-06-01

    Cryo-electron microscopy (cryo-EM) methods are now being used to determine structures at near-atomic resolution and have great promise in molecular pharmacology, especially in the context of mapping the binding of small-molecule ligands to protein complexes that display conformational flexibility. We illustrate this here using glutamate dehydrogenase (GDH), a 336-kDa metabolic enzyme that catalyzes the oxidative deamination of glutamate. Dysregulation of GDH leads to a variety of metabolic and neurologic disorders. Here, we report near-atomic resolution cryo-EM structures, at resolutions ranging from 3.2 Å to 3.6 Å for GDH complexes, including complexes for which crystal structures are not available. We show that the binding of the coenzyme NADH alone or in concert with GTP results in a binary mixture in which the enzyme is in either an "open" or "closed" state. Whereas the structure of NADH in the active site is similar between the open and closed states, it is unexpectedly different at the regulatory site. Our studies thus demonstrate that even in instances when there is considerable structural information available from X-ray crystallography, cryo-EM methods can provide useful complementary insights into regulatory mechanisms for dynamic protein complexes.

  17. Using Cryo-EM to Map Small Ligands on Dynamic Metabolic Enzymes: Studies with Glutamate Dehydrogenase

    PubMed Central

    Borgnia, Mario J.; Banerjee, Soojay; Merk, Alan; Matthies, Doreen; Bartesaghi, Alberto; Rao, Prashant; Pierson, Jason; Earl, Lesley A.; Falconieri, Veronica

    2016-01-01

    Cryo-electron microscopy (cryo-EM) methods are now being used to determine structures at near-atomic resolution and have great promise in molecular pharmacology, especially in the context of mapping the binding of small-molecule ligands to protein complexes that display conformational flexibility. We illustrate this here using glutamate dehydrogenase (GDH), a 336-kDa metabolic enzyme that catalyzes the oxidative deamination of glutamate. Dysregulation of GDH leads to a variety of metabolic and neurologic disorders. Here, we report near-atomic resolution cryo-EM structures, at resolutions ranging from 3.2 Å to 3.6 Å for GDH complexes, including complexes for which crystal structures are not available. We show that the binding of the coenzyme NADH alone or in concert with GTP results in a binary mixture in which the enzyme is in either an “open” or “closed” state. Whereas the structure of NADH in the active site is similar between the open and closed states, it is unexpectedly different at the regulatory site. Our studies thus demonstrate that even in instances when there is considerable structural information available from X-ray crystallography, cryo-EM methods can provide useful complementary insights into regulatory mechanisms for dynamic protein complexes. PMID:27036132

  18. Using Cryo-EM to Map Small Ligands on Dynamic Metabolic Enzymes: Studies with Glutamate Dehydrogenase.

    PubMed

    Borgnia, Mario J; Banerjee, Soojay; Merk, Alan; Matthies, Doreen; Bartesaghi, Alberto; Rao, Prashant; Pierson, Jason; Earl, Lesley A; Falconieri, Veronica; Subramaniam, Sriram; Milne, Jacqueline L S

    2016-06-01

    Cryo-electron microscopy (cryo-EM) methods are now being used to determine structures at near-atomic resolution and have great promise in molecular pharmacology, especially in the context of mapping the binding of small-molecule ligands to protein complexes that display conformational flexibility. We illustrate this here using glutamate dehydrogenase (GDH), a 336-kDa metabolic enzyme that catalyzes the oxidative deamination of glutamate. Dysregulation of GDH leads to a variety of metabolic and neurologic disorders. Here, we report near-atomic resolution cryo-EM structures, at resolutions ranging from 3.2 Å to 3.6 Å for GDH complexes, including complexes for which crystal structures are not available. We show that the binding of the coenzyme NADH alone or in concert with GTP results in a binary mixture in which the enzyme is in either an "open" or "closed" state. Whereas the structure of NADH in the active site is similar between the open and closed states, it is unexpectedly different at the regulatory site. Our studies thus demonstrate that even in instances when there is considerable structural information available from X-ray crystallography, cryo-EM methods can provide useful complementary insights into regulatory mechanisms for dynamic protein complexes. PMID:27036132

  19. Differential levels of glutamate dehydrogenase 1 (GLUD1) in Balb/c and C57BL/6 mice and the effects of overexpression of the Glud1 gene on glutamate release in striatum

    PubMed Central

    Hascup, Kevin N; Bao, Xiaodong; Hascup, Erin R; Hui, Dongwei; Xu, Wenhao; Pomerleau, Francois; Huettl, Peter; Michaelis, Mary L; Michaelis, Elias K; Gerhardt, Greg A

    2011-01-01

    We have previously shown that overexpression of the Glud1 (glutamate dehydrogenase 1) gene in neurons of C57BL/6 mice results in increased depolarization-induced glutamate release that eventually leads to selective neuronal injury and cell loss by 12 months of age. However, it is known that isogenic lines of Tg (transgenic) mice produced through back-crossing with one strain may differ in their phenotypic characteristics from those produced using another inbred mouse strain. Therefore, we decided to introduce the Glud1 transgene into the Balb/c strain that has endogenously lower levels of GLUD1 (glutamate dehydrogenase 1) enzyme activity in the brain as compared with C57BL/6. Using an enzyme-based MEA (microelectrode array) that is selective for measuring glutamate in vivo, we measured depolarization-induced glutamate release. Within a discrete layer of the striatum, glutamate release was significantly increased in Balb/c Tg mice compared with wt (wild-type) littermates. Furthermore, Balb/c mice released approx. 50–60% of the amount of glutamate compared with C57BL/6 mice. This is similar to the lower levels of endogenous GLUD1 protein in Balb/c compared with C57BL/6 mice. The development of these Glud1-overexpressing mice may allow for the exploration of key molecular events produced by chronic exposure of neurons to moderate, transient increases in glutamate release, a process hypothesized to occur in neurodegenerative disorders. PMID:21446915

  20. Urease-glutamic dehydrogenase biosensor for screening heavy metals in water and soil samples.

    PubMed

    Rodriguez, Belen Bello; Bolbot, John A; Tothill, Ibtisam E

    2004-09-01

    A screen-printed three-electrode amperometric biosensor based on urease and the nicotinamide adenine dinucleotide hydrogen (NADH)-glutamic dehydrogenase system was developed and applied to the screening of heavy metals in environmental samples. The development of an amperometric sensor for the monitoring of urease activity was feasible by coupling the urea breakdown reaction catalysed by urease to the reductive ammination of ketoglutarate catalysed by glutamic dehydrogenase (GLDH). The ammonia provided by the urea conversion is required for the conversion of ketoglutarate to glutamate with the concomitant oxidation of the NADH cofactor. NADH oxidation is monitored amperometrically at 0.3 V (vs. Ag/AgCl) after urease immobilization onto the screen-printed three-electrode configuration. Immobilization of urease on the surface of screen-printed electrodes was performed by entrapment in alginate gel and adsorption on the electrode in a nafion film. Low sensitivity to inactivation by metals was recorded after urease entrapment in alginate gel with detection limits of 2.9 and 29.8 mg L(-1) for Hg(II) and Cu(II), respectively. The use of the negatively charged nafion film created a more concentrated environment of cations in proximity to the enzyme, thus enhancing the urease inhibition when compared to gel entrapment. The calculated detection limits were 63.6 and 55.3 microg L(-1) for Hg(II) and Cu(II), respectively, and 4.3 mg L(-1) for Cd(II). A significant urease inactivation was recorded in the presence of trace amounts of metals (microg L(-1)) when the enzyme was used free in solution. Analysis of water and soil samples with the developed nafion-based sensor produced inhibition on urease activity according to their metal contents. The obtained results were in agreement with the standard methods employed for sample analysis. Nevertheless, the use of the amperometric assay (with free urease) proved more feasible for the screening of trace amounts of metals in polluted

  1. Importance of Glutamate Dehydrogenase (GDH) in Clostridium difficile Colonization In Vivo

    PubMed Central

    Girinathan, Brintha Parasumanna; Braun, Sterling; Sirigireddy, Apoorva Reddy; Lopez, Jose Espinola; Govind, Revathi

    2016-01-01

    Clostridium difficile is the principal cause of antibiotic-associated diarrhea. Major metabolic requirements for colonization and expansion of C. difficile after microbiota disturbance have not been fully determined. In this study, we show that glutamate utilization is important for C. difficile to establish itself in the animal gut. When the gluD gene, which codes for glutamate dehydrogenase (GDH), was disrupted, the mutant C. difficile was unable to colonize and cause disease in a hamster model. Further, from the complementation experiment it appears that extracellular GDH may be playing a role in promoting C. difficile colonization and disease progression. Quantification of free amino acids in the hamster gut during C. difficile infection showed that glutamate is among preferred amino acids utilized by C. difficile during its expansion. This study provides evidence of the importance of glutamate metabolism for C. difficile pathogenesis. PMID:27467167

  2. The kinetic mechanism of ox liver glutamate dehydrogenase in the presence of the allosteric effector ADP. The oxidative deamination of L-glutamate.

    PubMed

    Hornby, D P; Aitchison, M J; Engel, P C

    1984-10-01

    In steady-state kinetic studies of ox liver glutamate dehydrogenase in 0.11 M-potassium phosphate buffer, pH7, at 25 degrees C, the concentration of ADP was varied from 0.5 to 1000 microM. Inhibition was observed except when the concentrations of both glutamate and coenzyme were high, when activation was seen. With NAD+ or NADP+ as coenzyme, 200 microM-ADP was sufficient to saturate the enzyme with respect to the major effect of this nucleotide. In the presence of 210 microM-ADP, widely varied concentrations of coenzyme give linear Lineweaver-Burk plots, in marked contrast with results obtained previously for kinetics without ADP. This has allowed evaluation for the reaction with NAD+, NADP+ and acetylpyridine-adenine dinucleotide (315 microM-ADP in the last case) of all four initial rate parameters, i.e. the phi coefficients in the equation: (Formula: see text) where A is coenzyme and B is glutamate. The relative constancy of phi B and of phi AB/phi A with the different coenzymes point to a compulsory-order mechanism with glutamate as the leading substrate. This conclusion, though unexpected, agrees well with various previous observations on the binding of oxidized coenzyme.

  3. Deamination role of inducible glutamate dehydrogenase isoenzyme 7 in Brassica napus leaf protoplasts.

    PubMed

    Watanabe, Masami; Yumi, Ohnishi; Itoh, Yasuhiro; Yasuda, Kaori; Kamachi, Kazunari; Ratcliffe, R George

    2011-05-01

    Glutamate dehydrogenase (GDH) is a ubiquitous enzyme that catalyzes the reversible amination of 2-oxoglutarate to glutamate. In Brassica napus, GDH isoenzymes 1 and 7 are hexamers of β and α subunits, respectively and the isoenzyme profile in leaves is known to change on wounding. Here, parallels were sought between the effects of wounding and protoplast isolation because of the possible relevance of changes in GDH activity to the perturbed metabolism in recalcitrant B. napus protoplasts. When leaf protoplasts of B. napus were isolated, GDH7 isoforms predominated. Transcription of GDH2, which encodes the GDH α subunit, was activated and translation of the GDH2 mRNA was also activated to synthesize α subunit polypeptides. When detached leaves absorbed either acidic 5mM jasmonic acid or salicylic acid solutions via petioles, GDH7 isoenzymes were activated and the GDH isoenzyme patterns were similar to those of protoplasts. Salicylic acid β-glycosides were generated soon after treatment with the pectinase-cellulase enzyme solution and peaked at 1h. NMR spectroscopic analysis of protoplasts and unstressed leaves incubated with 5mM (15)NH(4)Cl showed that the change in GDH isoenzyme profile had no effect on ammonium assimilation. Protoplast isolation changed the redox state with NAD(P)H and oxidized glutathione levels increasing, and ascorbate, dehydroascorbate, NAD(P) and glutathione decreasing. ATP content in protoplasts declined to 2.6% of that in leaves, while that in wounded leaves increased by twofold. It is concluded that GDH7 does not support net amination in vivo and it is suggested that the increase in GDH7 activity is a response to oxidative stress during protoplast isolation.

  4. Regulation of Glutamate Dehydrogenase and Glutamine Synthetase in Avocado Fruit during Development and Ripening.

    PubMed Central

    Loulakakis, K. A.; Roubelakis-Angelakis, K. A.; Kanellis, A. K.

    1994-01-01

    The activity, protein, and isoenzymic profiles of glutamate de-hydrogenase (GDH) and glutamine synthetase (GS) were studied during development and ripening of avocado (Percea americana Mill. cv Hass) fruit. During fruit development, the activity and protein content of both GDH and GS remained relatively constant. In contrast, considerable changes in these enzymes were observed during ripening of avocado fruit. The specific activity of GDH increased about 4-fold, coincident with a similar increase in GDH protein content and mRNA levels. On the other hand, GS specific activity showed a decline at the end of the ripening process. On the isoenzymic profile of GDH, changes in the prevalence of the seven isoenzymes were found, with a predominance of the more cathodal isoenzymes in the unripe and of the most anodal isoenzymes in the ripe fruit. Two-dimensional electrophoresis revealed that avocado fruit GDH consists of two subunits whose association gives rise to seven isoenzymes. The results support the view that the predominance of the more anodal isoenzymes in the overripe fruit was due to the accumulation of the [alpha]-polypeptide. PMID:12232322

  5. Regulation of Glutamate Dehydrogenase and Glutamine Synthetase in Avocado Fruit during Development and Ripening.

    PubMed

    Loulakakis, K. A.; Roubelakis-Angelakis, K. A.; Kanellis, A. K.

    1994-09-01

    The activity, protein, and isoenzymic profiles of glutamate de-hydrogenase (GDH) and glutamine synthetase (GS) were studied during development and ripening of avocado (Percea americana Mill. cv Hass) fruit. During fruit development, the activity and protein content of both GDH and GS remained relatively constant. In contrast, considerable changes in these enzymes were observed during ripening of avocado fruit. The specific activity of GDH increased about 4-fold, coincident with a similar increase in GDH protein content and mRNA levels. On the other hand, GS specific activity showed a decline at the end of the ripening process. On the isoenzymic profile of GDH, changes in the prevalence of the seven isoenzymes were found, with a predominance of the more cathodal isoenzymes in the unripe and of the most anodal isoenzymes in the ripe fruit. Two-dimensional electrophoresis revealed that avocado fruit GDH consists of two subunits whose association gives rise to seven isoenzymes. The results support the view that the predominance of the more anodal isoenzymes in the overripe fruit was due to the accumulation of the [alpha]-polypeptide.

  6. Gene transcript accumulation and in situ mRNA hybridization of two putative glutamate dehydrogenase genes in etiolated Glycine max seedlings.

    PubMed

    Dimou, M; Tsaniklidis, G; Aivalakis, G; Katinakis, P

    2015-01-01

    Glutamate dehydrogenase (EC 1.4.1.2) is a multimeric enzyme that catalyzes the reversible amination of α-ketoglutarate to form glutamate. We characterized cDNA clones of two Glycine max sequences, GmGDH1 and GmGDH2, that code for putative α- and β-subunits, respectively, of the NADH dependent enzyme. Temporal and spatial gene transcript accumulation studies using semiquantitative RT-PCR and in situ hybridization have shown an overlapping gene transcript accumulation pattern with differences in relative gene transcript accumulation in the organs examined. Detection of NADH-dependent glutamate dehydrogenase activity in situ using a histochemical method showed concordance with the spatial gene transcript accumulation patterns. Our findings suggest that although the two gene transcripts are co-localized in roots of etiolated soybean seedlings, the ratio of the two subunits of the active holoenzyme may vary among tissues.

  7. Expression and in vitro assembly of recombinant glutamate dehydrogenase from the hyperthermophilic archaeon Pyrococcus furiosus.

    PubMed Central

    Diruggiero, J; Robb, F T

    1995-01-01

    The gdhA gene, encoding the hexameric glutamate dehydrogenase (GDH) from the hyperthermophilic archaeon Pyrococcus furiosus, was expressed in Escherichia coli by using the pET11-d system. The recombinant GDH was soluble and constituted 15% of the E. coli cell extract. The N-terminal amino acid sequence of the recombinant protein was identical to the sequence of the P. furiosus enzyme, except for the presence of an initial methionine which was absent from the enzyme purified from P. furiosus. By molecular exclusion chromatography we showed that the recombinant GDH was composed of equal amounts of monomeric and hexameric forms. Heat treatment of the recombinant protein triggered in vitro assembly of inactive monomers into hexamers, resulting in increased GDH activity. The specific activity of the recombinant enzyme, purified by heat treatment and affinity chromatography, was equivalent to that of the native enzyme from P. furiosus. The recombinant GDH displayed a slightly lower level of thermostability, with a half-life of 8 h at 100 degrees C, compared with 10.5 h for the enzyme purified from P. furiosus. PMID:7887598

  8. Coimmobilization of l-methioninase and glutamate dehydrogenase: Novel approach for L-homoalanine synthesis.

    PubMed

    El-Sayed, Ashraf S A; Yassin, Marwa A; Ibrahim, Hend

    2015-01-01

    L-Homoalanine, a nonnatural amino acid that is rarely found in human and microorganisms, is used in the synthesis of various medically pivotal antiepileptic drugs and antituberculosis compounds. l-Homoalanine can be synthesized by different enzymatic approaches. In this article, the synthesis of l-homoalanine from l-methionine was explored by coimmobilization of Aspergillus flavipes l-methioninase (AfMETase) and glutamate dehydrogenase (GDH) on polyacrylamide and chitosan. Polyacrylamide coimmobilized AfMETase and GDH displayed a maximum reactivity for the synthesis of homoalanine from l-methionine. The chitosan-coimmobilized AfMETase and GDH retain about 70% of their initial activity of l-homoalanine production by the fifth catalytic reusability cycle as compared with 50% for polyacrylamide coimmobilizate. Catalytic conditions were optimized for the maximum yield of homoalanine. Homoalanine was purified by cationic and anionic chromatographs and the proton nuclear magnetic resonance (H-NMR) analysis of the lyophilized sample displayed a unique chemical structure identical to the authentic homoalanine. Using dependable dual action of AfMETase and GDH immobilized on a solid support is a novel approach for in vitro enzymatic synthesis of l-homoalanine from l-methionine, and the immobilized enzymes can be reused many times without any significant loss of their activities. PMID:25273833

  9. α-Ketoglutarate Dehydrogenase and Glutamate Dehydrogenase Work in Tandem To Modulate the Antioxidant α-Ketoglutarate during Oxidative Stress in Pseudomonas fluorescens▿

    PubMed Central

    Mailloux, Ryan J.; Singh, Ranji; Brewer, Guy; Auger, Christopher; Lemire, Joseph; Appanna, Vasu D.

    2009-01-01

    α-Ketoglutarate (KG) is a crucial metabolite in all living organisms, as it participates in a variety of biochemical processes. We have previously shown that this keto acid is an antioxidant and plays a key role in the detoxification of reactive oxygen species (ROS). In an effort to further confirm this intriguing phenomenon, Pseudomonas fluorescens was exposed to menadione-containing media, with various amino acids as the sources of nitrogen. Here, we demonstrate that KG dehydrogenase (KGDH) and NAD-dependent glutamate dehydrogenase (GDH) work in tandem to modulate KG homeostasis. While KGDH was sharply decreased in cells challenged with menadione, GDH was markedly increased in cultures containing arginine (Arg), glutamate (Glu), and proline (Pro). When ammonium (NH4) was utilized as the nitrogen source, both KGDH and GDH levels were diminished. These enzymatic profiles were reversed when control cells were incubated in menadione media. 13C nuclear magnetic resonance and high-performance liquid chromatography studies revealed how KG was utilized to eliminate ROS with the concomitant formation of succinate. The accumulation of KG in the menadione-treated cells was dependent on the redox status of the lipoic acid residue in KGDH. Indeed, the treatment of cellular extracts from the menadione-exposed cells with dithiothreitol, a reducing agent, partially restored the activity of KGDH. Taken together, these data reveal that KG is pivotal to the antioxidative defense strategy of P. fluorescens and also point to the ROS-sensing role for KGDH. PMID:19376872

  10. Purification and side chain selective chemical modifications of glutamate dehydrogenase from Bacillus subtilis natto.

    PubMed

    Ni, Yinyun; Wang, Jiale; Qian, Bingjun; Song, Guangyan; Yao, Xiaomin; Zhang, Jian-hua

    2014-04-01

    Glutamate dehydrogenase (GDH) from Bacillus subtilis natto was purified to apparent homogeneity by ammonium sulfate precipitation, ion-exchange chromatography, size exclusion chromatography, and hydroxyapatite (HA) affinity chromatography. The GDH was purified 34-fold, with a yield of 41 % of total activity and a specific activity of 34.29 U/mg proteins. The molecular weight (Mr) of was measured at 47 kDa with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and 264 kDa with high-performance liquid chromatography (HPLC). The optimum pH and temperature for the deammoniation reaction were measured to be 7.5 and 30 °C, respectively. The active-site amino acid residues of GDH were investigated by chemical modification. The compounds 2,4,6-trinitrobenzenesulfonic acid (TNBS), phenylglyoxal (PG), and phenylmethanesulfonyl fluoride (PMSF) were used to modify lysine, arginine, and serine active site residues, respectively. After treatment with modifying reagents at concentrations of 1 mM, GDH activity fell to 10.7 % with TNBS, 83.3 % with PG, and 12.8 % with PMSF. However, with substrate protection, there was almost no loss in GDH activity following treatment with any modifying reagent. The kinetic parameters K m and V max were determined in each case. K m values for native GDH, 50 % TNBS-inactivated GDH, and 50 % PMSF-inactivated GDH were 0.037, 0.104, and 0.017 mM, respectively. V max values were 0.048, 0.022, and 0.031 mM/s, respectively. These results suggest that the active site contains one or more lysine residues that play a role in substrate binding and one or more serine residues that may maintain the enzyme conformation. However, arginine residues played less of a role in the activity of GDH. PMID:24557956

  11. Identification of lysine residue involved in inactivation of brain glutamate dehydrogenase isoproteins by o-phthalaldehyde.

    PubMed

    Ahn, J Y; Choi, S; Cho, S W

    1999-12-01

    Incubation of two types of glutamate dehydrogenase (GDH) isoproteins from bovine brain with o-phthalaldehyde resulted in a time-dependent loss of enzyme activity. The inactivation was partially prevented by preincubation of the GDH isoproteins with 2-oxoglutarate or NADH. Spectrophotometric studies indicated that the inactivation of GDH isoproteins with o-phthalaldehyde resulted in isoindole derivatives characterized by typical fluorescence emission spectra with a stoichiometry of one isoindole derivative per molecule of enzyme subunit. There were no differences between the two GDH isoproteins in sensitivities to inactivation by o-phthalaldehyde indicating that the microenvironmental structures of the GDH isoproteins are very similar to each other. Tryptic peptides of the isoproteins, modified with and without protection, identified a selective modification of one lysine as in the region containing the sequence L-Q-H-G-S-I-L-G-F-P-X-A-K for both GDH isoproteins. The symbol X indicates a position for which no phenylthiohydantoin-amino acid could be assigned. The missing residue, however, can be designated as an o-phthalaldehyde-labeled lysine since the sequences including the lysine residue in question have a complete identity with those of the other mammalian GDHs. Also, trypsin was unable to cleave the labeled peptide at this site. Both amino acid sequencing and compositional analysis identified Lys-306 as the site of o-phthalaldehyde binding within the brain GDH isoproteins. PMID:10607407

  12. Expression, purification and characterization of human glutamate dehydrogenase (GDH) allosteric regulatory mutations.

    PubMed Central

    Fang, Jie; Hsu, Betty Y L; MacMullen, Courtney M; Poncz, Mortimer; Smith, Thomas J; Stanley, Charles A

    2002-01-01

    Glutamate dehydrogenase (GDH) catalyses the reversible oxidative deamination of l-glutamate to 2-oxoglutarate in the mitochondrial matrix. In mammals, this enzyme is highly regulated by allosteric effectors. The major allosteric activator and inhibitor are ADP and GTP, respectively; allosteric activation by leucine may play an important role in amino acid-stimulated insulin secretion. The physiological significance of this regulation has been highlighted by the identification of children with an unusual hyperinsulinism/hyperammonaemia syndrome associated with dominant mutations in GDH that cause a loss in GTP inhibition. In order to determine the effects of these mutations on the function of the human GDH homohexamer, we studied the expression, purification and characterization of two of these regulatory mutations (H454Y, which affects the putative GTP-binding site, and S448P, which affects the antenna region) and a mutation designed to alter the putative binding site for ADP (R463A). The sensitivity to GTP inhibition was impaired markedly in the purified H454Y (ED(50), 210 microM) and S448P (ED(50), 3.1 microM) human GDH mutants compared with the wild-type human GDH (ED(50), 42 nM) or GDH isolated from heterozygous patient cells (ED(50), 290 and 280 nM, respectively). Sensitivity to ADP or leucine stimulation was unaffected by these mutations, confirming that they interfere specifically with the inhibitory GTP-binding site. Conversely, the R463A mutation completely eliminated ADP activation of human GDH, but had little effect on either GTP inhibition or leucine activation. The effects of these three mutations on ATP regulation indicated that this nucleotide inhibits human GDH through binding of its triphosphate tail to the GTP site and, at higher concentrations, activates the enzyme through binding of the nucleotide to the ADP site. These data confirm the assignment of the GTP and ADP allosteric regulatory sites on GDH based on X-ray crystallography and provide

  13. Structure of NADP(+)-dependent glutamate dehydrogenase from Escherichia coli--reflections on the basis of coenzyme specificity in the family of glutamate dehydrogenases.

    PubMed

    Sharkey, Michael A; Oliveira, Tânia F; Engel, Paul C; Khan, Amir R

    2013-09-01

    Glutamate dehydrogenases (GDHs; EC 1.4.1.2-4) catalyse the oxidative deamination of L-glutamate to α-ketoglutarate, using NAD(+) and/or NADP(+) as a cofactor. Subunits of homo-hexameric bacterial enzymes comprise a substrate-binding domain I followed by a nucleotide-binding domain II. The reaction occurs in a catalytic cleft between the two domains. Although conserved residues in the nucleotide-binding domains of various dehydrogenases have been linked to cofactor preferences, the structural basis for specificity in the GDH family remains poorly understood. Here, the refined crystal structure of Escherichia coli GDH in the absence of reactants is described at 2.5-Å resolution. Modelling of NADP(+) in domain II reveals the potential contribution of positively charged residues from a neighbouring α-helical hairpin to phosphate recognition. In addition, a serine that follows the P7 aspartate is presumed to form a hydrogen bond with the 2'-phosphate. Mutagenesis and kinetic analysis confirms the importance of these residues in NADP(+) recognition. Surprisingly, one of the positively charged residues is conserved in all sequences of NAD(+)-dependent enzymes, but the conformations adopted by the corresponding regions in proteins whose structure has been solved preclude their contribution to the coordination of the 2'-ribose phosphate of NADP(+). These studies clarify the sequence-structure relationships in bacterial GDHs, revealing that identical residues may specify different coenzyme preferences, depending on the structural context. Primary sequence alone is therefore not a reliable guide for predicting coenzyme specificity. We also consider how it is possible for a single sequence to accommodate both coenzymes in the dual-specificity GDHs of animals.

  14. Structure of NADP+-dependent glutamate dehydrogenase from Escherichia coli - reflections on the basis of coenzyme specificity in the family of glutamate dehydrogenases

    SciTech Connect

    Sharkey, Michael A.; Oliveira, Tânia F.; Engel, Paul C.; Khan, Amir R.

    2013-09-05

    Glutamate dehydrogenases catalyse the oxidative deamination of L-glutamate to α-ketoglutarate, using NAD+ and/or NADP+ as a cofactor. Subunits of homo-hexameric bacterial enzymes comprise a substrate-binding domain I followed by a nucleotide-binding domain II. The reaction occurs in a catalytic cleft between the two domains. Although conserved residues in the nucleotide-binding domains of various dehydrogenases have been linked to cofactor preferences, the structural basis for specificity in the GDH family remains poorly understood. Here, the refined crystal structure of Escherichia coli GDH in the absence of reactants is described at 2.5-Å resolution. Modelling of NADP+ in domain II reveals the potential contribution of positively charged residues from a neighbouring α-helical hairpin to phosphate recognition. In addition, a serine that follows the P7 aspartate is presumed to form a hydrogen bond with the 2'-phosphate. Mutagenesis and kinetic analysis confirms the importance of these residues in NADP+ recognition. Surprisingly, one of the positively charged residues is conserved in all sequences of NAD+-dependent enzymes, but the conformations adopted by the corresponding regions in proteins whose structure has been solved preclude their contribution to the coordination of the 2'-ribose phosphate of NADP+. These studies clarify the sequence–structure relationships in bacterial GDHs, revealing that identical residues may specify different coenzyme preferences, depending on the structural context. Primary sequence alone is therefore not a reliable guide for predicting coenzyme specificity. We also consider how it is possible for a single sequence to accommodate both coenzymes in the dual-specificity GDHs of animals.

  15. Regulation of accumulation and turnover of an inducible glutamate dehydrogenase in synchronous cultures of Chlorella.

    PubMed Central

    Israel, D W; Gronostajski, R M; Yeung, A T; Schmidt, R R

    1977-01-01

    Earlier studies indicated that the gene of an ammonium-inducible glutamate dehydrogenase (GDH) was inducible throughout the cell cycle and was expressible shortly after replication early in the S-phase in synchronous Chlorella cells growing at a rate of 13% per h in the absence of inducer. In the present study, synchronous cells cultured at the same growth rate in the continuous presence of inducer accumulated this enzyme in a linear manner, with a positive rate change observed late instead of early in the S-phase. At a growth rate of 26% per h, the positive rate change appeared to be displaced to 1.5 h before the S-phase in the next cell cycle. With 2'-deoxyadenosine, an in vivo inhibitor of deoxyribonucleic acid (DNA) synthesis, the magnitude of the positive rate change was shown to be proportional to the relative increase in DNA in the previous cell cycle. Collectively, these data support the idea that expression of newly replicated genes of this enzyme can be delayed into the subsequent cell cycle in cells in the continuous presence of inducer. Studies with cycloheximide indicated that the inducible GDH and another GDH isozyme were stable in fully induced cells in the absence of protein synthesis. However, after ammonium was removed from the culture medium, the activity of the inducible GDH decreased rapidly in vivo, with a half-time of 5 to 10 min at 38.5 degrees C, whereas the rate of accumulation of the other GDH isozyme did not change. Addition of cycloheximide, at the time of inducer removal, prevented this loss in activity of the inducible GDH. The inability to rescue the activity of the inducible GDH, by readdition of ammonium during the deinduction period, indicates that this enzyme probably underwent irreversible inactivation and/or proteolytic degradation. PMID:45486

  16. CcpA-dependent regulation of Bacillus subtilis glutamate dehydrogenase gene expression.

    PubMed

    Belitsky, Boris R; Kim, Hyun-Jin; Sonenshein, Abraham L

    2004-06-01

    The Bacillus subtilis rocG gene, encoding catabolic glutamate dehydrogenase, was found to be subject to direct CcpA-dependent glucose repression. The effect of CcpA required the presence of both the HPr and Crh proteins. The primary CcpA binding site was identified by mutational analysis and DNase I footprinting. In the absence of inducers of the Roc pathway, rocG was still expressed at a low level due to readthrough transcription. CcpA-dependent repression of rocG readthrough transcription proved to contribute to the slow growth rate of B. subtilis cells in glucose-glutamate medium. Increased readthrough expression of rocG was shown to be partially responsible for the growth defect of ccpA strains in glucose-ammonium medium.

  17. Reduced levels of NADH-dependent glutamate dehydrogenase decrease the glutamate content of ripe tomato fruit but have no effect on green fruit or leaves.

    PubMed

    Ferraro, Gisela; D'Angelo, Matilde; Sulpice, Ronan; Stitt, Mark; Valle, Estela M

    2015-06-01

    Glutamate (Glu) is a taste enhancer that contributes to the characteristic flavour of foods. In fruit of tomato (Solanum lycopersicum L.), the Glu content increases dramatically during the ripening process, becoming the most abundant free amino acid when the fruit become red. There is also a concomitant increase in NADH-dependent glutamate dehydrogenase (GDH) activity during the ripening transition. This enzyme is located in the mitochondria and catalyses the reversible amination of 2-oxoglutarate to Glu. To investigate the potential effect of GDH on Glu metabolism, the abundance of GDH was altered by artificial microRNA technology. Efficient silencing of all the endogenous SlGDH genes was achieved, leading to a dramatic decrease in total GDH activity. This decrease in GDH activity did not lead to any clear morphological or metabolic phenotype in leaves or green fruit. However, red fruit on the transgenic plants showed markedly reduced levels of Glu and a large increase in aspartate, glucose and fructose content in comparison to wild-type fruit. These results suggest that GDH is involved in the synthesis of Glu in tomato fruit during the ripening processes. This contrasts with the biological role ascribed to GDH in many other tissues and species. Overall, these findings suggest that GDH has a major effect on the control of metabolic composition during tomato fruit ripening, but not at other stages of development. PMID:25878356

  18. Reduced levels of NADH-dependent glutamate dehydrogenase decrease the glutamate content of ripe tomato fruit but have no effect on green fruit or leaves.

    PubMed

    Ferraro, Gisela; D'Angelo, Matilde; Sulpice, Ronan; Stitt, Mark; Valle, Estela M

    2015-06-01

    Glutamate (Glu) is a taste enhancer that contributes to the characteristic flavour of foods. In fruit of tomato (Solanum lycopersicum L.), the Glu content increases dramatically during the ripening process, becoming the most abundant free amino acid when the fruit become red. There is also a concomitant increase in NADH-dependent glutamate dehydrogenase (GDH) activity during the ripening transition. This enzyme is located in the mitochondria and catalyses the reversible amination of 2-oxoglutarate to Glu. To investigate the potential effect of GDH on Glu metabolism, the abundance of GDH was altered by artificial microRNA technology. Efficient silencing of all the endogenous SlGDH genes was achieved, leading to a dramatic decrease in total GDH activity. This decrease in GDH activity did not lead to any clear morphological or metabolic phenotype in leaves or green fruit. However, red fruit on the transgenic plants showed markedly reduced levels of Glu and a large increase in aspartate, glucose and fructose content in comparison to wild-type fruit. These results suggest that GDH is involved in the synthesis of Glu in tomato fruit during the ripening processes. This contrasts with the biological role ascribed to GDH in many other tissues and species. Overall, these findings suggest that GDH has a major effect on the control of metabolic composition during tomato fruit ripening, but not at other stages of development.

  19. Control of carbon flux to glutamate excretion in Klebsiella pneumoniae: the role of the indigenous plasmid and its encoded isocitrate dehydrogenase.

    PubMed

    El-Mansi, Mansi; Trappey, Francois; Clark, Ewan; Campbell, Malcolm

    2015-11-01

    Klebsiella pneumoniae (NCTC, CL687/80) harbors a large indigenous plasmid (p(C3)), which in addition to encoding for citrate utilization, proline synthesis and glutamate excretion, it uniquely carries the structural gene (icd); encoding isocitrate dehydrogenase (ICDH). Flux analysis revealed that ICDH, despite its role in the generation of NADPH required for glutamate dehydrogenase, is not rate-limiting (controlling) in central metabolism as evidenced by a negative flux control coefficient and an adverse effect of overexpression (14-fold) on glutamate excretion. More significantly, however, this paper presents, for the first time, clear evidence that the accumulation of glutamate and its subsequent excretion is associated with the C3 plasmid-encoded regulatory elements, which trigger a shift-down in the activity of α-ketoglutarate dehydrogenase, both in the K. pneumoniae parental strain as well as in the E. coli exconjugants strains. This finding opens the door for the exploitation of regulatory elements as a tool for manipulating flux in microbial cell factories.

  20. Reconfiguration of N Metabolism upon Hypoxia Stress and Recovery: Roles of Alanine Aminotransferase (AlaAT) and Glutamate Dehydrogenase (GDH)

    PubMed Central

    Diab, Houssein; Limami, Anis M.

    2016-01-01

    In the context of climatic change, more heavy precipitation and more frequent flooding and waterlogging events threaten the productivity of arable farmland. Furthermore, crops were not selected to cope with flooding- and waterlogging-induced oxygen limitation. In general, low oxygen stress, unlike other abiotic stresses (e.g., cold, high temperature, drought and saline stress), received little interest from the scientific community and less financial support from stakeholders. Accordingly, breeding programs should be developed and agronomical practices should be adapted in order to save plants’ growth and yield—even under conditions of low oxygen availability (e.g., submergence and waterlogging). The prerequisite to the success of such breeding programs and changes in agronomical practices is a good knowledge of how plants adapt to low oxygen stress at the cellular and the whole plant level. In the present paper, we summarized the recent knowledge on metabolic adjustment in general under low oxygen stress and highlighted thereafter the major changes pertaining to the reconfiguration of amino acids syntheses. We propose a model showing (i) how pyruvate derived from active glycolysis upon hypoxia is competitively used by the alanine aminotransferase/glutamate synthase cycle, leading to alanine accumulation and NAD+ regeneration. Carbon is then saved in a nitrogen store instead of being lost through ethanol fermentative pathway. (ii) During the post-hypoxia recovery period, the alanine aminotransferase/glutamate dehydrogenase cycle mobilizes this carbon from alanine store. Pyruvate produced by the reverse reaction of alanine aminotransferase is funneled to the TCA cycle, while deaminating glutamate dehydrogenase regenerates, reducing equivalent (NADH) and 2-oxoglutarate to maintain the cycle function. PMID:27258319

  1. Reconfiguration of N Metabolism upon Hypoxia Stress and Recovery: Roles of Alanine Aminotransferase (AlaAT) and Glutamate Dehydrogenase (GDH).

    PubMed

    Diab, Houssein; Limami, Anis M

    2016-01-01

    In the context of climatic change, more heavy precipitation and more frequent flooding and waterlogging events threaten the productivity of arable farmland. Furthermore, crops were not selected to cope with flooding- and waterlogging-induced oxygen limitation. In general, low oxygen stress, unlike other abiotic stresses (e.g., cold, high temperature, drought and saline stress), received little interest from the scientific community and less financial support from stakeholders. Accordingly, breeding programs should be developed and agronomical practices should be adapted in order to save plants' growth and yield-even under conditions of low oxygen availability (e.g., submergence and waterlogging). The prerequisite to the success of such breeding programs and changes in agronomical practices is a good knowledge of how plants adapt to low oxygen stress at the cellular and the whole plant level. In the present paper, we summarized the recent knowledge on metabolic adjustment in general under low oxygen stress and highlighted thereafter the major changes pertaining to the reconfiguration of amino acids syntheses. We propose a model showing (i) how pyruvate derived from active glycolysis upon hypoxia is competitively used by the alanine aminotransferase/glutamate synthase cycle, leading to alanine accumulation and NAD⁺ regeneration. Carbon is then saved in a nitrogen store instead of being lost through ethanol fermentative pathway. (ii) During the post-hypoxia recovery period, the alanine aminotransferase/glutamate dehydrogenase cycle mobilizes this carbon from alanine store. Pyruvate produced by the reverse reaction of alanine aminotransferase is funneled to the TCA cycle, while deaminating glutamate dehydrogenase regenerates, reducing equivalent (NADH) and 2-oxoglutarate to maintain the cycle function. PMID:27258319

  2. A kinetic study of the oxidative deamination of L-glutamate by Peptostreptococcus asaccharolyticus glutamate dehydrogenase using a variety of coenzymes.

    PubMed

    Hornby, D P; Engel, P C

    1984-09-17

    The NAD+-specific glutamate dehydrogenase from Peptostreptococcus asaccharolyticus follows Michaelis-Menten kinetics in contrast to the enzyme from several other sources, and thus gives linear double-reciprocal plots of initial-rate data. The initial-rate parameters have been determined for this bacterial dehyrogenase in the direction of oxidative deamination. The use of alternative coenzymes leads to some conclusions about the order of substrate addition. An investigation of the pH dependence of this reaction reveals that the binding of oxidised coenzyme is independent of pH over the range 6-9. The kinetic data are consistent with an ordered addition of coenzyme prior to glutamate, the reverse of the mechanism derived with ox glutamate dehydrogenase in the presence of ADP.

  3. Organ-specific expression of glutamate dehydrogenase (GDH) subunits in yellow lupine.

    PubMed

    Lehmann, Teresa; Dabert, Mirosława; Nowak, Witold

    2011-07-01

    Glutamate dehydrogenase (GDH, EC 1.4.2-4) is present in yellow lupine (Lupinus luteus cv. Juno) in many isoforms. The number and banding pattern of isoenzymes varies with respect to plant organ and developmental stage. To better understand the complex nature of GDH regulation in plants, the levels of GDH transcripts, enzyme activity and isoenzyme patterns in germinating seeds and roots of yellow lupine were examined. The analysis of GDH cDNA sequences in lupine revealed three mRNA types, of which two encoded the β-GDH subunit and one encoded the α-GDH subunit (corresponding to the GDH1(GDH3) and GDH2 genes, respectively). The relative expression of GDH1 and GDH2 genes was analyzed in various lupine organs by using quantitative real-time PCR. Our results indicate that different mRNA types were differently regulated depending on organ type. Although both genes appeared to be ubiquitously expressed in all lupine tissues, the GDH1 transcripts evidently predominated over those of GDH2. Immunochemical analyses confirmed that, during embryo development, varied expression of two GDH subunits takes place. The α-GDH subunit (43kDa) predominated in the early stages of germinating seeds, while the β-GDH subunit (44kDa) was the only GDH polypeptide present in lupine roots. These results firmly support the hypothesis that isoenzyme variability of GDH in yellow lupine is associated with the varied expression of α and β subunits into the complexes of hexameric GDH forms. The presence of several isogenes of GDH in yellow lupine may explain the high number (over 20) of its molecular forms in germinating lupine.

  4. Mechanism for the activation of glutamate receptors

    Cancer.gov

    Scientists at the NIH have used a technique called cryo-electron microscopy to determine a molecular mechanism for the activation and desensitization of ionotropic glutamate receptors, a prominent class of neurotransmitter receptors in the brain and spina

  5. Temperature dependence of kinetic parameters for hyperthermophilic glutamate dehydrogenase from Aeropyrum pernix K1.

    PubMed

    Bhuiya, Mohammad W; Sakuraba, Haruhiko; Ohshima, Toshihisa

    2002-04-01

    The temperature dependence of the steady-state kinetic parameters for a glutamate dehydrogenase from Aeropyrum pernix K1 was investigated. The enzyme showed a biphasic kinetic characteristic for L-glutamate and a monophasic one for NADP at 50-90 degrees C. At low concentrations of L-glutamate the Km decreased from 2.02 to 0.56 mM and the catalytic efficiency (Vmax/Km) markedly increased (4-150 micromol x mg(-1) x mM(-1)) along with the increase of temperature from 50 to 90 degrees C. At high concentrations of the substrate the Km was fairly high and approximately constant (around 225 mM), and the catalytic efficiency was low and its temperature-dependent change was small. The Km (0.039 mM) for NADP did not change with the increase of temperature. In the reductive amination, the Kms for 2-oxoglutarate (1.81 and 9.37 mM at low and high levels of ammonia, respectively) were independent on temperature, but the Kms for ammonia and NADPH rose from 86 to 185 mM and 0.050 to 0.175 mM, respectively.

  6. Evidence for the Degradation of Nicotinamide Adenine Dinucleotide Phosphate-Dependent Glutamate Dehydrogenase of Candida utilis During Rapid Enzyme Inactivation

    PubMed Central

    Hemmings, Brian A.

    1978-01-01

    The nicotinamide adenine dinucleotide phosphate-dependent glutamate dehydrogenase (NADP-GDH) from the food yeast Candida utilis was found to be rapidly inactivated when cultures were starved of a carbon source. The addition of glutamate or alanine to the starvation medium stimulated the rate of inactivation. Loss of enzyme activity was irreversible since the reappearance of enzyme activity, following the addition of glucose to carbon-starved cultures, was blocked by cycloheximide. A specific rabbit antibody was prepared against the NADP-GDH from C. utilis and used to quantitate the enzyme during inactivation promoted by carbon starvation. The amount of precipitable antigenic material paralleled the rapid decrease of enzyme activity observed after transition of cells from NH4+-glucose to glutamate medium. No additional small-molecular-weight protein was precipitated by the antibody as a result of the inactivation, suggesting that the enzyme is considerably altered during the primary steps of the inactivation process. Analysis by immunoprecipitation of the reappearance of enzyme activity after enzyme inactivation showed that increase of NADP-GDH activity was almost totally due to de novo synthesis, ruling out the possibility that enzyme activity modulation is achieved by reversible covalent modification. Enzyme degradation was also measured during steady-state growth and other changes in nitrogen and carbon status of the culture media. In all instances so far estimated, the enzyme was found to be very stable and not normally subject to high rates of degradation. Therefore, the possibility that inactivation was caused by a change in the ratio of synthesis to degradation can be excluded. Images PMID:24041

  7. Purification of glutamate dehydrogenase isoenzymes and characterization of their substrate specificities.

    PubMed

    Osuji, Godson O; Braithwaite, Cleantis; Fordjour, Kusi; Madu, Wenceslaus C; Beyene, Asefa; Roberts, Paul S; Wright, Victor

    2003-02-01

    Glutamate dehydrogenase (GDH) isoenzymes were purified from control, and ribonucleoside triphosphate (NTP)-treated peanut seedlings. GDH purification was by preparative-scale, free solution isoelectric focusing, followed by native PAGE, and the cryoelectrophoretic elution of the isoenzymes from the gel. SDS-PAGE of the purified GDH isoenzymes, followed by either silver staining of the gel, or western analysis using anti-GDH antibody, gave identical GDH polypeptide (a, alpha, and b) bands, thus, confirming the purity of the isoenzymes. The substrate specificities in the aminating activity of the GDH isoenzymes, or disaggregated polypeptides were determined by photometry, but the substrate specificities in the RNA synthesis activity were determined in cocktails containing 0.06-0.8 mM of each of UTP, ATP, GTP, and CTP, 0-100.0 mM NH4Cl, 0-50.0 mM alpha-ketoglutaratr (alpha-KG), 0-0.2 mM NADH, 0-10.0 mM CaCl2 5 units of DNase 1, antibiotics, and approximately 5 microg pure GDH isoenzymes or polypeptides at pH 8.0, and overnight at 16 degrees C. The GDH polypeptides were active only in amination reaction, but the GDH isoenzymes were active in both amination and RNA synthesis. Whereas, NADH, NH4Cl and alpha-KG served as the substrates for the amination reaction, and as modulators in the RNA synthetic reaction, ATP, GTP, UTP, and CTP served as substrates for theisoenzymes in RNA synthesis reaction. The product RNA was up to 2 microg microg(-1) GDH, and consisted of RNA species in the size ranges of 26, 16, and 5 S rRNAs. DNAse 1 in the assay cocktail ruled out transcription as the mechanism of the RNA synthesis. Addition of [alpha-32P] NTP led to the production of labeled RNA, thus confirming the specificity of NTPs as substrates, and that the RNA was not pre-existing in the reaction cocktail.

  8. Glutamate Dehydrogenase Is Required by Mycobacterium bovis BCG for Resistance to Cellular Stress

    PubMed Central

    Gallant, James L.; Viljoen, Albertus J.; van Helden, Paul D.; Wiid, Ian J. F.

    2016-01-01

    We recently reported on our success to generate deletion mutants of the genes encoding glutamate dehydrogenase (GDH) and glutamine oxoglutarate aminotransferase (GOGAT) in M. bovis BCG, despite their in vitro essentiality in M. tuberculosis. We could use these mutants to delineate the roles of GDH and GOGAT in mycobacterial nitrogen metabolism by using M. bovis BCG as a model for M. tuberculosis specifically. Here, we extended our investigation towards the involvement of GDH and GOGAT in other aspects of M. bovis BCG physiology, including the use of glutamate as a carbon source and resistance to known phagosomal stresses, as well as in survival inside macrophages. We find that gdh is indispensable for the utilization of glutamate as a major carbon source, in low pH environments and when challenged with nitric oxide. On the other hand, the gltBD mutant had increased viability under low pH conditions and was unaffected by a challenge with nitric oxide. Strikingly, GDH was required to sustain M. bovis BCG during infection of both murine RAW 264.7 and bone-marrow derived and macrophages, while GOGAT was not. We conclude that the catabolism of glutamate in slow growing mycobacteria may be a crucial function during infection of macrophage cells and demonstrate a novel requirement for M. bovis BCG GDH in the protection against acidic and nitrosative stress. These results provide strong clues on the role of GDH in intracellular survival of M. tuberculosis, in which the essentiality of the gdh gene complicates knock out studies making the study of the role of this enzyme in pathogenesis difficult. PMID:26824899

  9. Insights into the In Vivo Regulation of Glutamate Dehydrogenase from the Foot Muscle of an Estivating Land Snail

    PubMed Central

    Bell, Ryan A. V.; Dawson, Neal J.; Storey, Kenneth B.

    2012-01-01

    Land snails, Otala lactea, survive in seasonally hot and dry environments by entering a state of aerobic torpor called estivation. During estivation, snails must prevent excessive dehydration and reorganize metabolic fuel use so as to endure prolonged periods without food. Glutamate dehydrogenase (GDH) was hypothesized to play a key role during estivation as it shuttles amino acid carbon skeletons into the Krebs cycle for energy production and is very important to urea biosynthesis (a key molecule used for water retention). Analysis of purified foot muscle GDH from control and estivating conditions revealed that estivated GDH was approximately 3-fold more active in catalyzing glutamate deamination as compared to control. This kinetic difference appears to be regulated by reversible protein phosphorylation, as indicated by ProQ Diamond phosphoprotein staining and incubations that stimulate endogenous protein kinases and phosphatases. The increased activity of the high-phosphate form of GDH seen in the estivating land snail foot muscle correlates well with the increased use of amino acids for energy and increased synthesis of urea for water retention during prolonged estivation. PMID:22536484

  10. Abiotic Stress Generates ROS That Signal Expression of Anionic Glutamate Dehydrogenases to Form Glutamate for Proline Synthesis in Tobacco and Grapevine[W

    PubMed Central

    Skopelitis, Damianos S.; Paranychianakis, Nikolaos V.; Paschalidis, Konstantinos A.; Pliakonis, Eleni D.; Delis, Ioannis D.; Yakoumakis, Dimitris I.; Kouvarakis, Antonios; Papadakis, Anastasia K.; Stephanou, Euripides G.; Roubelakis-Angelakis, Kalliopi A.

    2006-01-01

    Glutamate dehydrogenase (GDH) may be a stress-responsive enzyme, as GDH exhibits considerable thermal stability, and de novo synthesis of the α-GDH subunit is induced by exogenous ammonium and senescence. NaCl treatment induces reactive oxygen species (ROS), intracellular ammonia, expression of tobacco (Nicotiana tabacum cv Xanthi) gdh-NAD;A1 encoding the α-subunit of GDH, increase in immunoreactive α-polypeptide, assembly of the anionic isoenzymes, and in vitro GDH aminating activity in tissues from hypergeous plant organs. In vivo aminating GDH activity was confirmed by gas chromatorgraphy–mass spectrometry monitoring of 15N-Glu, 15N-Gln, and 15N-Pro in the presence of methionine sulfoximine and amino oxyacetic acid, inhibitors of Gln synthetase and transaminases, respectively. Along with upregulation of α-GDH by NaCl, isocitrate dehydrogenase genes, which provide 2-oxoglutarate, are also induced. Treatment with menadione also elicits a severalfold increase in ROS and immunoreactive α-polypeptide and GDH activity. This suggests that ROS participate in the signaling pathway for GDH expression and protease activation, which contribute to intracellular hyperammonia. Ammonium ions also mimic the effects of salinity in induction of gdh-NAD;A1 expression. These results, confirmed in tobacco and grape (Vitis vinifera cv Sultanina) tissues, support the hypothesis that the salinity-generated ROS signal induces α-GDH subunit expression, and the anionic iso-GDHs assimilate ammonia, acting as antistress enzymes in ammonia detoxification and production of Glu for Pro synthesis. PMID:17041150

  11. Quantitative determination of glycine in aqueous solution using glutamate dehydrogenase-immobilized glyoxal agarose beads.

    PubMed

    Keskin, Semra Yilmazer; Keskin, Can Serkan

    2014-01-01

    In this study, an enzymatic procedure for the determination of glycine (Gly) was developed by using a column containing immobilized glutamate dehydrogenase (GDH) on glyoxal agarose beads. Ammonia is produced from the enzymatic reactions between Gly and GDH with NAD(+) in phosphate buffer medium. The indophenol blue method was used for ammonia detection based on the spectrophotometric measurements of blue-colored product absorbing at 640 nm. The calibration graph is linear in the range of 0.1-10 mM of Gly concentrations. The effect of pH, temperature, and time interval was studied to find column stability, and also the interference effects of other amino acids was investigated. The interaction between GDH and glyoxal agarose beads was analyzed by Fourier transform infrared (FTIR) spectroscopy. The morphology of the immobilized and non-immobilized agarose beads were characterized by atomic force microscopy (AFM).

  12. Purification, crystallization and preliminary X-ray diffraction analysis of NADP-dependent glutamate dehydrogenase from Aspergillus niger

    PubMed Central

    Prakash, Prem; Walvekar, Adhish S.; Punekar, Narayan S.; Bhaumik, Prasenjit

    2014-01-01

    Glutamate dehydrogenase (GDH) catalyzes the NAD-dependent or NADP-dependent oxidative deamination of l-glutamate to 2-oxoglutarate and ammonia. This important reversible reaction establishes the link between carbon and nitrogen metabolism. In this study, Aspergillus niger NADP-GDH (AnGDH) has been overexpressed and purified. Purified AnGDH, with a high specific activity of 631.1 units per milligram of protein, was crystallized and the crystal diffracted to 2.9 Å resolution using a home X-ray source. Preliminary analysis of the X-ray diffraction data showed that the crystal belonged to space group R32, with unit-cell parameters a = b = 173.8, c = 241.5 Å, α = β = 90, γ = 120°. The crystals exhibited an unusually high solvent content (83.0%) and had only one molecule in the asymmetric unit. Initial phases were obtained by molecular replacement, and model building and structure refinement of AnGDH are in progress. PMID:25372818

  13. Transgenic Expression of Glud1 (Glutamate Dehydrogenase 1) in Neurons: In Vivo Model of Enhanced Glutamate Release, Altered Synaptic Plasticity, and Selective Neuronal Vulnerability

    PubMed Central

    Bao, Xiaodong; Pal, Ranu; Hascup, Kevin N.; Wang, Yongfu; Wang, Wen-Tung; Xu, Wenhao; Hui, Dongwei; Agbas, Abdulbaki; Wang, Xinkun; Michaelis, Mary L.; Choi, In-Young; Belousov, Andrei B.; Gerhardt, Greg A.; Michaelis, Elias K.

    2010-01-01

    The effects of lifelong, moderate excess release of glutamate (Glu) in the CNS have not been previously characterized. We created a transgenic (Tg) mouse model of lifelong excess synaptic Glu release in the CNS by introducing the gene for glutamate dehydrogenase 1 (Glud1) under the control of the neuron-specific enolase promoter. Glud1 is, potentially, an important enzyme in the pathway of Glu synthesis in nerve terminals. Increased levels of GLUD protein and activity in CNS neurons of hemizygous Tg mice were associated with increases in the in vivo release of Glu after neuronal depolarization in striatum and in the frequency and amplitude of miniature EPSCs in the CA1 region of the hippocampus. Despite overexpression of Glud1 in all neurons of the CNS, the Tg mice suffered neuronal losses in select brain regions (e.g., the CA1 but not the CA3 region). In vulnerable regions, Tg mice had decreases in MAP2A labeling of dendrites and in synaptophysin labeling of presynaptic terminals; the decreases in neuronal numbers and dendrite and presynaptic terminal labeling increased with advancing age. In addition, the Tg mice exhibited decreases in long-term potentiation of synaptic activity and in spine density in dendrites of CA1 neurons. Behaviorally, the Tg mice were significantly more resistant than wild-type mice to induction and duration of anesthesia produced by anesthetics that suppress Glu neurotransmission. The Glud1 mouse might be a useful model for the effects of lifelong excess synaptic Glu release on CNS neurons and for age-associated neurodegenerative processes. PMID:19890003

  14. Novel Inhibitors Complexed with Glutamate Dehydrogenase: ALLOSTERIC REGULATION BY CONTROL OF PROTEIN DYNAMICS

    SciTech Connect

    Li, Ming; Smith, Christopher J.; Walker, Matthew T.; Smith, Thomas J.

    2009-12-01

    Mammalian glutamate dehydrogenase (GDH) is a homohexameric enzyme that catalyzes the reversible oxidative deamination of L-glutamate to 2-oxoglutarate using NAD(P){sup +} as coenzyme. Unlike its counterparts from other animal kingdoms, mammalian GDH is regulated by a host of ligands. The recently discovered hyperinsulinism/hyperammonemia disorder showed that the loss of allosteric inhibition of GDH by GTP causes excessive secretion of insulin. Subsequent studies demonstrated that wild-type and hyperinsulinemia/hyperammonemia forms of GDH are inhibited by the green tea polyphenols, epigallocatechin gallate and epicatechin gallate. This was followed by high throughput studies that identified more stable inhibitors, including hexachlorophene, GW5074, and bithionol. Shown here are the structures of GDH complexed with these three compounds. Hexachlorophene forms a ring around the internal cavity in GDH through aromatic stacking interactions between the drug and GDH as well as between the drug molecules themselves. In contrast, GW5074 and bithionol both bind as pairs of stacked compounds at hexameric 2-fold axes between the dimers of subunits. The internal core of GDH contracts when the catalytic cleft closes during enzymatic turnover. None of the drugs cause conformational changes in the contact residues, but all bind to key interfaces involved in this contraction process. Therefore, it seems likely that the drugs inhibit enzymatic turnover by inhibiting this transition. Indeed, this expansion/contraction process may play a major role in the inter-subunit communication and allosteric regulation observed in GDH.

  15. A marriage full of surprises: forty-five years living with glutamate dehydrogenase.

    PubMed

    Engel, Paul C

    2011-09-01

    Detailed kinetic studies of bovine glutamate dehydrogenase [GDH] from the 1960s revealed complexities that remain to be fully explained. In the absence of heterotropic nucleotide regulators the enzyme follows a random pathway of substrate addition but saturation with ADP enforces a compulsory-order mechanism in which glutamate is the leading substrate. The rate dependence on NAD(P)(+) concentration is complex and is probably only partly explained by negative binding cooperativity. Bovine GDH eluded successful analysis by crystallographers for 30 years but the final structural solution presented in this symposium at last provides a comprehensible framework for much of the heterotropic regulation, focussing attention on an antenna region in the C-terminal tail, a structure that is missing in the slightly smaller hexameric GDHs of lower organisms. Nonetheless, our studies with one such smaller (clostridial) GDH reveal that even without the antenna the underlying core structure still mediates homotropic cooperativity, and the ability to generate a variety of mutants has made it possible to start to dissect this machinery. In addition, this short personal review discusses a number of unresolved issues such as the significance of phospholipid inhibition and of specific interaction with mRNA, and above all the question of why it is necessary to regulate an enzyme reputedly maintaining its reactants at equilibrium and whether this might be in some way related to its coexistence with an energy-linked transhydrogenase.

  16. Glutamate and GABA-metabolizing enzymes in post-mortem cerebellum in Alzheimer's disease: phosphate-activated glutaminase and glutamic acid decarboxylase.

    PubMed

    Burbaeva, G Sh; Boksha, I S; Tereshkina, E B; Savushkina, O K; Prokhorova, T A; Vorobyeva, E A

    2014-10-01

    Enzymes of glutamate and GABA metabolism in postmortem cerebellum from patients with Alzheimer's disease (AD) have not been comprehensively studied. The present work reports results of original comparative study on levels of phosphate-activated glutaminase (PAG) and glutamic acid decarboxylase isoenzymes (GAD65/67) in autopsied cerebellum samples from AD patients and matched controls (13 cases in each group) as well as summarizes published evidence for altered levels of PAG and GAD65/67 in AD brain. Altered (decreased) levels of these enzymes and changes in links between amounts of these enzymes and other glutamate-metabolizing enzymes (such as glutamate dehydrogenase and glutamine synthetase-like protein) in AD cerebella suggest significantly impaired glutamate and GABA metabolism in this brain region, which was previously regarded as not substantially involved in AD pathogenesis.

  17. NADPH-dependent glutamate dehydrogenase in Penicillium chrysogenum is involved in regulation of beta-lactam production.

    PubMed

    Thykaer, Jette; Rueksomtawin, Kanchana; Noorman, Henk; Nielsen, Jens

    2008-04-01

    The interactions between the ammonium assimilatory pathways and beta-lactam production were investigated by disruption of the NADPH-dependent glutamate dehydrogenase gene (gdhA) in two industrial beta-lactam-producing strains of Penicillium chrysogenum. The strains used were an adipoyl-7-ADCA- and a penicillin-producing strain. The gdhA gene disruption caused a decrease in maximum specific growth rate of 26 % and 35 % for the adipoyl-7-ADCA-producing strain and the penicillin-producing strain, respectively, compared to the corresponding reference strains. Interestingly, no beta-lactam production was detected in either of the DeltagdhA strains. Supplementation with glutamate restored growth but no beta-lactam production was detected for the constructed strains. Cultures with high ammonium concentrations (repressing conditions) and with proline as nitrogen source (de-repressed conditions) showed continued beta-lactam production for the reference strains whereas the DeltagdhA strains remained non-productive under all conditions. By overexpressing the NAD-dependent glutamate dehydrogenase, the specific growth rate could be restored, but still no beta-lactam production was detected. The results indicate that the NADPH-dependent glutamate dehydrogenase may be directly or indirectly involved in the regulation of beta-lactam production in industrial strains of P. chrysogenum.

  18. Glutamate Mediated Astrocytic Filtering of Neuronal Activity

    PubMed Central

    Herzog, Nitzan; De Pittà, Maurizio; Jacob, Eshel Ben; Berry, Hugues; Hanein, Yael

    2014-01-01

    Neuron-astrocyte communication is an important regulatory mechanism in various brain functions but its complexity and role are yet to be fully understood. In particular, the temporal pattern of astrocyte response to neuronal firing has not been fully characterized. Here, we used neuron-astrocyte cultures on multi-electrode arrays coupled to Ca2+ imaging and explored the range of neuronal stimulation frequencies while keeping constant the amount of stimulation. Our results reveal that astrocytes specifically respond to the frequency of neuronal stimulation by intracellular Ca2+ transients, with a clear onset of astrocytic activation at neuron firing rates around 3-5 Hz. The cell-to-cell heterogeneity of the astrocyte Ca2+ response was however large and increasing with stimulation frequency. Astrocytic activation by neurons was abolished with antagonists of type I metabotropic glutamate receptor, validating the glutamate-dependence of this neuron-to-astrocyte pathway. Using a realistic biophysical model of glutamate-based intracellular calcium signaling in astrocytes, we suggest that the stepwise response is due to the supralinear dynamics of intracellular IP3 and that the heterogeneity of the responses may be due to the heterogeneity of the astrocyte-to-astrocyte couplings via gap junction channels. Therefore our results present astrocyte intracellular Ca2+ activity as a nonlinear integrator of glutamate-dependent neuronal activity. PMID:25521344

  19. High protein diet induces pericentral glutamate dehydrogenase and ornithine aminotransferase to provide sufficient glutamate for pericentral detoxification of ammonia in rat liver lobules.

    PubMed

    Boon, L; Geerts, W J; Jonker, A; Lamers, W H; Van Noorden, C J

    1999-06-01

    The liver plays a central role in nitrogen metabolism. Nitrogen enters the liver as free ammonia and as amino acids of which glutamine and alanine are the most important precursors. Detoxification of ammonia to urea involves deamination and transamination. By applying quantitative in situ hybridization, we found that mRNA levels of the enzymes involved are mainly expressed in periportal zones of liver lobules. Free ammonia, that is not converted periportally, is efficiently detoxified in the small rim of hepatocytes around the central veins by glutamine synthetase preventing it from entering the systemic circulation. Detoxification of ammonia by glutamine synthetase may be limited due to a shortage of glutamate when the nitrogen load is high. Adaptations in metabolism that prevent release of toxic ammonia from the liver were studied in rats that were fed diets with different amounts of protein, thereby varying the nitrogen load of the liver. We observed that mRNA levels of periportal deaminating and transaminating enzymes increased with the protein content in the diet. Similarly, mRNA levels of pericentral glutamate dehydrogenase and ornithine aminotransferase, the main producers of glutamate in this zone, and pericentral glutamine synthetase all increased with increasing protein levels in the diet. On the basis of these changes in mRNA levels, we conclude that: (a) glutamate is produced pericentrally in sufficient amounts to allow ammonia detoxification by glutamine synthetase and (b) in addition to the catalytic role of ornithine in the periportally localized ornithine cycle, pericentral ornithine degradation provides glutamate for ammonia detoxification.

  20. Amino acid residues involved in the catalytic mechanism of NAD-dependent glutamate dehydrogenase from Halobacterium salinarum.

    PubMed

    Pérez-Pomares, F; Ferrer, J; Camacho, M; Pire, C; LLorca, F; Bonete, M J

    1999-02-01

    The pH dependence of kinetic parameters for a competitive inhibitor (glutarate) was determined in order to obtain information on the chemical mechanism for NAD-dependent glutamate dehydrogenase from Halobacterium salinarum. The maximum velocity is pH dependent, decreasing at low pHs giving a pK value of 7.19+/-0.13, while the V/K for l-glutamate at 30 degrees C decreases at low and high pHs, yielding pK values of 7.9+/-0.2 and 9.8+/-0.2, respectively. The glutarate pKis profile decreases at high pHs, yielding a pK of 9. 59+/-0.09 at 30 degrees C. The values of ionization heat calculated from the change in pK with temperature are: 1.19 x 10(4), 5.7 x 10(3), 7 x 10(3), 6.6 x 10(3) cal mol-1, for the residues involved. All these data suggest that the groups required for catalysis and/or binding are lysine, histidine and tyrosine. The enzyme shows a time-dependent loss in glutamate oxidation activity when incubated with diethyl pyrocarbonate (DEPC). Inactivation follows pseudo-first-order kinetics with a second-order rate constant of 53 M-1min-1. The pKa of the titratable group was pK1=6.6+/-0.6. Inactivation with ethyl acetimidate also shows pseudo-first-order kinetics as well as inactivation with TNM yielding second-order constants of 1.2 M-1min-1 and 2.8 M-1min-1, and pKas of 8.36 and 9.0, respectively. The proposed mechanism involves hydrogen binding of each of the two carboxylic groups to tyrosyl residues; histidine interacts with one of the N-hydrogens of the l-glutamate amino group. We also corroborate the presence of a conservative lysine that has a remarkable ability to coordinate a water molecule that would act as general base.

  1. Fasciola gigantica: enzymes of the ornithine-proline-glutamate pathway--characterization of delta1-pyrroline-5-carboxylate dehydrogenase.

    PubMed

    Mohamed, Saleh A; Mohamed, Tarek M; Fahmy, Afaf S; El-Badry, Mohamed O; Abdel-Gany, Somia S

    2008-01-01

    Ornithine aminotransferase (OAT), proline oxidase (PO), Delta 1-pyrroline-5-carboxylate reductase (P5CR), and Delta 1-pyrroline-5-carboxylate dehydrogenase (P5CD) were assessed in Fasciola gigantica. All enzymes are involved in the conversion of ornithine into glutamate and proline. High levels of P5CD suggest that the direction of the metabolic flow from ornithine is more toward glutamate than proline. F. gigantica P5CD1 and P5CD2 were separated from the majority of contaminating proteins in crude homogenate using a CM-cellulose column. A Sephacryl S-200 column was employed for P5CD2 to obtain pure enzyme with increased specific activity. The molecular mass of P5CD2 was estimated to be 50kDa using a Sephacryl S-200 column and SDS-PAGE. It migrated as a single band on SDS-PAGE, indicating a monomeric enzyme. P5CD2 had Km values of 1.44mM and 0.37mM for NAD and P5C, respectively. P5CD2 oxidized a number of aliphatic and aromatic aldehydes, where the aromatic compounds had higher affinity toward the enzyme. All amino acids examined had partial inhibitory effects on the enzyme. While 3mM AMP caused 31% activation of enzyme, 3mM ADP and ATP inhibited activity by 18% and 23%, respectively. Apart from Cu2+, the divalent cations that were studied caused partial inhibitory effects on the enzyme. PMID:17655846

  2. Construction of a dimeric form of glutamate dehydrogenase from Clostridium symbiosum by site-directed mutagenesis.

    PubMed

    Pasquo, A; Britton, K L; Stillman, T J; Rice, D W; Cölfen, H; Harding, S E; Scandurra, R; Engel, P C

    1996-10-17

    By using site-directed mutagenesis, Phe-187, one of the amino-acid residues involved in hydrophobic interaction between the three identical dimers comprising the hexamer of Clostridium symbiosum glutamate dehydrogenase (GDH), has been replaced by an aspartic acid residue. Over-expression in Escherichia coli led to production of large amounts of a soluble protein which, though devoid of GDH activity, showed the expected subunit M(r) on SDS-PAGE, and cross-reacted with an anti-GDH antibody preparation in Western blots. The antibody was used to monitor purification of the inactive protein. F187D GDH showed altered mobility on non-denaturing electrophoresis, consistent with changed size and/or surface charge. Gel filtration on a calibrated column indicated an M(r) of 87000 +/- 3000. The mutant enzyme did not bind to the dye column routinely used in preparing wild-type GDH. Nevertheless suspicions of major misfolding were allayed by the results of chemical modification studies: as with wild-type GDH, NAD+ completely protected one-SH group against modification by DTNB, implying normal coenzyme binding. A significant difference, however, is that in the mutant enzyme both cysteine groups were modified by DTNB, rather than C320 only. The CD spectrum in the far-UV region indicated no major change in secondary structure in the mutant protein. The near-UV CD spectrum, however, was less intense and showed a pronounced Phe contribution, possibly reflecting the changed environment of Phe-199, which would be buried in the hexamer. Sedimentation velocity experiments gave corrected coefficients S20,W of 11.08 S and 5.29 S for the wild-type and mutant proteins. Sedimentation equilibrium gave weight average molar masses M(r,app) of 280000 +/- 5000 g/mol. consistent with the hexameric structure for the wild-type protein and 135000 +/- 3000 g/mol for F187D. The value for the mutant is intermediate between the values expected for a dimer (98000) and a trimer (147000). To investigate the

  3. E1 of α-ketoglutarate dehydrogenase defends Mycobacterium tuberculosis against glutamate anaplerosis and nitroxidative stress

    PubMed Central

    Maksymiuk, Christina; Balakrishnan, Anand; Bryk, Ruslana; Rhee, Kyu Y.; Nathan, Carl F.

    2015-01-01

    Enzymes of central carbon metabolism (CCM) in Mycobacterium tuberculosis (Mtb) make an important contribution to the pathogen’s virulence. Evidence is emerging that some of these enzymes are not simply playing the metabolic roles for which they are annotated, but can protect the pathogen via additional functions. Here, we found that deficiency of 2-hydroxy-3-oxoadipate synthase (HOAS), the E1 component of the α-ketoglutarate (α-KG) dehydrogenase complex (KDHC), did not lead to general metabolic perturbation or growth impairment of Mtb, but only to the specific inability to cope with glutamate anaplerosis and nitroxidative stress. In the former role, HOAS acts to prevent accumulation of aldehydes, including growth-inhibitory succinate semialdehyde (SSA). In the latter role, HOAS can participate in an alternative four-component peroxidase system, HOAS/dihydrolipoyl acetyl transferase (DlaT)/alkylhydroperoxide reductase colorless subunit gene (ahpC)-neighboring subunit (AhpD)/AhpC, using α-KG as a previously undescribed source of electrons for reductase action. Thus, instead of a canonical role in CCM, the E1 component of Mtb’s KDHC serves key roles in situational defense that contribute to its requirement for virulence in the host. We also show that pyruvate decarboxylase (AceE), the E1 component of pyruvate dehydrogenase (PDHC), can participate in AceE/DlaT/AhpD/AhpC, using pyruvate as a source of electrons for reductase action. Identification of these systems leads us to suggest that Mtb can recruit components of its CCM for reactive nitrogen defense using central carbon metabolites. PMID:26430237

  4. Diagnostic test accuracy of glutamate dehydrogenase for Clostridium difficile: Systematic review and meta-analysis.

    PubMed

    Arimoto, Jun; Horita, Nobuyuki; Kato, Shingo; Fuyuki, Akiko; Higurashi, Takuma; Ohkubo, Hidenori; Endo, Hiroki; Takashi, Nonaka; Kaneko, Takeshi; Nakajima, Atsushi

    2016-07-15

    We performed this systematic review and meta-analysis to assess the diagnostic accuracy of detecting glutamate dehydrogenase (GDH) for Clostridium difficile infection (CDI) based on the hierarchical model. Two investigators electrically searched four databases. Reference tests were stool cell cytotoxicity neutralization assay (CCNA) and stool toxigenic culture (TC). To assess the overall accuracy, we calculated the diagnostic odds ratio (DOR) using a DerSimonian-Laird random-model and area the under hierarchical summary receiver operating characteristics (AUC) using Holling's proportional hazard models. The summary estimate of the sensitivity and the specificity were obtained using the bivariate model. According to 42 reports consisting of 3055 reference positive comparisons, and 26188 reference negative comparisons, the DOR was 115 (95%CI: 77-172, I(2) = 12.0%) and the AUC was 0.970 (95%CI: 0.958-0.982). The summary estimate of sensitivity and specificity were 0.911 (95%CI: 0.871-0.940) and 0.912 (95%CI: 0.892-0.928). The positive and negative likelihood ratios were 10.4 (95%CI 8.4-12.7) and 0.098 (95%CI 0.066-0.142), respectively. Detecting GDH for the diagnosis of CDI had both high sensitivity and specificity. Considering its low cost and prevalence, it is appropriate for a screening test for CDI.

  5. Diagnostic test accuracy of glutamate dehydrogenase for Clostridium difficile: Systematic review and meta-analysis

    PubMed Central

    Arimoto, Jun; Horita, Nobuyuki; Kato, Shingo; Fuyuki, Akiko; Higurashi, Takuma; Ohkubo, Hidenori; Endo, Hiroki; Takashi, Nonaka; Kaneko, Takeshi; Nakajima, Atsushi

    2016-01-01

    We performed this systematic review and meta-analysis to assess the diagnostic accuracy of detecting glutamate dehydrogenase (GDH) for Clostridium difficile infection (CDI) based on the hierarchical model. Two investigators electrically searched four databases. Reference tests were stool cell cytotoxicity neutralization assay (CCNA) and stool toxigenic culture (TC). To assess the overall accuracy, we calculated the diagnostic odds ratio (DOR) using a DerSimonian-Laird random-model and area the under hierarchical summary receiver operating characteristics (AUC) using Holling’s proportional hazard models. The summary estimate of the sensitivity and the specificity were obtained using the bivariate model. According to 42 reports consisting of 3055 reference positive comparisons, and 26188 reference negative comparisons, the DOR was 115 (95%CI: 77–172, I2 = 12.0%) and the AUC was 0.970 (95%CI: 0.958–0.982). The summary estimate of sensitivity and specificity were 0.911 (95%CI: 0.871–0.940) and 0.912 (95%CI: 0.892–0.928). The positive and negative likelihood ratios were 10.4 (95%CI 8.4–12.7) and 0.098 (95%CI 0.066–0.142), respectively. Detecting GDH for the diagnosis of CDI had both high sensitivity and specificity. Considering its low cost and prevalence, it is appropriate for a screening test for CDI. PMID:27418431

  6. Surface Induced Dissociation Yields Quaternary Substructure of Refractory Noncovalent Phosphorylase B and Glutamate Dehydrogenase Complexes

    NASA Astrophysics Data System (ADS)

    Ma, Xin; Zhou, Mowei; Wysocki, Vicki H.

    2014-03-01

    Ion mobility (IM) and tandem mass spectrometry (MS/MS) coupled with native MS are useful for studying noncovalent protein complexes. Collision induced dissociation (CID) is the most common MS/MS dissociation method. However, some protein complexes, including glycogen phosphorylase B kinase (PHB) and L-glutamate dehydrogenase (GDH) examined in this study, are resistant to dissociation by CID at the maximum collision energy available in the instrument. Surface induced dissociation (SID) was applied to dissociate the two refractory protein complexes. Different charge state precursor ions of the two complexes were examined by CID and SID. The PHB dimer was successfully dissociated to monomers and the GDH hexamer formed trimeric subcomplexes that are informative of its quaternary structure. The unfolding of the precursor and the percentages of the distinct products suggest that the dissociation pathways vary for different charge states. The precursors at lower charge states (+21 for PHB dimer and +27 for GDH hexamer) produce a higher percentage of folded fragments and dissociate more symmetrically than the precusors at higher charge states (+29 for PHB dimer and +39 for GDH hexamer). The precursors at lower charge state may be more native-like than the higher charge state because a higher percentage of folded fragments and a lower percentage of highly charged unfolded fragments are detected. The combination of SID and charge reduction is shown to be a powerful tool for quaternary structure analysis of refractory noncovalent protein complexes, as illustrated by the data for PHB dimer and GDH hexamer.

  7. Gene expression patterns in the hippocampus during the development and aging of Glud1 (Glutamate Dehydrogenase 1) transgenic and wild type mice

    PubMed Central

    2014-01-01

    Background Extraneuronal levels of the neurotransmitter glutamate in brain rise during aging. This is thought to lead to synaptic dysfunction and neuronal injury or death. To study the effects of glutamate hyperactivity in brain, we created transgenic (Tg) mice in which the gene for glutamate dehydrogenase (Glud1) is over-expressed in neurons and in which such overexpression leads to excess synaptic release of glutamate. In this study, we analyzed whole genome expression in the hippocampus, a region important for learning and memory, of 10 day to 20 month old Glud1 and wild type (wt) mice. Results During development, maturation and aging, both Tg and wt exhibited decreases in the expression of genes related to neurogenesis, neuronal migration, growth, and process elongation, and increases in genes related to neuro-inflammation, voltage-gated channel activity, and regulation of synaptic transmission. Categories of genes that were differentially expressed in Tg vs. wt during development were: synaptic function, cytoskeleton, protein ubiquitination, and mitochondria; and, those differentially expressed during aging were: synaptic function, vesicle transport, calcium signaling, protein kinase activity, cytoskeleton, neuron projection, mitochondria, and protein ubiquitination. Overall, the effects of Glud1 overexpression on the hippocampus transcriptome were greater in the mature and aged than the young. Conclusions Glutamate hyperactivity caused gene expression changes in the hippocampus at all ages. Some of these changes may result in premature brain aging. The identification of these genomic expression differences is important in understanding the effects of glutamate dysregulation on neuronal function during aging or in neurodegenerative diseases. PMID:24593767

  8. Production of gamma-aminobutyric acid from glucose by introduction of synthetic scaffolds between isocitrate dehydrogenase, glutamate synthase and glutamate decarboxylase in recombinant Escherichia coli.

    PubMed

    Pham, Van Dung; Lee, Seung Hwan; Park, Si Jae; Hong, Soon Ho

    2015-08-10

    Escherichia coli were engineered for the direct production of gamma-aminobutyric acid from glucose by introduction of synthetic protein scaffold. In this study, three enzymes consisting GABA pathway (isocitrate dehydrogenase, glutamate synthase and glutamate decarboxylase) were connected via synthetic protein scaffold. By introduction of scaffold, 0.92g/L of GABA was produced from 10g/L of glucose while no GABA was produced in wild type E. coli. The optimum pH and temperature for GABA production were 4.5 and 30°C, respectively. When competing metabolic network was inactivated by knockout mutation, maximum GABA concentration of 1.3g/L was obtained from 10g/L glucose. The recombinant E. coli strain which produces GABA directly from glucose was successfully constructed by introduction of protein scaffold.

  9. Activity of select dehydrogenases with Sepharose-immobilized N6-carboxymethyl-NAD

    PubMed Central

    Beauchamp, Justin; Vieille, Claire

    2015-01-01

    N6-carboxymethyl-NAD (N6-CM-NAD) can be used to immobilize NAD onto a substrate containing terminal primary amines. We previously immobilized N6-CM-NAD onto sepharose beads and showed that Thermotoga maritima glycerol dehydrogenase could use the immobilized cofactor with cofactor recycling. We now show that Saccharomyces cerevisiae alcohol dehydrogenase, rabbit muscle L-lactate dehydrogenase (type XI), bovine liver L-glutamic dehydrogenase (type III), Leuconostoc mesenteroides glucose-6-phosphate dehydro-genase, and Thermotoga maritima mannitol dehydrogenase are active with soluble N6-CM-NAD. The products of all enzymes but 6-phospho-D-glucono-1,5-lactone were formed when sepharose-immobilized N6-CM-NAD was recycled by T. maritima glycerol dehydrogenase, indicating that N6-immobilized NAD is suitable for use by a variety of different dehydrogenases. Observations of the enzyme active sites suggest that steric hindrance plays a greater role in limiting or allowing activity with the modified cofactor than do polarity and charge of the residues surrounding the N6-amine group on NAD. PMID:25611453

  10. Activity of select dehydrogenases with sepharose-immobilized N(6)-carboxymethyl-NAD.

    PubMed

    Beauchamp, Justin; Vieille, Claire

    2015-01-01

    N(6)-carboxymethyl-NAD (N(6)-CM-NAD) can be used to immobilize NAD onto a substrate containing terminal primary amines. We previously immobilized N(6)-CM-NAD onto sepharose beads and showed that Thermotoga maritima glycerol dehydrogenase could use the immobilized cofactor with cofactor recycling. We now show that Saccharomyces cerevisiae alcohol dehydrogenase, rabbit muscle L-lactate dehydrogenase (type XI), bovine liver L-glutamic dehydrogenase (type III), Leuconostoc mesenteroides glucose-6-phosphate dehydro-genase, and Thermotoga maritima mannitol dehydrogenase are active with soluble N(6)-CM-NAD. The products of all enzymes but 6-phospho-D-glucono-1,5-lactone were formed when sepharose-immobilized N(6)-CM-NAD was recycled by T. maritima glycerol dehydrogenase, indicating that N(6)-immobilized NAD is suitable for use by a variety of different dehydrogenases. Observations of the enzyme active sites suggest that steric hindrance plays a greater role in limiting or allowing activity with the modified cofactor than do polarity and charge of the residues surrounding the N(6)-amine group on NAD.

  11. Affinity chromatography of the Neurospora NADP-specific glutamate dehydrogenase, its mutational variants and hybrid hexamers.

    PubMed Central

    Watson, D H; Wootton, J C

    1977-01-01

    The synthesis of an affinity adsorbent, 8-(6-aminohexyl)aminoadenosine 2'-phosphate-Sepharose 4B, is described. The assembly of the 2'-AMP ligand and the hexanediamide spacer arm was synthesized in free solution before its attachment to the Sepharose matrix. This adsorbent retarded the hexameric NADP-specific glutamate dehydrogenase of Neurospora crassa, showing a capacity for this enzyme similar to that of comparable coenzyme-analogue adsorbents for other dehydrogenases. The enzyme was eluted either at pH 6.8 in a concentration gradient of NADP+, or at pH 8.5 in the presence of NADP+ in concentration gradients of either dicarboxylates or NaCl. Anomalous effects of dicarboxylates in facilitating elution are discussed. 2'-AMP and its derivatives, 8-bromoadenosine 2'-phosphate and 8-(l-aminohexyl)aminoadenosine 2'-phosphate, which were used in the synthesis of the adsorbent, all acted as enzyme inhibitors competitive with NADP+. The chromatographic properties of the wild-type enzyme were compared with those of mutationally modified variants containing defined amino acid substitutions. This approach was used to assess the biospecificity of adsorption and elution and the contribution of non-specific binding. The adsorbent showed a low capacity for the enzyme from mutant am1 (Ser-336 replaced by Phe), a variant that has a localized defect in NADP binding, but an otherwise almost normal conformation, suggesting that non-specific interactions are at most weak. The enzyme from mutant am3, a variant modified in a conformational equilibrium, was fully retarded by the adsorbent, but showed a significantly earlier elution position than the wild-type enzyme. This is consistent with measurements in free solution that showed the am3 enzyme to have a higher Ki for 2'-AMP than the wild-type enzyme. The enzyme from mutant am19 was eluted as two distinct peaks at both pH 6.8 and 8.5. The adsorbent was used to separate hybrid hexamers constructed in vitro by a freeze-thaw procedure

  12. Preliminary crystallographic analysis of an extremely thermostable glutamate dehydrogenase from the archaeon Pyrococcus woesei.

    PubMed

    Knapp, S; Karshikoff, A; Waldkötter, K; Rüdiger, A; Antranikian, G; Ladenstein, R

    1995-05-01

    The extremely heat-stable glutamate dehydrogenase (GluDH) from Pyrococcus woesei was crystallized by the hanging-drop vapour-diffusion method. Crystals suitable for X-ray crystallographic investigations were obtained using polyethylene glycol (PEG) 4000 and ammonium acetate as precipitating agents. The crystals obtained diffract to a resolution of 2.8 A, have a prismatic shape and grow up to 0.5 mm in their maximal dimension. They belong to the triclinic system (space group P1; a = 90.9, b = 92.8, c = 107.1 A, alpha = 69.1, beta = 80.7, Vgamma = 65.0 degrees ) with a unit-cell volume of 765052 A(3) which accommodates one GluDH hexamer of 276 kDa. The averaged density of the crystal determined by Ficoll-gradient centrifugation is 1.15 g cm(-3), which corresponds to a molecular mass of 255 kDa in the unit cell. A native data set has been collected on an MAR image-plate system using Cu Kalpha radiation. The completeness of the data set in the range 316-3 A is 74%, and contains 64% of the data in the outer shell (3.4-2.8 A), with an average R(merge) value of 9%. Calculation of self-rotation functions revealed the 32 symmetry of the hexamer; 3 non-crystallographic twofold axes were found at a distance of 120 degrees in a plane perpendicular to the non-crystallographic threefold axis. PMID:15299309

  13. Carbon source-dependent modulation of NADP-glutamate dehydrogenases in isophthalate-degrading Pseudomonas aeruginosa strain PP4, Pseudomonas strain PPD and Acinetobacter lwoffii strain ISP4.

    PubMed

    Vamsee-Krishna, C; Phale, Prashant S

    2008-11-01

    Acinetobacter lwoffii strain ISP4 metabolizes isophthalate rapidly compared with Pseudomonas aeruginosa strain PP4 and Pseudomonas strain PPD. Isophthalate has been reported to be a potent competitive inhibitor of glutamate dehydrogenase (GDH). Exogenous supplementation of isophthalate with glutamate or alpha-ketoglutarate at 1 mM concentration caused strains PP4 and PPD to grow faster than in the presence of isophthalate alone; however, no such effect was observed in strain ISP4. When grown on isophthalate, all strains showed activity of NADP-dependent GDH (NADP-GDH), while cells grown on glucose, 2x yeast extract-tryptone broth (2YT) or glutamate showed activities of both NAD-dependent GDH (NAD-GDH) and NADP-GDH. Activity staining, inhibition and thermal stability studies indicated the carbon source-dependent presence of two (GDH(I) and GDH(II)), three (GDH(A), GDH(B) and GDH(C)) and one (GDH(P)) forms of NADP-GDH in strains PP4, PPD and ISP4, respectively. The results demonstrate the carbon source-dependent modulation of different forms of NADP-GDH in these bacterial strains. This modulation may help the efficient utilization of isophthalate as a carbon source by overcoming the inhibitory effect on GDH.

  14. Physiological Characterization of Pseudomonas aeruginosa during Exotoxin A Synthesis: Glutamate, Iron Limitation, and Aconitase Activity

    PubMed Central

    Somerville, Greg; Mikoryak, Carole Ann; Reitzer, Larry

    1999-01-01

    Glutamate enhances the yield of exotoxin A (ETA), which is induced by iron limitation, from Pseudomonas aeruginosa. We tested the possibility that glutamate affects growth during iron restriction. We confirmed that iron limitation caused early entry into stationary phase but had no effect on the exponential growth rate. We showed that glutamate, as well as citrate and isocitrate, partially overcame this growth limitation. Glutamate had no effect on toxA (ETA-encoding) transcription, which implies that glutamate primarily increases the number of toxin-producing cells. In contrast, citrate and isocitrate diminished toxA transcription. Since glutamate, citrate, and isocitrate stimulated growth, we suspected a block in the citric acid cycle. Iron limitation reduced the activity of the iron-containing aconitase 12-fold but had no effect on isocitrate dehydrogenase activity, which was assayed as a control. There is a reciprocal relationship between aconitase activity and ETA synthesis, and this correlation does not appear to be coincidental because aconitase-specific effectors affect ETA synthesis. We tested whether a metabolic block is sufficient to induce ETA synthesis, but an aconitase-specific inhibitor diminished ETA production, which argues against this possibility. Finally, we present preliminary evidence that iron limitation may reversibly and posttranslationally inactivate aconitase in vivo. In summary, the environmental factors that stimulate ETA synthesis are related: glutamate bypasses an iron limitation-dependent metabolic block that causes entry into stationary phase. We speculate that one or more of the aconitases in P. aeruginosa may contribute to the control of virulence factor synthesis. PMID:9973331

  15. Neuronal Activity and Glutamate Uptake Decrease Mitochondrial Mobility in Astrocytes and Position Mitochondria Near Glutamate Transporters

    PubMed Central

    Jackson, Joshua G.; O'Donnell, John C.; Takano, Hajime; Coulter, Douglas A.

    2014-01-01

    Within neurons, mitochondria are nonuniformly distributed and are retained at sites of high activity and metabolic demand. Glutamate transport and the concomitant activation of the Na+/K+-ATPase represent a substantial energetic demand on astrocytes. We hypothesized that mitochondrial mobility within astrocytic processes might be regulated by neuronal activity and glutamate transport. We imaged organotypic hippocampal slice cultures of rat, in which astrocytes maintain their highly branched morphologies and express glutamate transporters. Using time-lapse confocal microscopy, the mobility of mitochondria within individual astrocytic processes and neuronal dendrites was tracked. Within neurons, a greater percentage of mitochondria were mobile than in astrocytes. Furthermore, they moved faster and farther than in astrocytes. Inhibiting neuronal activity with tetrodotoxin (TTX) increased the percentage of mobile mitochondria in astrocytes. Mitochondrial movement in astrocytes was inhibited by vinblastine and cytochalasin D, demonstrating that this mobility depends on both the microtubule and actin cytoskeletons. Inhibition of glutamate transport tripled the percentage of mobile mitochondria in astrocytes. Conversely, application of the transporter substrate d-aspartate reversed the TTX-induced increase in the percentage of mobile mitochondria. Inhibition of reversed Na+/Ca2+ exchange also increased the percentage of mitochondria that were mobile. Last, we demonstrated that neuronal activity increases the probability that mitochondria appose GLT-1 particles within astrocyte processes, without changing the proximity of GLT-1 particles to VGLUT1. These results imply that neuronal activity and the resulting clearance of glutamate by astrocytes regulate the movement of astrocytic mitochondria and suggest a mechanism by which glutamate transporters might retain mitochondria at sites of glutamate uptake. PMID:24478345

  16. Use of primary deuterium and /sup 15/N isotope effects to deduce the relative rates of steps in the mechanisms of alanine and glutamate dehydrogenases

    SciTech Connect

    Weiss, P.M.; Chen, C.Y.; Cleland, W.W.; Cook, P.F.

    1988-06-28

    The authors have used deuterium and /sup 15/N isotope effects to study the relative rates of the steps in the mechanisms of alanine and glutamate dehydrogenases. The proposed chemical mechanisms for these enzymes involve carbinolamine formation, imine formation, and reduction of the imine to the amino acid. These steps are almost equally rate limiting for V/K/sub ammonia/ with alanine dehydrogenase, while with glutamate dehydrogenase carbinolamine formation, imine formation, and release of glutamate after hydride transfer provide most of the rate limitation of V/K/sub ammonia/. Release of oxidized nucleotide is largely rate limiting for V/sub max/ for both enzymes. When ..beta..-hydroxypyruvate replaces pyruvate, or 3-acetylpyridine NADH (Acpyr-NADH) or thio-NADH replaces NADH with alanine dehydrogenase, nucleotide release no longer limits V/sub max/, and hydride transfer becomes more rate limiting. With glutamate dehydrogenase, replacement of ..cap alpha..-ketoglutarate by ..cap alpha..-ketovalerate makes hydride transfer more rate limiting. Use of Acpyr-NADPH has a minimal effect with ..cap alpha..-ketoglutarate but causes an 8-fold decrease in V/sub max/ with ..cap alpha..-ketovalerate, with hydride transfer the major rate-limiting step. In contrast, thio-NADPH with either ..cap alpha..-keto acid causes carbinolamide formation to become almost completely rate limiting. These studies show the power of multiple isotope effects in deducing details of the chemistry and changes in rate-limiting step(s) in complicated reaction mechanisms such as those of alanine and glutamate dehydrogenases.

  17. Glutamate 190 is a general acid catalyst in the 6-phosphogluconate-dehydrogenase-catalyzed reaction.

    PubMed

    Karsten, W E; Chooback, L; Cook, P F

    1998-11-10

    Site-directed mutagenesis was used to change E190 of sheep liver 6-phosphogluconate dehydrogenase to A, D, H, K, Q, and R to probe its possible role as a general acid catalyst. Each of the mutant proteins was characterized with respect to the pH dependence of kinetic parameters. Mutations that eliminate a titrable group at position 190, result in pH-rate profiles with no observable pK on the basic side of the V/K6PG profile. Mutations that change the pK of the group at position 190 result in the expected pK perturbations in the V/K6PG profile. Kinetic parameters obtained at the pH optimum in the pH-rate profiles are consistent with a rate-limiting tautomerization of the 1,2-enediol of ribulose 5-phosphate consistent with the proposed role of E190. Data are also consistent with some participation of E190 in an isomerization required to form the active Michaelis complex.

  18. Amino acid changes within antenna helix are responsible for different regulatory preferences of human glutamate dehydrogenase isozymes.

    PubMed

    Choi, Myung-Min; Kim, Eun-A; Yang, Seung-Ju; Choi, Soo Young; Cho, Sung-Woo; Huh, Jae-Wan

    2007-07-01

    Human glutamate dehydrogenase (hGDH) exists in hGDH1 (housekeeping isozyme) and in hGDH2 (nerve-specific isozyme), which differ markedly in their allosteric regulation. Because they differ in only 16 of their 505 amino acids, the regulatory preferences must arise from amino acid residues that are not common between hGDH1 and hGDH2. To our knowledge none of the mutagenesis studies on the hGDH isozymes to date have identified the amino acid residues fully responsible for the different regulatory preferences between hGDH1 and hGDH2. In this study we constructed hGDH1(hGDH2(390-448))hGDH1 (amino acid segment 390-448 of hGDH1 replaced by the corresponding hGDH2 segment) and hGDH2(hGDH1(390-448))hGDH2 (amino acid segment 390-448 of hGDH2 replaced by the corresponding hGDH1 segment) by swapping the corresponding amino acid segments in hGDH1 and hGDH2. The chimeric enzymes by reciprocal swapping resulted in double mutations in amino acid sequences at 415 and 443 residues that are not common between hGDH1 and hGDH2 and are located in the C-terminal 48-residue "antenna" helix, which is thought to be part of the regulatory domain of mammalian GDHs. Functional analyses revealed that the doubly mutated chimeric enzymes almost completely acquired most of the different regulatory preferences between hGDH1 and hGDH2 for electrophoretic mobility, heat-stability, ADP activation, palmitoyl-CoA inhibition, and l-leucine activation, except for GTP inhibition. Our results indicate that substitutions of the residues in the antenna region may be important evolutionary changes that led to the adaptation of hGDH2 to the unique metabolic needs of the nerve tissue.

  19. Radiochemical assay for a NADP+-specific gamma-glutamate semialdehyde dehydrogenase extracted from mitochondrial membrane of rat intestinal epithelial cells

    SciTech Connect

    Kramer, J.J.; Gooding, R.C.; Jones, M.E.

    1988-02-01

    A radiochemical assay has been developed for a NADP+-specific gamma-glutamate semialdehyde dehydrogenase from rat intestinal epithelial cells. The spectrophotometric assay utilized to measure the enzyme in bacterial cell homogenates is not sensitive enough for homogenates from rat mitochondria, which require an assay that can measure as little as 0.5 nmol NADPH formed/min/ml extract. The assay described here is sensitive to 0.1 nmol product formed/min/ml of extract and employs the use of (/sup 3/H)pyrroline 5-carboxylate which is phosphorylated and oxidized by the enzyme to gamma-(/sup 3/H)glutamyl phosphate, a product that decomposes to (/sup 3/H)pyrrolidone 5-carboxylate. The latter product is separated from the substrate by ion-exchange chromatography. In order to correct for any product loss during separation by ion-exchange (/sup 14/C)pyrrolidone 5-carboxylate is added as an internal standard to the deproteinized assay mixture. Under the assay conditions described mammalian gamma-glutamate semialdehyde dehydrogenase activity is linear with respect to time and protein concentration. Comparison between the kinetic parameters reported for the bacterial enzyme and those reported here for the mammalian enzyme indicate similarities in the pH optima as well as a requirement for phosphate. Kinetic studies on mammalian enzyme yield apparent Km values of 1.8 mM for pyrroline 5-carboxylate, 0.2 mM for NADP+, and 11.3 mM for phosphate.

  20. [Glutamate dehydrogenase activity in platelets of patients with endogenous psychosis].

    PubMed

    Prokhorova, T A; Boksha, I S; Savushkina, O K; Tereshkina, E B; Vorob'eva, E A; Pomytkin, A N; Kaleda, V G; Burbaeva, G Sh

    2016-01-01

    Цель исследования — сравнение уровня ферментативной активности тромбоцитарной глутаматдегидрогеназы (ГДГ) у больных с эндогенными психозами и в контрольной группе здоровых и выявление возможной связи активности ГДГ с клинико-психопатологической оценкой состояния больных. Материал и методы. Группу больных составили 69 мужчин, госпитализированных с приступом эндогенного психоза (шизофрения у 48 больных, шизоаффективное расстройство у 21); 34 пациента были с первым психотическим приступом (ПП) и 35 — c повторными приступами заболевания (ППЗ). Больные обследовались с использованием шкалы PANSS до и после курса антипсихотической терапии. Контрольные образцы крови были взяты у 34 мужчин без психической патологии. Изо всех образцов крови были выделены тромбоциты, в белковых экстрактах которых была определена удельная активность ГДГ. Результаты и заключение. Активность ГДГ в группе больных в целом до лечения была достоверно ниже, чем в контрольной группе. Активность ГДГ достоверно различалась в контроле и до лечения в подгруппах больных с ПП и с ППЗ. У больных с ПП (но не ППЗ) до лечения активность ГДГ достоверно коррелировала с баллами по PANSS. У больных с ПП активность ГДГ до лечения достоверно была связана с баллами PANSS после лечения: чем выше активность ГДГ у больного, тем ниже оценка по PANSS после лечения. Сделан вывод, что определение исходных значений активности тромбоцитарной ГДГ может иметь практическую ценность для прогноза эффективности антипсихотической фармакотерапии у пациентов с ПП эндогенного психоза.

  1. Effects of herbal infusions, tea and carbonated beverages on alcohol dehydrogenase and aldehyde dehydrogenase activity.

    PubMed

    Li, Sha; Gan, Li-Qin; Li, Shu-Ke; Zheng, Jie-Cong; Xu, Dong-Ping; Li, Hua-Bin

    2014-01-01

    Various alcoholic beverages containing different concentrations of ethanol are widely consumed, and excessive alcohol consumption may result in serious health problems. The consumption of alcoholic beverages is often accompanied by non-alcoholic beverages, such as herbal infusions, tea and carbonated beverages to relieve drunk symptoms. The aim of this study was to supply new information on the effects of these beverages on alcohol metabolism for nutritionists and the general public, in order to reduce problems associated with excessive alcohol consumption. The effects of 57 kinds of herbal infusions, tea and carbonated beverages on alcohol dehydrogenase and aldehyde dehydrogenase activity were evaluated. Generally, the effects of these beverages on alcohol dehydrogenase and aldehyde dehydrogenase activity are very different. The results suggested that some beverages should not be drank after excessive alcohol consumption, and several beverages may be potential dietary supplements for the prevention and treatment of problems related to excessive alcohol consumption.

  2. Glutamate excitotoxicity and Ca2+-regulation of respiration: Role of the Ca2+ activated mitochondrial transporters (CaMCs).

    PubMed

    Rueda, Carlos B; Llorente-Folch, Irene; Traba, Javier; Amigo, Ignacio; Gonzalez-Sanchez, Paloma; Contreras, Laura; Juaristi, Inés; Martinez-Valero, Paula; Pardo, Beatriz; Del Arco, Araceli; Satrustegui, Jorgina

    2016-08-01

    Glutamate elicits Ca(2+) signals and workloads that regulate neuronal fate both in physiological and pathological circumstances. Oxidative phosphorylation is required in order to respond to the metabolic challenge caused by glutamate. In response to physiological glutamate signals, cytosolic Ca(2+) activates respiration by stimulation of the NADH malate-aspartate shuttle through Ca(2+)-binding to the mitochondrial aspartate/glutamate carrier (Aralar/AGC1/Slc25a12), and by stimulation of adenine nucleotide uptake through Ca(2+) binding to the mitochondrial ATP-Mg/Pi carrier (SCaMC-3/Slc25a23). In addition, after Ca(2+) entry into the matrix through the mitochondrial Ca(2+) uniporter (MCU), it activates mitochondrial dehydrogenases. In response to pathological glutamate stimulation during excitotoxicity, Ca(2+) overload, reactive oxygen species (ROS), mitochondrial dysfunction and delayed Ca(2+) deregulation (DCD) lead to neuronal death. Glutamate-induced respiratory stimulation is rapidly inactivated through a mechanism involving Poly (ADP-ribose) Polymerase-1 (PARP-1) activation, consumption of cytosolic NAD(+), a decrease in matrix ATP and restricted substrate supply. Glutamate-induced Ca(2+)-activation of SCaMC-3 imports adenine nucleotides into mitochondria, counteracting the depletion of matrix ATP and the impaired respiration, while Aralar-dependent lactate metabolism prevents substrate exhaustion. A second mechanism induced by excitotoxic glutamate is permeability transition pore (PTP) opening, which critically depends on ROS production and matrix Ca(2+) entry through the MCU. By increasing matrix content of adenine nucleotides, SCaMC-3 activity protects against glutamate-induced PTP opening and lowers matrix free Ca(2+), resulting in protracted appearance of DCD and protection against excitotoxicity in vitro and in vivo, while the lack of lactate protection during in vivo excitotoxicity explains increased vulnerability to kainite-induced toxicity in Aralar

  3. ROLE OF GLUTAMATE DEHYDROGENASE AND GLUTAMINE SYNTHETASE IN CHLORELLA VULGARIS DURING ASSIMILATION OF AMMONIUM WHEN JOINTLY IMMOBILIZED WITH THE MICROALGAE-GROWTH-PROMOTING BACTERIUM AZOSPIRILLUM BRASILENSE(1).

    PubMed

    De-Bashan, Luz E; Magallon, Paola; Antoun, Hani; Bashan, Yoav

    2008-10-01

    Enzymatic activities of glutamate dehydrogenase (GDH) and glutamine synthetase (GS) participating in the nitrogen metabolism and related ammonium absorption were assayed after the microalga Chlorella vulgaris Beij. was jointly immobilized with the microalgae-growth-promoting bacterium Azospirillum brasilense. At initial concentrations of 3, 6, and 10 mg · L(-1)  NH4 (+) , joint immobilization enhances growth of C. vulgaris but does not affect ammonium absorption capacity of the microalga. However, at 8 mg · L(-1)  NH4 (+) , joint immobilization enhanced ammonium absorption by the microalga without affecting the growth of the microalgal population. Correlations between absorption of ammonium per cell and per culture showed direct (negative and positive) linear correlations between these parameters and microalga populations at 3, 6, and 10 mg · L(-1)  NH4 (+) , but not at 8 mg · L(-1)  NH4 (+) , where the highest absorption of ammonium occurred. In all cultures, immobilized and jointly immobilized, having the four initial ammonium concentrations, enzymatic activities of Chlorella are affected by A. brasilense. Regardless of the initial concentration of ammonium, GS activity in C. vulgaris was always higher when jointly immobilized and determined on a per-cell basis. When jointly immobilized, only at an initial concentration of 8 mg · L(-1)  NH4 (+) was GDH activity per cell higher.

  4. Characterization of Yersinia enterocolitica, Y. intermedia, Y. aldovae, Y. frederiksenii, Y. kristensenii and Y. pseudotuberculosis by electrophoretic polymorphism of acid phosphatase, esterases, and glutamate and malate dehydrogenases.

    PubMed

    Goullet, P; Picard, B

    1988-02-01

    Acid phosphatase, esterases, and glutamate and malate dehydrogenases of 192 strains of Yersinia enterocolitica, Y. intermedia, Y. aldovae, Y. frederiksenii, Y. kristensenii and Y. pseudotuberculosis were analysed by horizontal polyacrylamide agarose gel electrophoresis and by isoelectrofocusing in thin-layer polyacrylamide gels. The six species were clearly separated from each other by their distinct enzyme electrophoretic polymorphism. For Y. enterocolitica, the strains of biotype 5 were differentiated from the other biotypes by the mobility of glutamate dehydrogenase. For Y. frederiksenii, six zymotypes were delineated by pI and by the mobility of the enzymes. Variation in number or mobility of esterases within each species could represent a marker for epidemiological and ecological analyses. A linear relationship was obtained between the mean genetic diversity coefficient of enzymes and the mean percentage DNA-DNA relatedness of Y. intermedia, Y. aldovae, Y. enterocolitica and Y. frederiksenii.

  5. Trehalose and sorbitol alter the kinetic pattern of inactivation of glutamate dehydrogenase during drying in levitated microdroplets.

    PubMed

    Lorenzen, Elke; Lee, Geoffrey

    2013-12-01

    A single-droplet acoustic levitator was used to determine the drying rate and the kinetics of inactivation of glutamate dehydrogenase in the presence of added trehalose or sorbitol. The solution was also spray dried under the same process condition of drying gas temperature on a bench-top machine. Both trehalose and sorbitol delay the point of onset of enzyme inactivation which lies after the critical point of drying. Both carbohydrates also reduce the apparent rate constant of inactivation calculated during the subsequent inactivation phase. The carbohydrates stabilise, therefore, the enzyme during droplet drying and particle formation mainly during the falling rate drying period. There is no difference between the stabilising effects of the two carbohydrates when examined as levitated single droplets. This suggests the importance of water replacement as a stabilising mechanism in the levitated droplets/particles. On spray drying, the trehalose stabilises the enzyme better than does the sorbitol at a drying gas (outlet) temperature of 60°C. This suggests glass formation with the trehalose but not the sorbitol during the very rapid drying process of small-atomised droplets in the spray dryer.

  6. Deletion of glutamate dehydrogenase in beta-cells abolishes part of the insulin secretory response not required for glucose homeostasis.

    PubMed

    Carobbio, Stefania; Frigerio, Francesca; Rubi, Blanca; Vetterli, Laurène; Bloksgaard, Maria; Gjinovci, Asllan; Pournourmohammadi, Shirin; Herrera, Pedro L; Reith, Walter; Mandrup, Susanne; Maechler, Pierre

    2009-01-01

    Insulin exocytosis is regulated in pancreatic ss-cells by a cascade of intracellular signals translating glucose levels into corresponding secretory responses. The mitochondrial enzyme glutamate dehydrogenase (GDH) is regarded as a major player in this process, although its abrogation has not been tested yet in animal models. Here, we generated transgenic mice, named betaGlud1(-/-), with ss-cell-specific GDH deletion. Our results show that GDH plays an essential role in the full development of the insulin secretory response. In situ pancreatic perfusion revealed that glucose-stimulated insulin secretion was reduced by 37% in betaGlud1(-/-). Furthermore, isolated islets with either constitutive or acute adenovirus-mediated knock-out of GDH showed a 49 and 38% reduction in glucose-induced insulin release, respectively. Adenovirus-mediated re-expression of GDH in betaGlud1(-/-) islets fully restored glucose-induced insulin release. Thus, GDH appears to account for about 40% of glucose-stimulated insulin secretion and to lack redundant mechanisms. In betaGlud1(-/-) mice, the reduced secretory capacity resulted in lower plasma insulin levels in response to both feeding and glucose load, while body weight gain was preserved. The results demonstrate that GDH is essential for the full development of the secretory response in beta-cells. However, maximal secretory capacity is not required for maintenance of glucose homeostasis in normo-caloric conditions.

  7. Characterization of a NADH-dependent glutamate dehydrogenase mutant of Arabidopsis demonstrates the key role of this enzyme in root carbon and nitrogen metabolism.

    PubMed

    Fontaine, Jean-Xavier; Tercé-Laforgue, Thérèse; Armengaud, Patrick; Clément, Gilles; Renou, Jean-Pierre; Pelletier, Sandra; Catterou, Manuella; Azzopardi, Marianne; Gibon, Yves; Lea, Peter J; Hirel, Bertrand; Dubois, Frédéric

    2012-10-01

    The role of NADH-dependent glutamate dehydrogenase (GDH) was investigated by studying the physiological impact of a complete lack of enzyme activity in an Arabidopsis thaliana plant deficient in three genes encoding the enzyme. This study was conducted following the discovery that a third GDH gene is expressed in the mitochondria of the root companion cells, where all three active GDH enzyme proteins were shown to be present. A gdh1-2-3 triple mutant was constructed and exhibited major differences from the wild type in gene transcription and metabolite concentrations, and these differences appeared to originate in the roots. By placing the gdh triple mutant under continuous darkness for several days and comparing it to the wild type, the evidence strongly suggested that the main physiological function of NADH-GDH is to provide 2-oxoglutarate for the tricarboxylic acid cycle. The differences in key metabolites of the tricarboxylic acid cycle in the triple mutant versus the wild type indicated that, through metabolic processes operating mainly in roots, there was a strong impact on amino acid accumulation, in particular alanine, γ-aminobutyrate, and aspartate in both roots and leaves. These results are discussed in relation to the possible signaling and physiological functions of the enzyme at the interface of carbon and nitrogen metabolism.

  8. Postsynaptic activation at the squid giant synapse by photolytic release of L-glutamate from a 'caged' L-glutamate.

    PubMed Central

    Corrie, J E; DeSantis, A; Katayama, Y; Khodakhah, K; Messenger, J B; Ogden, D C; Trentham, D R

    1993-01-01

    1. Pharmacological evidence suggests L-glutamate is a strong candidate as a transmitter at the giant synapse of the squid. Postsynaptic activation at the giant synapse cannot be effected by conventional application of putative neurotransmitters by iontophoresis or perfusion, apparently because the complex structure of the synapse prevents a sufficiently rapid change in concentration at the postsynaptic membrane. Flash photolytic release of L-glutamate from a pharmacologically inert 'caged' L-glutamate pre-equilibrated in the stellate ganglion of Alloteuthis or Loligo was used to determine whether L-glutamate can produce postsynaptic activation when released rapidly in the synaptic clefts. 2. The preparation, reaction mechanism and properties of the caged L-glutamate, N-1-(2-nitrophenyl)ethoxycarbonyl-L-glutamate, are described. The product quantum yield on photolysis was 0.65 (+/- 0.05). On flash photolysis glutamate release followed a single exponential time-course in the pH range 5.5-7.8. The rate constant was proportional to [H+] and was 93 s-1 at pH 5.5 and 16 degrees C in artificial sea water (ionic strength, I = 0.68 M). 3. At pH 7.8 flash photolysis of caged glutamate pre-equilibrated in the synapse caused only a slow depolarization. A second photolytic release of L-glutamate or transsynaptic activation produced no further depolarization, suggesting desensitization and inactivation of postsynaptic mechanisms by the initial pulse of L-glutamate. 4. Synaptic transmission in the giant synapse was normal at pH 5.5. Flash photolysis at pH 5.5 caused rapid production of L-glutamate within the synaptic cleft and a fast postsynaptic depolarization which generated postsynaptic action potentials.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7901400

  9. [Glutamine synthetase-like protein, glutamate dehydrogenase, and cytochrome c-oxidase in platelets of patients with the first episode psychosis in the course of treatment].

    PubMed

    Burbaeva, G Sh; Boksha, I S; Kaleda, V G; Barkhatova, A N; Turishcheva, M S; Omel'chenko, M A; Tereshkina, E B; Savushkina, O K; Starodubtseva, L I; Prokhorova, T A; Vorob'eva, E A

    2011-01-01

    The authors searched for correlations between amounts of platelet proteins and results of psychometric tests in patients with the first episode psychosis (schizophrenia, schizoaffective psychosis) in the course of their combined antipsychotic treatment with haloperidol and clozapine. Psychometric evaluations (PANSS, BPRS) and analyses of platelet enzymes - glutamine synthetase-like protein (GSLP), glutamate dehydrogenase (GDH), and cytochrome c-oxidase (COX) - were carried out before, during, and after the treatment. These proteins were also analyzed in matched controls. All the parameters comprised a database, followed by statistical data processing using Statistica 6.0 (StatSoft) software, nonparametric statistics module. The patients before the treatment, when compared with controls, demonstrated significantly decreased COX activity (p=0,0000001) and increased GSLP amount (p=0,006) with a positive correlation between GSLP amount and PANSSneg (R=0,34, p<0,01). Those patients who displayed initially low COX activity (below median) demonstrated significant increase in COX activity after the treatment. Negative correlations were revealed between COX activity and PANSS, PANSSpsy scores during the treatment, i.e. the lower was COX activity, the more severe syndromes were observed. Negative correlations were found between the initial COX activity and PANSS, PANSSpsy, BPRS scores after the treatment, i.e., the higher was COX before the treatment, the less prominent syndromes were observed after the treatment. Significantly more "non-responders" by PANSSneg were found among the patients with low GSLP level (below median) than their calculated expected amount. The COX activity measured before the treatment was significantly lower in patients with schizophrenia than in patients with schizoaffective disorder (SAD) (p=0,038). In SAD patients, the initial COX activity was negatively correlated with PANSSpsy and BPRS scores after the treatment (R=-0,5, p=0,02), i.e. the lower

  10. Different rates of synthesis and degradation of two chloroplastic ammonium-inducible NADP-specific glutamate dehydrogenase isoenzymes during induction and deinduction in Chlorella sorokiniana cells

    SciTech Connect

    Bascomb, N.F.; Prunkard, D.E.; Schmidt, R.R.

    1987-01-01

    The kinetics of accumulation (per milliliter of culture) of the ..cap alpha..- and ..beta..-subunits, associated with chloroplast-localized ammonium inducible nicotinamide adenine dinucleotide phosphate-specific glutamate dehydrogenase (NADP-GDH) isoenzymes, were measured during a 3 hour induction of synchronized daughter cells of Chlorella sorokiniana in 29 millimolar ammonium medium under photoautotrophic conditions. The ..beta..-subunit holoenzyme(s) accumulated in a linear manner for 3 hours without an apparent induction lag. A 40 minute induction lag preceded the accumulation of the ..cap alpha..-subunit holoenzyme(s). After 120 minutes, the ..cap alpha..-subunit ceased accumulating and thereafter remained at a constant level. From pulse-chase experiments, using /sup 35/SO/sub 4/ and immunochemical procedures, the rate of synthesis of the ..cap alpha..-subunit was shown to be greater than the ..beta..-subunit during the first 80 minutes of induction. The ..cap alpha..- and ..beta..-subunits had different rates of degradation during the induction period (t/sub 1/2/ = 50 versus 150 minutes, respectively) and during the deinduction period (t/sub 1/2/ = 5 versus 13.5 minutes) after removal of ammonium from the culture. During deinduction, total NADP-GDH activity decreased with a half-time of 9 minutes. Cycloheximide completely inhibited the synthesis and degradation of both subunits. A model for regulation of expression of the NADP-GDH gene was proposed.

  11. Evidence for messenger ribonucleic acid of an ammonium-inducible glutamate dehydrogenase and synthesis, covalent modification, and degradation of enzyme subunits in uninduced Chlorella sorokiniana cells.

    PubMed Central

    Turner, K J; Bascomb, N F; Lynch, J J; Molin, W T; Thurston, C F; Schmidt, R R

    1981-01-01

    The cells of Chlorella sorokiniana cultured in nitrate medium contain no detectable catalytic activity of an ammonium-inducible nicotinamide adenine dinucleotide phosphate-specific glutamate dehydrogenase (NADP-GDH). However, several lines of experimental evidence indicated that the NADP-GDH messenger ribonucleic acid was present at high levels and was being translated in uninduced cells. First, binding studies with 125I-labeled anti-NADP-GDH immunoglobulin G and total polysomes isolated from uninduced and induced cells showed that NADP-GDH subunits were being synthesized on polysomes from both types of cells. Second, when polyadenylic acid-containing ribonucleic acid was extracted from polysomes from uninduced and induced cells and placed into a messenger ribonucleic acid-dependent in vitro translation system, NADP-GDH subunits were synthesized from the ribonucleic acid from both sources. Third, when ammonia was added to uninduced cells, NADP-GDH antigen accumulated without an apparent induction lag. Fourth, by use of a specific immunoprecipitation procedure coupled to pulse-chase studies with [35S]sulfate, it was shown that the NADP-GDH subunits are rapidly synthesized, covalently modified, and then degraded in uninduced cells. PMID:7217012

  12. Among the twenty classical L-amino acids, only glutamate directly activates metabotropic glutamate receptors.

    PubMed

    Frauli, Mélanie; Neuville, Pascal; Vol, Claire; Pin, Jean-Philippe; Prézeau, Laurent

    2006-02-01

    Under pathophysiological conditions, cellular amino acids can be profusely released from cells into the cerebral interstitial space. Because several class-C G protein coupled receptors (GPCRs) display a broad natural ligand spectrum, being sensitive to more than one endogenous ligand, we wondered whether the related metabotropic glutamate (mGlu) receptors could be modulated by various types of L-amino acids, allowing them to sense large increase in extracellular amino acid concentration. Here, the agonist, antagonist and allosteric effects of the twenty classical L-amino acids were evaluated on the eight mGlu receptor subtypes. We show that, in addition to glutamate (Glu), cysteine, aspartate and asparagine also lead to the activation of mGlu3, 4 and 5. Interestingly, our data demonstrate that the effect of these three amino acids did not result from a direct activation of the receptors, but from an indirect action involving Glu-transporters/exchangers. These data first demonstrate that mGlu receptors, unlike other class-C GPCRs, display an extremely high selectivity towards one ligand. Moreover, our results also show that Glu transport systems allow mGlu receptors to sense large increase in the extracellular concentration of some amino acids. Such a system will certainly lead to a large increase in some mGlu receptor activity under pathological conditions, such as seizure, ischemia or other brain injuries. PMID:16310227

  13. Dehydrogenase activity of forest soils depends on the assay used

    NASA Astrophysics Data System (ADS)

    Januszek, Kazimierz; Długa, Joanna; Socha, Jarosław

    2015-01-01

    Dehydrogenases are exclusively intracellular enzymes, which play an important role in the initial stages of oxidation of soil organic matter. One of the most frequently used methods to estimate dehydrogenase activity in soil is based on the use of triphenyltetrazolium chloride as an artificial electron acceptor. The purpose of this study was to compare the activity of dehydrogenases of forest soils with varied physicochemical properties using different triphenyltetrazolium chloride assays. The determination was carried out using the original procedure by Casida et al., a modification of the procedure which involves the use of Ca(OH)2 instead of CaCO3, the Thalmann method, and the assay by Casida et al. without addition of buffer or any salt. Soil dehydrogenase activity depended on the assay used. Dehydrogenase determined by the Casida et al. method without addition of buffer or any salt correlated with the pH values of soils. The autoclaved strongly acidic samples of control soils showed high concentrations of triphenylformazan, probably due to chemical reduction of triphenyltetrazolium chloride. There is, therefore, a need for a sterilization method other than autoclaving, ie a process that results in significant changes in soil properties, thus helping to increase the chemical reduction of triphenyltetrazolium chloride.

  14. Glutamate dehydrogenase and Na+-K+ ATPase expression and growth response of Litopenaeus vannamei to different salinities and dietary protein levels

    NASA Astrophysics Data System (ADS)

    Li, Erchao; Arena, Leticia; Lizama, Gabriel; Gaxiola, Gabriela; Cuzon, Gerard; Rosas, Carlos; Chen, Liqiao; van Wormhoudt, Alain

    2011-03-01

    Improvement in the osmoregulation capacity via nutritional supplies is vitally important in shrimp aquaculture. The effects of dietary protein levels on the osmoregulation capacity of the Pacific white shrimp ( L. vannamei) were investigated. This involved an examination of growth performance, glutamate dehydrogenase (GDH) and Na+-K+ ATPase mRNA expression,, and GDH activity in muscles and gills. Three experimental diets were formulated, containing 25%, 40%, and 50% dietary protein, and fed to the shrimp at a salinity of 25. After 20 days, no significant difference was observed in weight gain, though GDH and Na+-K+ ATPase gene expression and GDH activity increased with higher dietary protein levels. Subsequently, shrimp fed diets with 25% and 50% dietary protein were transferred into tanks with salinities of 38 and 5, respectively, and sampled at weeks 1 and 2. Shrimp fed with 40% protein at 25 in salinity (optimal conditions) were used as a control. Regardless of the salinities, shrimp fed with 50% dietary protein had significantly higher growth performance than other diets; no significant differences were found in comparison with the control. Shrimp fed with 25% dietary protein and maintained at salinities of 38 and 5 had significantly lower weight gain values after 2 weeks. Ambient salinity change also stimulated the hepatosomatic index, which increased in the first week and then recovered to a relatively normal level, as in the control, after 2 weeks. These findings indicate that in white shrimp, the specific protein nutrient and energy demands related to ambient salinity change are associated with protein metabolism. Increased dietary protein level could improve the osmoregulation capacity of L. vannamei with more energy resources allocated to GDH activity and expression.

  15. Interaction of carbohydrates with alcohol dehydrogenase: Effect on enzyme activity.

    PubMed

    Jadhav, Swati B; Bankar, Sandip B; Granström, Tom; Ojamo, Heikki; Singhal, Rekha S; Survase, Shrikant A

    2015-09-01

    Alcohol dehydrogenase was covalently conjugated with three different oxidized carbohydrates i.e., glucose, starch and pectin. All the carbohydrates inhibited the enzyme. The inhibition was studied with respect to the inhibition rate constant, involvement of thiol groups in the binding, and structural changes in the enzyme. The enzyme activity decreased to half of its original activity at the concentration of 2 mg/mL of pectin, 4 mg/mL of glucose and 10 mg/mL of starch within 10 min at pH 7. This study showed oxidized pectin to be a potent inhibitor of alcohol dehydrogenase followed by glucose and starch. Along with the aldehyde-amino group interaction, thiol groups were also involved in the binding between alcohol dehydrogenase and carbohydrates. The structural changes occurring on binding of alcohol dehydrogenase with oxidized carbohydrates was also confirmed by fluorescence spectrophotometry. Oxidized carbohydrates could thus be used as potential inhibitors of alcohol dehydrogenase.

  16. Alpha helical structures in the leader sequence of human GLUD2 glutamate dehydrogenase responsible for mitochondrial import.

    PubMed

    Kotzamani, Dimitra; Plaitakis, Andreas

    2012-09-01

    Human glutamate dehydrogenase (hGDH) exists in two highly homologous isoforms with a distinct regulatory and tissue expression profile: a housekeeping hGDH1 isoprotein encoded by the GLUD1 gene and an hGDH2 isoenzyme encoded by the GLUD2 gene. There is evidence that both isoenzymes are synthesized as pro-enzymes containing a 53 amino acid long N-terminal leader peptide that is cleaved upon translocation into the mitochondria. However, this GDH signal peptide is substantially larger than that of most nuclear DNA-encoded mitochondrial proteins, the leader sequence of which typically contains 17-35 amino acids and they often form a single amphipathic α-helix. To decode the structural elements that are essential for the mitochondrial targeting of human GDHs, we performed secondary structure analyses of their leader sequence. These analyses predicted, with 82% accuracy, that both leader peptides are positively charged and that they form two to three α-helices, separated by intermediate loops. The first α-helix of hGDH2 is strongly amphipathic, displaying both a positively charged surface and a hydrophobic plane. We then constructed GLUD2-EGFP deletion mutants and used them to transfect three mammalian cell lines (HEK293, COS 7 and SHSY-5Y). Confocal laser scanning microscopy, following co-transfection with pDsRed2-Mito mitochondrial targeting vector, revealed that deletion of the entire leader sequence prevented the enzyme from entering the mitochondria, resulting in its retention in the cytoplasm. Deletion of the first strongly amphipathic α-helix only was also sufficient to prevent the mitochondrial localization of the truncated protein. Moreover, truncated leader sequences, retaining the second and/or the third putative α-helix, failed to restore the mitochondrial import of hGDH2. As such, the first N-terminal alpha helical structure is crucial for the mitochondrial import of hGDH2 and these findings may have implications in understanding the evolutionary

  17. Changes in nitrogen assimilation, metabolism, and growth in transgenic rice plants expressing a fungal NADP(H)-dependent glutamate dehydrogenase (gdhA).

    PubMed

    Abiko, Tomomi; Wakayama, Masataka; Kawakami, Akira; Obara, Mitsuhiro; Kisaka, Hiroaki; Miwa, Tetsuya; Aoki, Naohiro; Ohsugi, Ryu

    2010-07-01

    In plants, glutamine synthetase (GS) is the enzyme that is mainly responsible for the assimilation of ammonium. Conversely, in microorganisms such as bacteria and Ascomycota, NADP(H)-dependent glutamate dehydrogenase (GDH) and GS both have important roles in ammonium assimilation. Here, we report the changes in nitrogen assimilation, metabolism, growth, and grain yield of rice plants caused by an ectopic expression of NADP(H)-GDH (gdhA) from the fungus Aspergillus niger in the cytoplasm. An investigation of the kinetic properties of purified recombinant protein showed that the fungal gdhA had 5.4-10.2 times higher V(max) value and 15.9-43.1 times higher K(m) value for NH(4)(+), compared with corresponding values for rice cytosolic GS as reported in the literature. These results suggested that the introduction of fungal GDH into rice could modify its ammonium assimilation pathway. We therefore expressed gdhA in the cytoplasm of rice plants. NADP(H)-GDH activities in the gdhA-transgenic lines were markedly higher than those in a control line. Tracer experiments by feeding with (15)NH(4)(+) showed that the introduced gdhA, together with the endogenous GS, directly assimilated NH(4)(+) absorbed from the roots. Furthermore, in comparison with the control line, the transgenic lines showed an increase in dry weight and nitrogen content when sufficient nitrogen was present, but did not do so under low-nitrogen conditions. Under field condition, the transgenic line examined showed a significant increase in grain yield in comparison with the control line. These results suggest that the introduction of fungal gdhA into rice plants could lead to better growth and higher grain yield by enhancing the assimilation of ammonium.

  18. Identification and characterization of kinetically competent carbinolamine and alpha-iminoglutarate complexes in the glutamate dehydrogenase-catalyzed oxidation of L-glutamate using a multiwavelength transient state approach.

    PubMed

    Maniscalco, S J; Saha, S K; Fisher, H F

    1998-10-13

    A highly constrained and heavily overdetermined multiwavelength transient state kinetic approach has been used to study the oxidative deamination of L-glutamate catalyzed by beef liver glutamate dehydrogenase. Spectra generated using the known enzyme-reduced coenzyme-substrate spectrum served as models for deconvolution of kinetic scan data. Deconvolution of the multiwavelength time course array shows formation of three distinguishable intermediates in the reaction sequence, an ultrablue-shifted complex, an ultrared-shifted complex, and a blue-shifted complex. The ultrablue-shifted entity is identified as the enzyme-NADPH-alpha-iminoglutarate complex (ERI) and the ultrared as the enzyme-NADPH-alpha-carbinolamine complex (ERC). The blue-shifted complex is characterized as the E-NADPH-ketoglutarate species (ERK). The location of these species along the reaction coordinate has been determined and their kinetic competency in the reaction sequence has been established by fitting the concentration time courses of the components for both the alpha-deuterio- and the alpha-protio-L-glutamate reactions to the now highly constrained differential equations derived from a kinetic scheme involving the sequential formation of alpha-iminoglutarate, alpha-carbinolamine, and alpha-ketoglutarate-reduced coenzyme complexes, following the formation of two prehydride transfer complexes. PMID:9772187

  19. Spatial variability of the dehydrogenase activity in forest soils

    NASA Astrophysics Data System (ADS)

    Błońska, Ewa; Lasota, Jarosław

    2014-05-01

    The aim of this study was to assess the spatial variability of the dehydrogenase activity (DH) in forest soils using geostatistics. We have studied variability soil dehydrogenase and their relationship with variability of some physic-chemical properties. Two study areas (A and B) were set up in southern Poland in the Zlotoryja Forest District. Study areas were covered by different types of vegetation (A- broadleaf forest with beech, ash and sycamore), B- coniferous forest with Norway spruce). The soils were classified as Dystric Cambisols (WRB 2006). The samples for laboratory testing were collected from 49 places on each areas. 15 cm of surface horizon of soil were taken (with previously removed litter). Dehydrogenase activity was marked with Lenhard's method according to the Casida procedure. Soil pH, nitrogen (N) and soil organic carbon (C) content (by LECO CNS 2000 carbon analyzer) was marked. C/N ratio was calculated. Particle size composition was determined using laser diffraction. Statistical analysis were performed using STATISTICA 10 software. Geostatistical analysis and mapping were done by application of GS 9+ (Gamma Design) and Surfer 11 (Golden Software). The activity of DH ranged between 5,02 and 71,20 mg TPP• kg-1 •24 h-1 on the A area and between 0,94 and 16,47 mg TPP• kg-1 •24 h-1. Differences in spatial variability of the analised features were noted. The variability of dehydrogenase activity on the A study area was described by an exponential model, whereas on the B study area the spatial correlation has not been noted. The relationship of dehydrogenase activity with the remaining parameters of soil was noted only in the case of A study area. The variability of organic carbon content on the A and B study areas were described by an exponential model. The variability of nitrogen content on both areas were described by an spherical model.

  20. Electrogenic glutamate uptake in glial cells is activated by intracellular potassium

    NASA Astrophysics Data System (ADS)

    Barbour, Boris; Brew, Helen; Attwell, David

    1988-09-01

    Uptake of glutamate into glial cells in the CNS maintains the extracellular glutamate concentration below neurotoxic levels and helps terminate its action as a neurotransmitter 1. The co-transport of two sodium ions on the glutamate carrier is thought to provide the energy needed to transport glutamate into cells2,3. We have shown recently that glutamate uptake can be detected electrically because the excess of Na+ ions transported with each glutamate anion results in a net current flow into the cell4. We took advantage of the control of the environment, both inside and outside the cell, provided by whole-cell patch-clamping and now report that glutamate uptake is activated by intracellular potassium and inhibited by extracellular potassium. Our results indicate that one K+ ion is transported out of the cell each time a glutamate anion and three Na+ ions are transported in. A carrier with this stoichiometry can accumulate glutamate against a much greater concentration gradient than a carrier co-transporting one glutamate anion and two Na+ ions. Pathological rises in extracellular potassium concentration will inhibit glutamate uptake by depolarizing glial cells and by preventing the loss of K+ from the glutamate carrier. This will facilitate a rise in the extracellular glutamate concentration to neurotoxic levels and contribute to the neuronal death occurring in brain anoxia and ischaemia.

  1. An examination of aspartate decarboxylase and glutamate decarboxylase activity in mosquitoes

    PubMed Central

    Richardson, Graham; Ding, Haizhen; Rocheleau, Tom; Mayhew, George; Reddy, Erin; Han, Qian; Christensen, Bruce M.; Li, Jianyong

    2010-01-01

    A major pathway of beta-alanine synthesis in insects is through the alpha-decarboxylation of aspartate, but the enzyme involved in the decarboxylation of aspartate has not been clearly defined in mosquitoes and characterized in any insect species. In this study, we expressed two putative mosquito glutamate decarboxylase-like enzymes of mosquitoes and critically analyzed their substrate specificity and biochemical properties. Our results provide clear biochemical evidence establishing that one of them is an aspartate decarboxylase and the other is a glutamate decarboxylase. The mosquito aspartate decarboxylase functions exclusively on the production of beta-alanine with no activity with glutamate. Likewise the mosquito glutamate decarboxylase is highly specific to glutamate with essentially no activity with aspartate. Although insect aspartate decarboxylase shares high sequence identity with glutamate decarboxylase, we are able to closely predict aspartate decarboxylase from glutamate decarboxylase based on the difference of their active site residues. PMID:19842059

  2. Metabolic pathways and activity-dependent modulation of glutamate concentration in the human brain.

    PubMed

    Mangia, Silvia; Giove, Federico; Dinuzzo, Mauro

    2012-11-01

    Glutamate is one of the most versatile molecules present in the human brain, involved in protein synthesis, energy production, ammonia detoxification, and transport of reducing equivalents. Aside from these critical metabolic roles, glutamate plays a major part in brain function, being not only the most abundant excitatory neurotransmitter, but also the precursor for γ-aminobutyric acid, the predominant inhibitory neurotransmitter. Regulation of glutamate levels is pivotal for normal brain function, as abnormal extracellular concentration of glutamate can lead to impaired neurotransmission, neurodegeneration and even neuronal death. Understanding how the neuron-astrocyte functional and metabolic interactions modulate glutamate concentration during different activation status and under physiological and pathological conditions is a challenging task, and can only be tentatively estimated from current literature. In this paper, we focus on describing the various metabolic pathways which potentially affect glutamate concentration in the brain, and emphasize which ones are likely to produce the variations in glutamate concentration observed during enhanced neuronal activity in human studies.

  3. The activity of alcohol dehydrogenase (ADH) isoenzymes and aldehyde dehydrogenase (ALDH) in the sera of patients with brain cancer.

    PubMed

    Jelski, Wojciech; Laniewska-Dunaj, Magdalena; Orywal, Karolina; Kochanowicz, Jan; Rutkowski, Robert; Szmitkowski, Maciej

    2014-12-01

    Human brain tissue contains various alcohol dehydrogenase (ADH) isoenzymes and possess also aldehyde dehydrogenase (ALDH) activity. In our last experiments we have shown that ADH and ALDH are present also in the brain tumour cells. Moreover the activities of total ADH and class I isoenzymes were significantly higher in cancer tissue than healthy cells. It can suggests that these changes may be reflected by enzyme activity in the serum of patients with brain cancer. Serum samples were taken for routine biochemical investigation from 62 patients suffering from brain cancer (36 glioblastoma, 26 meningioma). For the measurement of the activity of class I and II ADH isoenzymes and ALDH activity, the fluorometric methods were used. The total ADH activity and activity of class III and IV isoenzymes were measured by the photometric method. A statistically significant increase of class I alcohol dehydrogenase isoenzymes was found in the sera of patients with brain cancer. The median activity of this class isoenzyme in the patients group increased about 24 % in the comparison to the control level. The total alcohol dehydrogenase activity was also significantly higher (26 %) among patients with brain tumour than healthy ones. The activities of other tested ADH isoenzymes and total ALDH were unchanged. The increase of the activity of total ADH and class I alcohol dehydrogenase isoenzyme in the sera of patients with brain cancer seems to be caused by the release of this isoenzyme from tumour's cells.

  4. Activity-Dependent Plasticity of Astroglial Potassium and Glutamate Clearance

    PubMed Central

    Cheung, Giselle; Sibille, Jérémie; Zapata, Jonathan; Rouach, Nathalie

    2015-01-01

    Recent evidence has shown that astrocytes play essential roles in synaptic transmission and plasticity. Nevertheless, how neuronal activity alters astroglial functional properties and whether such properties also display specific forms of plasticity still remain elusive. Here, we review research findings supporting this aspect of astrocytes, focusing on their roles in the clearance of extracellular potassium and glutamate, two neuroactive substances promptly released during excitatory synaptic transmission. Their subsequent removal, which is primarily carried out by glial potassium channels and glutamate transporters, is essential for proper functioning of the brain. Similar to neurons, different forms of short- and long-term plasticity in astroglial uptake have been reported. In addition, we also present novel findings showing robust potentiation of astrocytic inward currents in response to repetitive stimulations at mild frequencies, as low as 0.75 Hz, in acute hippocampal slices. Interestingly, neurotransmission was hardly affected at this frequency range, suggesting that astrocytes may be more sensitive to low frequency stimulation and may exhibit stronger plasticity than neurons to prevent hyperexcitability. Taken together, these important findings strongly indicate that astrocytes display both short- and long-term plasticity in their clearance of excess neuroactive substances from the extracellular space, thereby regulating neuronal activity and brain homeostasis. PMID:26346563

  5. Plant NAD(H)-Glutamate Dehydrogenase Consists of Two Subunit Polypeptides and Their Participation in the Seven Isoenzymes Occurs in an Ordered Ratio

    PubMed Central

    Loulakakis, Konstantinos A.; Roubelakis-Angelakis, Kalliopi A.

    1991-01-01

    The structure and function of NAD(H)-glutamate dehydrogenase in plants was studied by using grapevine (Vitis vinifera L. cv Sultanina) callus grown under different nitrogen sources. The enzyme consists of two subunit-polypeptides, α and β, with similar antigenic properties but with different molecular mass and charge. The two polypeptides have molecular masses of 43.0 and 42.5 kilodaltons, respectively. The holoenzyme is hexameric and is resolved into seven isoenzymes by native gel electrophoresis. Two-dimensional native/SDS-PAGE revealed that the 1 and 7 isoenzymes are homohexamers and the isoenzymes 2 through 6 are hybrids of the two polypeptides following an ordered ratio. The total quantity of α- and β-polypeptides and the isoenzymic pattern was altered by the exogenous nitrogen source. The sample derived from callus grown on nitrate or glutamic acid contained a slightly greater amount of β-polypeptide and of the more cathodal isoenzymes, whereas α-polypeptide and the more anodal isoenzymes predominated in callus grown in the presence of either ammonium or glutamine. The anabolic reaction was correlated with the α- and the catabolic reaction with the β-polypeptide; this could suggest that each isoenzyme exhibits anabolic and catabolic function of different magnitude. The isoenzymic patterns did not obey the expected binomial distribution proportions. ImagesFigure 1Figure 2Figure 3Figure 4 PMID:16668355

  6. Inhibition of glutamine synthesis induces glutamate dehydrogenase-dependent ammonia fixation into alanine in co-cultures of astrocytes and neurons.

    PubMed

    Dadsetan, Sherry; Bak, Lasse K; Sørensen, Michael; Keiding, Susanne; Vilstrup, Hendrik; Ott, Peter; Leke, Renata; Schousboe, Arne; Waagepetersen, Helle S

    2011-09-01

    It has been previously demonstrated that ammonia exposure of neurons and astrocytes in co-culture leads to net synthesis not only of glutamine but also of alanine. The latter process involves the concerted action of glutamate dehydrogenase (GDH) and alanine aminotransferase (ALAT). In the present study it was investigated if the glutamine synthetase (GS) inhibitor methionine sulfoximine (MSO) would enhance alanine synthesis by blocking the GS-dependent ammonia scavenging process. Hence, co-cultures of neurons and astrocytes were incubated for 2.5h with [U-(13)C]glucose to monitor de novo synthesis of alanine and glutamine in the absence and presence of 5.0 mM NH(4)Cl and 10 mM MSO. Ammonia exposure led to increased incorporation of label but not to a significant increase in the amount of these amino acids. However, in the presence of MSO, glutamine synthesis was blocked and synthesis of alanine increased leading to an elevated content intra- as well as extracellularly of this amino acid. Treatment with MSO led to a dramatic decrease in glutamine content and increased the intracellular contents of glutamate and aspartate. The large increase in alanine during exposure to MSO underlines the importance of the GDH and ALAT biosynthetic pathway for ammonia fixation, and it points to the use of a GS inhibitor to ameliorate the brain toxicity and edema induced by hyperammonemia, events likely related to glutamine synthesis.

  7. Lactate Dehydrogenase B Controls Lysosome Activity and Autophagy in Cancer.

    PubMed

    Brisson, Lucie; Bański, Piotr; Sboarina, Martina; Dethier, Coralie; Danhier, Pierre; Fontenille, Marie-Joséphine; Van Hée, Vincent F; Vazeille, Thibaut; Tardy, Morgane; Falces, Jorge; Bouzin, Caroline; Porporato, Paolo E; Frédérick, Raphaël; Michiels, Carine; Copetti, Tamara; Sonveaux, Pierre

    2016-09-12

    Metabolic adaptability is essential for tumor progression and includes cooperation between cancer cells with different metabolic phenotypes. Optimal glucose supply to glycolytic cancer cells occurs when oxidative cancer cells use lactate preferentially to glucose. However, using lactate instead of glucose mimics glucose deprivation, and glucose starvation induces autophagy. We report that lactate sustains autophagy in cancer. In cancer cells preferentially to normal cells, lactate dehydrogenase B (LDHB), catalyzing the conversion of lactate and NAD(+) to pyruvate, NADH and H(+), controls lysosomal acidification, vesicle maturation, and intracellular proteolysis. LDHB activity is necessary for basal autophagy and cancer cell proliferation not only in oxidative cancer cells but also in glycolytic cancer cells. PMID:27622334

  8. Glucose metabolism in perfused skeletal muscle. Pyruvate dehydrogenase activity in starvation, diabetes and exercise.

    PubMed Central

    Hagg, S A; Taylor, S I; Ruberman, N B

    1976-01-01

    1. The interconversion of pyruvate dehydrogenase between its inactive phosphorylated and active dephosphorylated forms was studied in skeletal muscle. 2. Exercise, induced by electrical stimulation of the sciatic nerve (5/s), increased the measured activity of (active) pyruvate dehydrogenase threefold in intact anaesthetized rated within 2 min. No further increase was seen after 15 min of stimulation. 3. In the perfused rat hindquarter, (active) pyruvate dehydrogenase activity was decreased by 50% in muscle of starved and diabetic rats. Exercise produced a twofold increase in its activity in all groups; however, the relative differences between fed, starved and diabetic groups persisted. 4. Perfusion of muslce with acetoacetate (2 mM) decreased (active) pyruvate dehydrogenase activity by 50% at rest but not during exercise. 5. Whole-tissue concentrations of pyruvate and citrate, inhibitors of (active) pyruvate dehydrogenase kinase and (inactive) pyruvate dehydrogenase phosphate phosphatase respectively, were not altered by excerise. A decrease in the ATP/ADP ratio was observed, but did not appear to be sufficient to account for the increase in (active) pyruvate dehydrogenase activity. 6. The results suggest that interconversion of the phosphorylated and dephosphorylated forms of pyruvate dehydrogenase plays a major role in the regulation of pyruvate oxidation by eomparison of enzyme activity with measurements of lactate oxidation in the perfused hindquarter [see the preceding paper, Berger et al. (1976)] suggest that pyruvate oxidation is also modulated by the concentrations of substrates, cofactors and inhibitors of (active) pyruvate dehydrogenase activity. PMID:825112

  9. Neuronal activity regulates remyelination via glutamate signalling to oligodendrocyte progenitors

    PubMed Central

    Gautier, Hélène O. B.; Evans, Kimberley A.; Volbracht, Katrin; James, Rachel; Sitnikov, Sergey; Lundgaard, Iben; James, Fiona; Lao-Peregrin, Cristina; Reynolds, Richard; Franklin, Robin J. M.; Káradóttir, Ragnhildur T

    2015-01-01

    Myelin regeneration can occur spontaneously in demyelinating diseases such as multiple sclerosis (MS). However, the underlying mechanisms and causes of its frequent failure remain incompletely understood. Here we show, using an in-vivo remyelination model, that demyelinated axons are electrically active and generate de novo synapses with recruited oligodendrocyte progenitor cells (OPCs), which, early after lesion induction, sense neuronal activity by expressing AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid)/kainate receptors. Blocking neuronal activity, axonal vesicular release or AMPA receptors in demyelinated lesions results in reduced remyelination. In the absence of neuronal activity there is a ∼6-fold increase in OPC number within the lesions and a reduced proportion of differentiated oligodendrocytes. These findings reveal that neuronal activity and release of glutamate instruct OPCs to differentiate into new myelinating oligodendrocytes that recover lost function. Co-localization of OPCs with the presynaptic protein VGluT2 in MS lesions implies that this mechanism may provide novel targets to therapeutically enhance remyelination. PMID:26439639

  10. Effect of ageing and ischemia on enzymatic activities linked to Krebs' cycle, electron transfer chain, glutamate and aminoacids metabolism of free and intrasynaptic mitochondria of cerebral cortex.

    PubMed

    Villa, Roberto Federico; Gorini, Antonella; Hoyer, Siegfried

    2009-12-01

    The effect of ageing and the relationships between the catalytic properties of enzymes linked to Krebs' cycle, electron transfer chain, glutamate and aminoacid metabolism of cerebral cortex, a functional area very sensitive to both age and ischemia, were studied on mitochondria of adult and aged rats, after complete ischemia of 15 minutes duration. The maximum rate (Vmax) of the following enzyme activities: citrate synthase, malate dehydrogenase, succinate dehydrogenase for Krebs' cycle; NADH-cytochrome c reductase as total (integrated activity of Complex I-III), rotenone sensitive (Complex I) and cytochrome oxidase (Complex IV) for electron transfer chain; glutamate dehydrogenase, glutamate-oxaloacetate-and glutamate-pyruvate transaminases for glutamate metabolism were assayed in non-synaptic, perikaryal mitochondria and in two populations of intra-synaptic mitochondria, i.e., the light and heavy mitochondrial fraction. The results indicate that in normal, steady-state cerebral cortex, the value of the same enzyme activity markedly differs according (a) to the different populations of mitochondria, i.e., non-synaptic or intra-synaptic light and heavy, (b) and respect to ageing. After 15 min of complete ischemia, the enzyme activities of mitochondria located near the nucleus (perikaryal mitochondria) and in synaptic structures (intra-synaptic mitochondria) of the cerebral tissue were substantially modified by ischemia. Non-synaptic mitochondria seem to be more affected by ischemia in adult and particularly in aged animals than the intra-synaptic light and heavy mitochondria. The observed modifications in enzyme activities reflect the metabolic state of the tissue at each specific experimental condition, as shown by comparative evaluation with respect to the content of energy-linked metabolites and substrates. The derangements in enzyme activities due to ischemia is greater in aged than in adult animals and especially the non-synaptic and the intra-synaptic light

  11. [Inhibition of glutamine synthetase activity by biologically active derivatives of glutamic acid].

    PubMed

    Firsova, N A; Selivanova, K M; Alekseeva, L V; Evstigneeva, Z G

    1986-05-01

    The inhibition of activity of glutamine synthetase from Chlorella and porcine brain by 4-hydroxy-D-4-fluoro-D,L- and 4-amino-D,L-glutamic acids diastereoisomers was studied. Each compound was shown to exert the same inhibiting effect on glutamine synthetase from both sources. In case of threo-4-hydroxy-D-glutamic acid the inhibition of the Chlorella enzyme was of a competitive and of a completely mixed type. The enzyme inhibition by 4-fluoro-D, L-glutamic acids seemed to be of a completely non-competitive type. The Ki values for all inhibition reactions were determined. A comparison of biochemical parameters and biological activity revealed that the most effective inhibitors of the enzyme exert a most potent antitumour and antiviral action.

  12. Microbial metabolic activity in soil as measured by dehydrogenase determinations

    NASA Technical Reports Server (NTRS)

    Casida, L. E., Jr.

    1977-01-01

    The dehydrogenase technique for measuring the metabolic activity of microorganisms in soil was modified to use a 6-h, 37 C incubation with either glucose or yeast extract as the electron-donating substrate. The rate of formazan production remained constant during this time interval, and cellular multiplication apparently did not occur. The technique was used to follow changes in the overall metabolic activities of microorganisms in soil undergoing incubation with a limiting concentration of added nutrient. The sequence of events was similar to that obtained by using the Warburg respirometer to measure O2 consumption. However, the major peaks of activity occurred earlier with the respirometer. This possibly is due to the lack of atmospheric CO2 during the O2 consumption measurements.

  13. [Activity of NADP-dependent glycerol-3-phosphate dehydrogenase in skeletal muscles of animals].

    PubMed

    Epifanova, Iu E; Glushankov, E P; Kolotilova, A I

    1978-01-01

    The NADP-dependent glycerol-3-phosphate dehydrogenase activity was studied in sketetal muscles of the rat, rabbit and frog. The dehydrogenase activity in the skeletal muscles of the rat and rabbit was higher than that of the frog. The enzyme activity was found to depend upon the buffer, being higher in tris-HCl buffer than in triethanolamine buffer.

  14. Resolving the Role of Plant NAD-Glutamate Dehydrogenase: III. Overexpressing Individually or Simultaneously the Two Enzyme Subunits Under Salt Stress Induces Changes in the Leaf Metabolic Profile and Increases Plant Biomass Production.

    PubMed

    Tercé-Laforgue, Thérèse; Clément, Gilles; Marchi, Laura; Restivo, Francesco M; Lea, Peter J; Hirel, Bertrand

    2015-10-01

    NAD-dependent glutamate dehydrogenase (NAD-GDH) of higher plants has a central position at the interface between carbon and nitrogen metabolism due to its ability to carry out the deamination of glutamate. In order to obtain a better understanding of the physiological function of NAD-GDH under salt stress conditions, transgenic tobacco (Nicotiana tabacum L.) plants that overexpress two genes from Nicotiana plumbaginifolia individually (GDHA and GDHB) or simultaneously (GDHA/B) were grown in the presence of 50 mM NaCl. In the different GDH overexpressors, the NaCl treatment induced an additional increase in GDH enzyme activity, indicating that a post-transcriptional mechanism regulates the final enzyme activity under salt stress conditions. A greater shoot and root biomass production was observed in the three types of GDH overexpressors following growth in 50 mM NaCl, when compared with the untransformed plants subjected to the same salinity stress. Changes in metabolites representative of the plant carbon and nitrogen status were also observed. They were mainly characterized by an increased amount of starch present in the leaves of the GDH overexpressors as compared with the wild type when plants were grown in 50 mM NaCl. Metabolomic analysis revealed that overexpressing the two genes GDHA and GDHB, individually or simultaneously, induced a differential accumulation of several carbon- and nitrogen-containing molecules involved in a variety of metabolic, developmental and stress-responsive processes. An accumulation of digalactosylglycerol, erythronate and porphyrin was found in the GDHA, GDHB and GDHA/B overexpressors, suggesting that these molecules could contribute to the improved performance of the transgenic plants under salinity stress conditions.

  15. Resolving the Role of Plant NAD-Glutamate Dehydrogenase: III. Overexpressing Individually or Simultaneously the Two Enzyme Subunits Under Salt Stress Induces Changes in the Leaf Metabolic Profile and Increases Plant Biomass Production.

    PubMed

    Tercé-Laforgue, Thérèse; Clément, Gilles; Marchi, Laura; Restivo, Francesco M; Lea, Peter J; Hirel, Bertrand

    2015-10-01

    NAD-dependent glutamate dehydrogenase (NAD-GDH) of higher plants has a central position at the interface between carbon and nitrogen metabolism due to its ability to carry out the deamination of glutamate. In order to obtain a better understanding of the physiological function of NAD-GDH under salt stress conditions, transgenic tobacco (Nicotiana tabacum L.) plants that overexpress two genes from Nicotiana plumbaginifolia individually (GDHA and GDHB) or simultaneously (GDHA/B) were grown in the presence of 50 mM NaCl. In the different GDH overexpressors, the NaCl treatment induced an additional increase in GDH enzyme activity, indicating that a post-transcriptional mechanism regulates the final enzyme activity under salt stress conditions. A greater shoot and root biomass production was observed in the three types of GDH overexpressors following growth in 50 mM NaCl, when compared with the untransformed plants subjected to the same salinity stress. Changes in metabolites representative of the plant carbon and nitrogen status were also observed. They were mainly characterized by an increased amount of starch present in the leaves of the GDH overexpressors as compared with the wild type when plants were grown in 50 mM NaCl. Metabolomic analysis revealed that overexpressing the two genes GDHA and GDHB, individually or simultaneously, induced a differential accumulation of several carbon- and nitrogen-containing molecules involved in a variety of metabolic, developmental and stress-responsive processes. An accumulation of digalactosylglycerol, erythronate and porphyrin was found in the GDHA, GDHB and GDHA/B overexpressors, suggesting that these molecules could contribute to the improved performance of the transgenic plants under salinity stress conditions. PMID:26251210

  16. [Effect Of Polyelectrolytes on Catalytic Activity of Alcohol Dehydrogenase].

    PubMed

    Dubrovsky, A V; Musina, E V; Kim, A L; Tikhonenko, S A

    2016-01-01

    Fluorescent and optical spectroscopy were used to study the interaction of alcohol dehydrogenase (ADH) with negatively charged polystyrene sulfonate (PSS) and dextran sulfate (DS), as well as positively charged poly(diallyldimethylammonium) (PDADMA). As found, DS and PDADMA did not affect the structural and catalytic enzyme properties. In contrast, PSS slightly decreased the protein self-fluorescence over 1 h of incubation, which is associated with partial destruction of its quaternary (globular) structure. Investigation of the ADH activity with and without PSS showed its dependency on the incubation time and the PSS presence. Sodium chloride (2.0 M and 0.2 M) or ammonium sulfate (0.1 M) added to the reaction mixture did not completely protect the enzyme quaternary structure from the PSS action. However ammonium sulfate or 0.2 M sodium chloride stabilized the enzyme and partially inhibited the negative PSS effect. PMID:27266256

  17. Evaluation of a rapid membrane enzyme immunoassay for the simultaneous detection of glutamate dehydrogenase and toxin for the diagnosis of Clostridium difficile infection.

    PubMed

    Kim, Heejung; Kim, Wan Hee; Kim, Myungsook; Jeong, Seok Hoon; Lee, Kyungwon

    2014-05-01

    We evaluated the new C. DIFF QUIK CHEK COMPLETE (CD COMPLETE; TechLab, USA), which is a rapid membrane enzyme immunoassay that uses a combination of glutamate dehydrogenase (GDH) antigen and toxin A and B detection. A total of 608 consecutive loose stool specimens collected from the patients with suspected Clostridium difficile infection (CDI) from August to December 2012 were subjected to the CD COMPLETE and VIDAS Clostridium difficile A & B (VIDAS CDAB; bioMérieux, France). Their performances were compared with a toxigenic culture as a reference. Stool specimens that were culture-negative and CD COMPLETE- or VIDAS CDAB-positive were analyzed by using an enrichment procedure. In comparison to the toxigenic cultures, sensitivity, specificity, positive predictive values (PPV), and negative predictive values (NPV) were 63.6%, 98.0%, 76.1%, and 96.4%, respectively, for the CD COMPLETE-toxin and 75.5%, 97.4%, 72.5%, and 97.8%, respectively, for the VIDAS CDAB. In comparison to the enriched C. difficile cultures, the sensitivity, specificity, PPV, and NPV for the CD COMPLETE-GDH were 91.0%, 92.4%, 70.5%, and 98.1%, respectively. The CD COMPLETE is a reliable method for the diagnosis of CDI and provides greater sensitivity than toxin enzyme immunoassay alone. Furthermore, the CD COMPLETE-GDH has advantages over direct culture in detecting C. difficile.

  18. RECIPIENT PRETRANSPLANT INOSINE MONOPHOSPHATE DEHYDROGENASE ACTIVITY IN NONMYELOABLATIVE HCT

    PubMed Central

    Bemer, Meagan J.; Risler, Linda J.; Phillips, Brian R.; Wang, Joanne; Storer, Barry E.; Sandmaier, Brenda M.; Duan, Haichuan; Raccor, Brianne S.; Boeckh, Michael J.; McCune, Jeannine S.

    2014-01-01

    Mycophenolic acid, the active metabolite of mycophenolate mofetil (MMF), inhibits inosine monophosphate dehydrogenase (IMPDH) activity. IMPDH is the rate-limiting enzyme involved in de novo synthesis of guanosine nucleotides and catalyzes the oxidation of inosine 5’- monophosphate (IMP) to xanthosine 5’-monophosphate (XMP). We developed a highly sensitive liquid chromatography–mass spectrometry method to quantitate XMP concentrations in peripheral blood mononuclear cells (PMNC) isolated from the recipient pretransplant and used this method to determine IMPDH activity in 86 nonmyeloablative allogeneic hematopoietic cell transplantation (HCT) patients. The incubation procedure and analytical method yielded acceptable within-sample and within-individual variability. Considerable between-individual variability was observed (12.2-fold). Low recipient pretransplant IMPDH activity was associated with increased day +28 donor T-cell chimerism, more acute graft-versus-host disease (GVHD), lower neutrophil nadirs, and more cytomegalovirus reactivation, but not with chronic GVHD, relapse, non-relapse mortality, or overall mortality. We conclude that quantitation of the recipient’s pretransplant IMPDH activity in PMNC lysate could provide a useful biomarker to evaluate a recipient’s sensitivity to MMF, but confirmatory studies are needed. Further trials should be conducted to confirm our findings and to optimize postgrafting immunosuppression in nonmyeloablative HCT recipients. PMID:24923537

  19. Aldehyde dehydrogenase activity promotes survival of human muscle precursor cells

    PubMed Central

    Jean, Elise; Laoudj-Chenivesse, Dalila; Notarnicola, Cécile; Rouger, Karl; Serratrice, Nicolas; Bonnieu, Anne; Gay, Stéphanie; Bacou, Francis; Duret, Cédric; Carnac, Gilles

    2011-01-01

    Abstract Aldehyde dehydrogenases (ALDH) are a family of enzymes that efficiently detoxify aldehydic products generated by reactive oxygen species and might therefore participate in cell survival. Because ALDH activity has been used to identify normal and malignant cells with stem cell properties, we asked whether human myogenic precursor cells (myoblasts) could be identified and isolated based on their levels of ALDH activity. Human muscle explant-derived cells were incubated with ALDEFLUOR, a fluorescent substrate for ALDH, and we determined by flow cytometry the level of enzyme activity. We found that ALDH activity positively correlated with the myoblast-CD56+ fraction in those cells, but, we also observed heterogeneity of ALDH activity levels within CD56-purified myoblasts. Using lentiviral mediated expression of shRNA we demonstrated that ALDH activity was associated with expression of Aldh1a1 protein. Surprisingly, ALDH activity and Aldh1a1 expression levels were very low in mouse, rat, rabbit and non-human primate myoblasts. Using different approaches, from pharmacological inhibition of ALDH activity by diethylaminobenzaldehyde, an inhibitor of class I ALDH, to cell fractionation by flow cytometry using the ALDEFLUOR assay, we characterized human myoblasts expressing low or high levels of ALDH. We correlated high ALDH activity ex vivo to resistance to hydrogen peroxide (H2O2)-induced cytotoxic effect and in vivo to improved cell viability when human myoblasts were transplanted into host muscle of immune deficient scid mice. Therefore detection of ALDH activity, as a purification strategy, could allow non-toxic and efficient isolation of a fraction of human myoblasts resistant to cytotoxic damage. PMID:19840193

  20. Analysis of rat cytosolic 9-cis-retinol dehydrogenase activity and enzymatic characterization of rat ADHII.

    PubMed

    Popescu, G; Napoli, J L

    2000-01-01

    We report the characterization of two enzymes that catalyze NAD(+)-dependent 9-cis-retinol dehydrogenase activity in rat liver cystol. Alcohol dehydrogenase class I (ADHI) contributes > 80% of the NA D+-dependent 9-cis-retinol dehydrogenase activity recovered, whereas alcohol dehydrogenase class II (ADHII), not identified previously at the protein level, nor characterized enzymatically in rat, accounts for approximately 2% of the activity. Rat ADHII exhibits properties different from those described for human ADHII. Moreover, rat ADHII-catalyzed rates of ethanol dehydrogenation are markedly lower than octanol or retinoid dehydrogenation rates. Neither ethanol nor 4-methylpyrazole inhibits the 9-cis-retinol dehydrogenase activity of rat ADHII. We propose that ADHII represents the previously observed additional retinoid oxidation activity of rat liver cytosol which occurred in the presence of either ethanol or 4-methylpyrazole. We also show that human and rat ADHII differ considerably in enzymatic properties. PMID:10606766

  1. Accelerated Lactate Dehydrogenase Activity Potentiates Osteoclastogenesis via NFATc1 Signaling.

    PubMed

    Ahn, Heejin; Lee, Kyunghee; Kim, Jin Man; Kwon, So Hyun; Lee, Seoung Hoon; Lee, Soo Young; Jeong, Daewon

    2016-01-01

    Osteoclasts seem to be metabolic active during their differentiation and bone-resorptive activation. However, the functional role of lactate dehydrogenase (LDH), a tetrameric enzyme consisting of an A and/or B subunit that catalyzes interconversion of pyruvate to lactate, in RANKL-induced osteoclast differentiation is not known. In this study, RANKL treatment induced gradual gene expression and activation of the LDH A2B2 isotype during osteoclast differentiation as well as the LDH A1B3 and B4 isotypes during osteoclast maturation after pre-osteoclast formation. Glucose consumption and lactate production in growth media were accelerated during osteoclast differentiation, together with enhanced expression of H+-lactate co-transporter and increased extracellular acidification, demonstrating that glycolytic metabolism was stimulated during differentiation. Further, oxygen consumption via mitochondria was stimulated during osteoclast differentiation. On the contrary, depletion of LDH-A or LDH-B subunit suppressed both glycolytic and mitochondrial metabolism, resulting in reduced mature osteoclast formation via decreased osteoclast precursor fusion and down-regulation of the osteoclastogenic critical transcription factor NFATc1 and its target genes. Collectively, our findings suggest that RANKL-induced LDH activation stimulates glycolytic and mitochondrial respiratory metabolism, facilitating mature osteoclast formation via osteoclast precursor fusion and NFATc1 signaling. PMID:27077737

  2. Accelerated Lactate Dehydrogenase Activity Potentiates Osteoclastogenesis via NFATc1 Signaling

    PubMed Central

    Kim, Jin Man; Kwon, So Hyun; Lee, Seoung Hoon; Lee, Soo Young; Jeong, Daewon

    2016-01-01

    Osteoclasts seem to be metabolic active during their differentiation and bone-resorptive activation. However, the functional role of lactate dehydrogenase (LDH), a tetrameric enzyme consisting of an A and/or B subunit that catalyzes interconversion of pyruvate to lactate, in RANKL-induced osteoclast differentiation is not known. In this study, RANKL treatment induced gradual gene expression and activation of the LDH A2B2 isotype during osteoclast differentiation as well as the LDH A1B3 and B4 isotypes during osteoclast maturation after pre-osteoclast formation. Glucose consumption and lactate production in growth media were accelerated during osteoclast differentiation, together with enhanced expression of H+-lactate co-transporter and increased extracellular acidification, demonstrating that glycolytic metabolism was stimulated during differentiation. Further, oxygen consumption via mitochondria was stimulated during osteoclast differentiation. On the contrary, depletion of LDH-A or LDH-B subunit suppressed both glycolytic and mitochondrial metabolism, resulting in reduced mature osteoclast formation via decreased osteoclast precursor fusion and down-regulation of the osteoclastogenic critical transcription factor NFATc1 and its target genes. Collectively, our findings suggest that RANKL-induced LDH activation stimulates glycolytic and mitochondrial respiratory metabolism, facilitating mature osteoclast formation via osteoclast precursor fusion and NFATc1 signaling. PMID:27077737

  3. Inhibitory effects of ionic liquids on the lactic dehydrogenase activity.

    PubMed

    Dong, Xing; Fan, Yunchang; Zhang, Heng; Zhong, Yingying; Yang, Yang; Miao, Juan; Hua, Shaofeng

    2016-05-01

    Ionic liquids (ILs) were widely used in scientific and industrial application and have been reported to possess potential toxicity to the environment and human health. The effects of six typical N-methylimidazolium-based ILs ([Cnmim]X, n=4, 6, 8; X=Br(-), Cl(-), BF4(-), CF3SO3(-)) on the lactic dehydrogenase (LDH) activity and the molecular interaction mechanism of ILs and the LDH were investigated with the aid of spectroscopic techniques. Experimental results showed that the LDH activity was inhibited in the presence of ILs. For the ILs with the same anion but different cations, their inhibitory ability on the LDH activity increased with increasing the alkyl chain length on the IL cation. Thermodynamic parameters, enthalpy change (ΔH) and entropy change (ΔS) were obtained by analyzing the fluorescence behavior of LDH with the addition of ILs. Both positive ΔH and ΔS suggested that hydrophobicity was the major driven force in the interaction process as expected. PMID:26802246

  4. On the regulative role of the glutamate receptor in mitochondria.

    PubMed

    Selin, Alexey A; Lobysheva, Natalia V; Nesterov, Semen V; Skorobogatova, Yulia A; Byvshev, Ivan M; Pavlik, Lyubov L; Mikheeva, Irina B; Moshkov, Dmitry A; Yaguzhinsky, Lev S; Nartsissov, Yaroslav R

    2016-05-01

    The purpose of this work was to study the regulative role of the glutamate receptor found earlier in the brain mitochondria. In the present work a glutamate-dependent signaling system with similar features was detected in mitochondria of the heart. The glutamate-dependent signaling system in the heart mitochondria was shown to be suppressed by γ-aminobutyric acid (GABA). The GABA receptor presence in the heart mitochondria was shown by golding with the use of antibodies to α- and β-subunits of the receptor. The activity of glutamate receptor was assessed according to the rate of synthesis of hydrogen peroxide. The glutamate receptor in mitochondria could be activated only under conditions of hypoxic stress, which in model experiments was imitated by blocking Complex I by rotenone or fatty acids. The glutamate signal in mitochondria was shown to be calcium- and potential-dependent and the activation of the glutamate cascade was shown to be accompanied by production of hydrogen peroxide. It was discovered that H2O2 synthesis involves two complexes of the mitochondrial electron transfer system - succinate dehydrogenase (SDH) and fatty acid dehydrogenase (ETF:QO). Thus, functions of the glutamate signaling system are associated with the system of respiration-glycolysis switching (the Pasteur-Crabtree) under conditions of hypoxia. PMID:26812870

  5. 2-Oxoglutarate dehydrogenase and pyruvate dehydrogenase activities in plant mitochondria: interaction via a common coenzyme a pool.

    PubMed

    Dry, I B; Wiskich, J T

    1987-08-15

    2-Oxoglutarate (2-OG)-dependent O2 uptake by washed or purified turnip (Brassica rapa L.) and pea (Pisum sativum L. cv. Massey Gem) leaf mitochondria, in the presence of malonate, was inhibited between 65 and 90% by micromolar levels of pyruvate. The inhibition was not observed in the absence of malonate and was reversed by alpha-cyano-4-hydroxycinnamic acid. The inhibition was also reversed by oxaloacetate or by malate, but not by any other tricarboxylic acid cycle intermediates. The stimulation of O2 uptake by oxaloacetate was half maximal at 8-9 microM and was transient, indicating its action was not mediated through the complete metabolic removal of pyruvate. Pyruvate had not effect on 2-OG oxidation under conditions in which pyruvate dehydrogenase was not active, indicating that pyruvate metabolism, rather than pyruvate itself, was responsible for producing the inhibition of 2-OG oxidation. Similar results were obtained with detergent-treated mitochondrial extracts with the exception that the inhibition of 2-OG oxidation by pyruvate could also be reversed by coenzyme A. The results suggest that pyruvate inhibits 2-oxoglutarate oxidation, in intact plant mitochondria, by sequestering intramitochondrial CoA as acetyl-CoA and, in the absence of citrate synthase activity, reduces the amount of free coenzyme A available for 2-oxoglutarate dehydrogenase. These results indicate that pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase share a common CoA pool within plant mitochondria and that the turnover of the acyl-CoA product of one enzyme will dramatically influence the activity of the other.

  6. Enzyme-based flow injection analysis system for glutamine and glutamate in mammalian cell culture media.

    PubMed

    Mayer, C; Frauer, A; Schalkhammer, T; Pittner, F

    1999-03-01

    We present the setup of a flow injection analysis system designed for on-line monitoring of glutamate and glutamine. These amino acids represent a major energy source in mammalian cell culture. A cycling assay consisting of glutamate dehydrogenase and aspartate aminotransferase produces NADH proportional to the glutamate concentration in the sample. NADH is then measured spectrophotometrically. Glutamine is determined by conversion to glutamate which is fed into the cycling assay. The conversion of glutamine to glutamate is catalyzed by asparaginase. Asparaginase was used in place of glutaminase due to its relatively high reactivity with glutamine and a pH optimum similar to that of glutamate dehydrogenase. The enzymes were immobilized covalently to activated controlled pore glass beads and integrated into the flow injection analysis system. The application of the immobilized enzymes and the technical setup are presented in this paper.

  7. Structural Studies of Yeast Δ1-Pyrroline-5-carboxylate Dehydrogenase (ALDH4A1): Active Site Flexibility and Oligomeric State

    PubMed Central

    2015-01-01

    The proline catabolic enzyme Δ1-pyrroline-5-carboxylate dehydrogenase (ALDH4A1) catalyzes the NAD+-dependent oxidation of γ-glutamate semialdehyde to l-glutamate. In Saccharomyces cerevisiae, ALDH4A1 is encoded by the PUT2 gene and known as Put2p. Here we report the steady-state kinetic parameters of the purified recombinant enzyme, two crystal structures of Put2p, and the determination of the oligomeric state and quaternary structure from small-angle X-ray scattering and sedimentation velocity. Using Δ1-pyrroline-5-carboxylate as the substrate, catalytic parameters kcat and Km were determined to be 1.5 s–1 and 104 μM, respectively, with a catalytic efficiency of 14000 M–1 s–1. Although Put2p exhibits the expected aldehyde dehydrogenase superfamily fold, a large portion of the active site is disordered in the crystal structure. Electron density for the 23-residue aldehyde substrate-binding loop is absent, implying substantial conformational flexibility in solution. We furthermore report a new crystal form of human ALDH4A1 (42% identical to Put2p) that also shows disorder in this loop. The crystal structures provide evidence of multiple active site conformations in the substrate-free form of the enzyme, which is consistent with a conformational selection mechanism of substrate binding. We also show that Put2p forms a trimer-of-dimers hexamer in solution. This result is unexpected because human ALDH4A1 is dimeric, whereas some bacterial ALDH4A1s are hexameric. Thus, global sequence identity and domain of life are poor predictors of the oligomeric states of ALDH4A1. Mutation of a single Trp residue that forms knob-in-hole interactions across the dimer–dimer interface abrogates hexamer formation, suggesting that this residue is the center of a protein–protein association hot spot. PMID:24502590

  8. Acute Modulation of Cortical Glutamate and GABA Content by Physical Activity.

    PubMed

    Maddock, Richard J; Casazza, Gretchen A; Fernandez, Dione H; Maddock, Michael I

    2016-02-24

    Converging evidence demonstrates that physical activity evokes a brain state characterized by distinctive changes in brain metabolism and cortical function. Human studies have shown that physical activity leads to a generalized increase in electroencephalography power across regions and frequencies, and a global increase in brain nonoxidative metabolism of carbohydrate substrates. This nonoxidative consumption of carbohydrate has been hypothesized to include increased de novo synthesis of amino acid neurotransmitters, especially glutamate and GABA. Here, we conducted a series of proton magnetic resonance spectroscopy studies in human volunteers before and after vigorous exercise (≥80% of predicted maximal heart rate). Results showed that the resonance signals of both glutamate and GABA increased significantly in the visual cortex following exercise. We further demonstrated a similar increase in glutamate following exercise in an executive region, the anterior cingulate cortex. The increase in glutamate was similar when using echo times of 30 and 144 ms, indicating that exercise-related T2 relaxation effects across this range of relaxation times did not account for the findings. In addition, we found preliminary evidence that more physical activity during the preceding week predicts higher resting glutamate levels. Overall, the results are consistent with an exercise-induced expansion of the cortical pools of glutamate and GABA, and add to a growing understanding of the distinctive brain state associated with physical activity. A more complete understanding of this brain state may reveal important insights into mechanisms underlying the beneficial effects of physical exercise in neuropsychiatric disorders, neurorehabilitation, aging, and cognition.

  9. Activation of muscarinic receptors inhibits glutamate-induced GSK-3β overactivation in PC12 cells

    PubMed Central

    Ma, Ke; Yang, Li-min; Chen, Hong-zhuan; Lu, Yang

    2013-01-01

    Aim: To investigate the actions of the muscarinic agonist carbachol on glutamate-induced neurotoxicity in PC12 cells, and the underlying mechanisms. Methods: PC12 cells were treated with different concentrations of glutamate for 24 or 48 h. The cell viability was measured using MTT assay, and the expression and activation of GSK-3β were detected with Western blot. β-Catenin translocation was detected using immunofluorescence. Luciferase reporter assay and real-time PCR were used to analyze the transcriptional activity of β-catenin. Results: Glutamate (1, 3, and 10 mmol/L) induced PC12 cell death in a dose-dependent manner. Moreover, treatment of the cells with glutamate (1 mmol/L) caused significant overactivation of GSK-3β and prevented β-catenin translocation to the nucleus. Pretreatment with carbachol (0.01 μmol/L) blocked glutamate-induced cell death and GSK-3β overactivation, and markedly enhanced β-catenin transcriptional activity. Conclusion: Activation of muscarinic receptors exerts neuroprotection in PC12 cells by attenuating glutamate-induced GSK-3β overactivation, suggesting potential benefits of muscarinic agonists for Alzheimer's disease. PMID:23685950

  10. Furosemide and 11beta-hydroxysteroid dehydrogenase activity, in man.

    PubMed

    Palermo, M; Armanini, D; Shackleton, C H L; Sorba, G; Cossu, M; Roitman, E; Scaroni, C; Delitala, G

    2002-09-01

    Mineralocorticoid receptors possess the same affinity for aldosterone and for cortisol and preferential binding of aldosterone is modulated by the 11 beta-hydroxysteroid dehydrogenase (11 beta-OHSD) enzyme, which converts cortisol to its inactive metabolite cortisone. Several endogenous or exogenous compounds able to inhibit the enzyme have been described and, as a consequence, produce the syndrome of apparent mineralocorticoid excess (AME) characterized by hypertension, hypokalemia, volume repletion and suppression of the renin-angiotensin-aldosterone system. High doses of furosemide, a diuretic that works in the luminal surface of the thick ascending limb of Henle's loop, have been reported to inhibit 11 beta-OHSD activity to the same extent as licorice in vivo and in vitro, in rat. The aim of our study was to verify the effect of the drug on 11 beta-OHSD activity in man at the doses currently used in clinical practice. We tested the activity of 11 beta-OHSD following both acute and protracted administration of furosemide. In the acute study, the drug was administered at low (40 mg i.v. in bolo) and high doses (infusion of 10 mg/kg bw i.v for six hours); the protracted furosemide administration consisted in 50 mg/day for 20 days, by mouth. The ratios between the cortisol metabolites tetrahydrocortisol plus allo-tetrahydrocortisol to tetra-hydrocortisone and urinary free cortisol to urinary free cortisone were used to measure the activity of 11 beta-OHSD. Urinary cortisol, cortisone and their metabolites were tested by a gas-chromatographic/mass spectrometric method. Neither acute nor prolonged administration of furosemide did affect the activity of 11 beta-OHSD although the drug was able to modify plasma aldosterone and PRA secretion and to determine hypokalemia. Our results suggest that furosemide does not play a significant role in 11 beta-OHSD modulation in humans, at least at the dosage used in clinical practice. PMID:12373630

  11. Diagnosing Clostridium difficile-associated diarrhea using enzyme immunoassay: the clinical significance of toxin negativity in glutamate dehydrogenase-positive patients

    PubMed Central

    Yuhashi, Kazuhito; Yagihara, Yuka; Misawa, Yoshiki; Sato, Tomoaki; Saito, Ryoichi; Okugawa, Shu; Moriya, Kyoji

    2016-01-01

    Purpose The enzyme immunoassay (EIA) has lower sensitivity for Clostridium difficile toxins A and B than the polymerase chain reaction in the diagnosis of C. difficile-associated diarrhea (CDAD). Furthermore, toxin positivity with EIA performed on C. difficile isolates from stool cultures may be observed even in patients with EIA glutamate dehydrogenase (GDH)-positive and toxin-negative stool specimens. It is unclear whether such patients should be treated as having CDAD. Methods The present study retrospectively compared patient characteristics, treatment, and diarrhea duration among three groups of patients who underwent stool EIA testing for CDAD diagnosis: a toxin-positive stool group (positive stool group; n=39); a toxin-negative stool/toxin-positive isolate group (discrepant negative/positive group, n=14); and a dual toxin-negative stool and isolate group (dual negative group, n=15). All cases included were confirmed to be GDH positive on EIA test. Results Patients’ backgrounds and comorbidities were not significantly different among three groups. No difference was observed among the three groups with regard to antimicrobial drug use before diarrhea onset. Treatment was received by 82.1% of the positive stool group compared to 7.1% of the discrepant positive/negative group and 0% of the dual negative group, while mean diarrhea duration was 10.6 days compared to 7.9 days (P=0.6006) and 3.4 days (P=0.0312), respectively. Conclusion Even without treatment, patients with toxin-negative stool specimens had shorter diarrhea duration than those with toxin-positive stool specimens even with toxin-positive isolates. These findings may suggest a limited need for CDAD treatment for GDH-positive patients and toxin-negative stool specimens. PMID:27313472

  12. Acute and chronic ethanol exposure differentially alters alcohol dehydrogenase and aldehyde dehydrogenase activity in the zebrafish liver.

    PubMed

    Tran, Steven; Nowicki, Magda; Chatterjee, Diptendu; Gerlai, Robert

    2015-01-01

    Chronic ethanol exposure paradigms have been successfully used in the past to induce behavioral and central nervous system related changes in zebrafish. However, it is currently unknown whether chronic ethanol exposure alters ethanol metabolism in adult zebrafish. In the current study we examine the effect of acute ethanol exposure on adult zebrafish behavioral responses, as well as alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) activity in the liver. We then examine how two different chronic ethanol exposure paradigms (continuous and repeated ethanol exposure) alter behavioral responses and liver enzyme activity during a subsequent acute ethanol challenge. Acute ethanol exposure increased locomotor activity in a dose-dependent manner. ADH activity was shown to exhibit an inverted U-shaped curve and ALDH activity was decreased by ethanol exposure at all doses. During the acute ethanol challenge, animals that were continuously housed in ethanol exhibited a significantly reduced locomotor response and increased ADH activity, however, ALDH activity did not change. Zebrafish that were repeatedly exposed to ethanol demonstrated a small but significant attenuation of the locomotor response during the acute ethanol challenge but ADH and ALDH activity was similar to controls. Overall, we identified two different chronic ethanol exposure paradigms that differentially alter behavioral and physiological responses in zebrafish. We speculate that these two paradigms may allow dissociation of central nervous system-related and liver enzyme-dependent ethanol induced changes in zebrafish.

  13. Gene Cloning and mRNA Expression of Glutamate Dehydrogenase in the Liver, Brain, and Intestine of the Swamp Eel, Monopterus albus (Zuiew), Exposed to Freshwater, Terrestrial Conditions, Environmental Ammonia, or Salinity Stress

    PubMed Central

    Tok, Chia Y.; Chew, Shit F.; Ip, Yuen K.

    2011-01-01

    The swamp eel, Monopterus albus, is an obligatory air-breathing teleost which can undergo long period of emersion, has high environmental and tissue ammonia tolerance, and can survive in brackish water. We obtained a cDNA sequence of glutamate dehydrogenase (gdh), which consisted of a 133-bp 5′ UTR, a complete coding sequence region spanning 1629 bp and a 3′ UTR of approximately 717 bp, from the liver, intestine, and brain of M. albus. The translated Gdh amino acid sequence had 542 residues, and it formed a monophyletic clade with Bostrychus sinensis Gdh1a, Tetraodon nigroviridis Gdh1a, Chaenocephalus aceratus Gdh1a, Salmo salar Gdh1a1 and Gdh1a2, and O. mykiss Gdh1a. One day of exposure to terrestrial conditions or 75 mmol l−1 NH4Cl, but not to water at salinity 20, resulted in a significant increase in mRNA expression of gdh1a and Gdh amination activity in the liver of M. albus. However, exposure to brackish water, but not to terrestrial conditions or 75 mmol l−1 NH4Cl, led to a significant increase in the mRNA expression of gdh1a and Gdh amination activity in the intestine. By contrast, all the three experimental conditions had no significant effects on the mRNA expression of gdh1a in the brain of M. albus, despite a significant decrease in the Gdh amination activity in the brain of fish exposed to 75 mmol l−1 NH4Cl for 6 days. Our results indicate for the first time that the mRNA expression of gdh1a was differentially up-regulated in the liver and intestine of M. albus in response to ammonia toxicity and salinity stress, respectively. The increases in mRNA expression of gdh1a and Gdh amination activity would probably lead to an increase in glutamate production in support of increased glutamine synthesis for the purpose of ammonia detoxification or cell volume regulation under these two different environmental conditions. PMID:22319499

  14. Activation of dehydrogenase activity and cardiac respiration: A sup 31 P-NMR study

    SciTech Connect

    Katz, L.A.; Koretsky, A.P.; Balaban, R.S. )

    1988-07-01

    {sup 31}P-NMR studies were performed to determine the tissue phosphate and oxygen consumption effects of known maneuvers on the activation of pyruvate dehydrogenase during work jumps in the perfused rat heart. In control studies of the glucose-perfused heart, work jumps, with pacing, resulted in a 32% increase in oxygen consumption ({dot char}Qo{sub 2}) from 1.72 {plus minus} 0.09 to 2.29 {plus minus} 0.12 mmol O{sub 2}{center dot}h{sup {minus}1}{center dot}g dry wt{sup {minus}1}. During this transition no significant change in the high energy phosphates were detected. In contrast, work jumps did cause changes in the phosphates when the activation of pyruvate dehydrogenase was blocked with 2.5 {mu}g of ruthenium red per milliliter or maximally stimulated with 11 mM pyruvate before the increase in work. The observed increase in {dot char}Qo{sub 2} and inorganic phosphate and calculated increase in ADP are consistent with these phosphates controlling mitochondrial respiration under these conditions. These results suggest that the activation of pyruvate dehydrogenase and/or other dehydrogenases may be an important step in the orchestration of work and {dot char}Qo{sub 2}.

  15. Allosteric activation of membrane-bound glutamate receptors using coordination chemistry within living cells

    NASA Astrophysics Data System (ADS)

    Kiyonaka, Shigeki; Kubota, Ryou; Michibata, Yukiko; Sakakura, Masayoshi; Takahashi, Hideo; Numata, Tomohiro; Inoue, Ryuji; Yuzaki, Michisuke; Hamachi, Itaru

    2016-10-01

    The controlled activation of proteins in living cells is an important goal in protein-design research, but to introduce an artificial activation switch into membrane proteins through rational design is a significant challenge because of the structural and functional complexity of such proteins. Here we report the allosteric activation of two types of membrane-bound neurotransmitter receptors, the ion-channel type and the G-protein-coupled glutamate receptors, using coordination chemistry in living cells. The high programmability of coordination chemistry enabled two His mutations, which act as an artificial allosteric site, to be semirationally incorporated in the vicinity of the ligand-binding pockets. Binding of Pd(2,2‧-bipyridine) at the allosteric site enabled the active conformations of the glutamate receptors to be stabilized. Using this approach, we were able to activate selectively a mutant glutamate receptor in live neurons, which initiated a subsequent signal-transduction pathway.

  16. Allosteric activation of membrane-bound glutamate receptors using coordination chemistry within living cells.

    PubMed

    Kiyonaka, Shigeki; Kubota, Ryou; Michibata, Yukiko; Sakakura, Masayoshi; Takahashi, Hideo; Numata, Tomohiro; Inoue, Ryuji; Yuzaki, Michisuke; Hamachi, Itaru

    2016-10-01

    The controlled activation of proteins in living cells is an important goal in protein-design research, but to introduce an artificial activation switch into membrane proteins through rational design is a significant challenge because of the structural and functional complexity of such proteins. Here we report the allosteric activation of two types of membrane-bound neurotransmitter receptors, the ion-channel type and the G-protein-coupled glutamate receptors, using coordination chemistry in living cells. The high programmability of coordination chemistry enabled two His mutations, which act as an artificial allosteric site, to be semirationally incorporated in the vicinity of the ligand-binding pockets. Binding of Pd(2,2'-bipyridine) at the allosteric site enabled the active conformations of the glutamate receptors to be stabilized. Using this approach, we were able to activate selectively a mutant glutamate receptor in live neurons, which initiated a subsequent signal-transduction pathway. PMID:27657873

  17. Blending foundry sands with soil: Effect on dehydrogenase activity.

    PubMed

    Dungan, Robert S; Kukier, Urzsula; Lee, Brad

    2006-03-15

    Each year U.S. foundries landfill several million tons of sand that can no longer be used to make metalcasting molds and cores. A possible use for these materials is as an ingredient in manufactured soils; however, potentially harmful metals and resin binders (used to make cores) may adversely impact the soil microbial community. In this study, the dehydrogenase activity (DHA) of soil amended with molding sand (clay-coated sand known as "green sand") or core sands at 10%, 30%, and 50% (dry wt.) was determined. The green sands were obtained from iron, aluminum, and brass foundries; the core sands were made with phenol-formaldehyde or furfuryl alcohol based resins. Overall, incremental additions of these sands resulted in a decrease in the DHA which lasted throughout the 12-week experimental period. A brass green sand, which contained high concentrations of Cu, Pb, and Zn, severely impacted the DHA. By week 12 no DHA was detected in the 30% and 50% treatments. In contrast, the DHA in soil amended with an aluminum green sand was 2.1 times higher (all blending ratios), on average, at week 4 and 1.4 times greater (30% and 50% treatments only) than the controls by week 12. In core sand-amended soil, the DHA results were similar to soils amended with aluminum and iron green sands. Increased activity in some treatments may be a result of the soil microorganisms utilizing the core resins as a carbon source. The DHA assay is a sensitive indicator of environmental stress caused by foundry sand constituents and may be useful to assess which foundry sands are suitable for beneficial use in the environment. PMID:15975632

  18. Separation of NADH-fumarate reductase and succinate dehydrogenase activities in Trypanosoma cruzi.

    PubMed

    Christmas, P B; Turrens, J F

    2000-02-15

    A recent review suggested that the activity of NADH-fumarate reductase from trypanosomatids could be catalyzed by succinate dehydrogenase working in reverse (Tielens and van Hellemond, Parasitol. Today 14, 265-271, 1999). The results reported in this study demonstrate that the two activities can easily be separated without any loss in either activity, suggesting that fumarate reductase and succinate dehydrogenase are separate enzymes.

  19. Maternal inflammation leads to impaired glutamate homeostasis and up-regulation of glutamate carboxypeptidase II in activated microglia in the fetal/newborn rabbit brain.

    PubMed

    Zhang, Zhi; Bassam, Bassam; Thomas, Ajit G; Williams, Monica; Liu, Jinhuan; Nance, Elizabeth; Rojas, Camilo; Slusher, Barbara S; Kannan, Sujatha

    2016-10-01

    Astrocyte dysfunction and excessive activation of glutamatergic systems have been implicated in a number of neurologic disorders, including periventricular leukomalacia (PVL) and cerebral palsy (CP). However, the role of chorioamnionitis on glutamate homeostasis in the fetal and neonatal brains is not clearly understood. We have previously shown that intrauterine endotoxin administration results in intense microglial 'activation' and increased pro-inflammatory cytokines in the periventricular region (PVR) of the neonatal rabbit brain. In this study, we assessed the effect of maternal inflammation on key components of the glutamate pathway and its relationship to astrocyte and microglial activation in the fetal and neonatal New Zealand white rabbit brain. We found that intrauterine endotoxin exposure at gestational day 28 (G28) induced acute and prolonged glutamate elevation in the PVR of fetal (G29, 1day post-injury) and postnatal day 1 (PND1, 3days post-injury) brains along with prominent morphological changes in the astrocytes (soma hypertrophy and retracted processes) in the white matter tracts. There was a significant increase in glutaminase and N-Methyl-d-Aspartate receptor (NMDAR) NR2 subunit expression along with decreased glial L-glutamate transporter 1 (GLT-1) in the PVR at G29, that would promote acute dysregulation of glutamate homeostasis. This was accompanied with significantly decreased TGF-β1 at PND1 in CP kits indicating ongoing neuroinflammation. We also show for the first time that glutamate carboxypeptidase II (GCPII) was significantly increased in the activated microglia at the periventricular white matter area in both G29 and PND1 CP kits. This was confirmed by in vitro studies demonstrating that LPS activated primary microglia markedly upregulate GCPII enzymatic activity. These results suggest that maternal intrauterine endotoxin exposure results in early onset and long-lasting dysregulation of glutamate homeostasis, which may be mediated by

  20. Evolution of D-lactate dehydrogenase activity from glycerol dehydrogenase and its utility for D-lactate production from lignocellulose.

    PubMed

    Wang, Qingzhao; Ingram, Lonnie O; Shanmugam, K T

    2011-11-22

    Lactic acid, an attractive, renewable chemical for production of biobased plastics (polylactic acid, PLA), is currently commercially produced from food-based sources of sugar. Pure optical isomers of lactate needed for PLA are typically produced by microbial fermentation of sugars at temperatures below 40 °C. Bacillus coagulans produces L(+)-lactate as a primary fermentation product and grows optimally at 50 °C and pH 5, conditions that are optimal for activity of commercial fungal cellulases. This strain was engineered to produce D(-)-lactate by deleting the native ldh (L-lactate dehydrogenase) and alsS (acetolactate synthase) genes to impede anaerobic growth, followed by growth-based selection to isolate suppressor mutants that restored growth. One of these, strain QZ19, produced about 90 g L(-1) of optically pure D(-)-lactic acid from glucose in < 48 h. The new source of D-lactate dehydrogenase (D-LDH) activity was identified as a mutated form of glycerol dehydrogenase (GlyDH; D121N and F245S) that was produced at high levels as a result of a third mutation (insertion sequence). Although the native GlyDH had no detectable activity with pyruvate, the mutated GlyDH had a D-LDH specific activity of 0.8 μmoles min(-1) (mg protein)(-1). By using QZ19 for simultaneous saccharification and fermentation of cellulose to D-lactate (50 °C and pH 5.0), the cellulase usage could be reduced to 1/3 that required for equivalent fermentations by mesophilic lactic acid bacteria. Together, the native B. coagulans and the QZ19 derivative can be used to produce either L(+) or D(-) optical isomers of lactic acid (respectively) at high titers and yields from nonfood carbohydrates. PMID:22065761

  1. Neuroprotective Activity of Pongamia pinnata in Monosodium Glutamate-induced Neurotoxicity in Rats

    PubMed Central

    Swamy, A. H. M. VISWANATHA; Patel, N. L.; Gadad, P. C.; Koti, B. C.; Patel, U. M.; Thippeswamy, A. H. M.; Manjula, D. V.

    2013-01-01

    This study was designed to evaluate the neuroprotective activity of ethanol extract of Pongamia pinnata stem bark in monosodium glutamate-induced neurotoxicity in rats. Neurotoxicity was induced by intraperitoneal injection of monosodium glutamate 2 g per kg body weight daily for 7 days. Ethanol extract of Pongamia pinnata stem bark (200 and 400 mg/kg) was administered orally after 1 h of monosodium glutamate treatment. Dextromethorphan (30 mg/kg, p.o.) was used as standard drug for the comparison. The degree of protection was determined by various behavioural, locomotor, muscle grip activity, lipid peroxidation and measurement of antioxidant status of glutathione, catalase and superoxide dismutase. Estimation of calcium, sodium and potassium ions in brain tissue and gamma aminobutyric acid level in serum was carried out. The histopathological study of brain tissue was also carried out. Treatment with Pongamia pinnata significantly improved monosodium glutamate-induced alteration in behavioural and locomotor activity and muscle strength. Significant decrease in lipid peroxidation and increase in glutathione, superoxide dismutase and catalase was observed in Pongamia pinnata treated group. Further, Pongamia pinnata also significantly reduced the monosodium glutamate-induced excitotoxicity by decreasing the level of Ca+2 and Na+ with concomitant increase in the level of K+. Serum gamma aminobutyric acid level was also increased in Pongamia pinnata treated animals. Further, the histopathological evidence supports the neuroprotective activity of Pongamia pinnata. In conclusion, the present study suggests that the ethanol extract of stem bark of Pongamia pinnata possesses significant neuroprotective activity in albino rats. PMID:24591740

  2. Neuroprotective Activity of Pongamia pinnata in Monosodium Glutamate-induced Neurotoxicity in Rats.

    PubMed

    Swamy, A H M Viswanatha; Patel, N L; Gadad, P C; Koti, B C; Patel, U M; Thippeswamy, A H M; Manjula, D V

    2013-11-01

    This study was designed to evaluate the neuroprotective activity of ethanol extract of Pongamia pinnata stem bark in monosodium glutamate-induced neurotoxicity in rats. Neurotoxicity was induced by intraperitoneal injection of monosodium glutamate 2 g per kg body weight daily for 7 days. Ethanol extract of Pongamia pinnata stem bark (200 and 400 mg/kg) was administered orally after 1 h of monosodium glutamate treatment. Dextromethorphan (30 mg/kg, p.o.) was used as standard drug for the comparison. The degree of protection was determined by various behavioural, locomotor, muscle grip activity, lipid peroxidation and measurement of antioxidant status of glutathione, catalase and superoxide dismutase. Estimation of calcium, sodium and potassium ions in brain tissue and gamma aminobutyric acid level in serum was carried out. The histopathological study of brain tissue was also carried out. Treatment with Pongamia pinnata significantly improved monosodium glutamate-induced alteration in behavioural and locomotor activity and muscle strength. Significant decrease in lipid peroxidation and increase in glutathione, superoxide dismutase and catalase was observed in Pongamia pinnata treated group. Further, Pongamia pinnata also significantly reduced the monosodium glutamate-induced excitotoxicity by decreasing the level of Ca(+2) and Na(+) with concomitant increase in the level of K(+). Serum gamma aminobutyric acid level was also increased in Pongamia pinnata treated animals. Further, the histopathological evidence supports the neuroprotective activity of Pongamia pinnata. In conclusion, the present study suggests that the ethanol extract of stem bark of Pongamia pinnata possesses significant neuroprotective activity in albino rats.

  3. Glutamine synthetase activity and glutamate uptake in hippocampus and frontal cortex in portal hypertensive rats

    PubMed Central

    Acosta, Gabriela Beatriz; Fernández, María Alejandra; Roselló, Diego Martín; Tomaro, María Luján; Balestrasse, Karina; Lemberg, Abraham

    2009-01-01

    AIM: To study glutamine synthetase (GS) activity and glutamate uptake in the hippocampus and frontal cortex (FC) from rats with prehepatic portal vein hypertension. METHODS: Male Wistar rats were divided into sham-operated group and a portal hypertension (PH) group with a regulated stricture of the portal vein. Animals were sacrificed by decapitation 14 d after portal vein stricture. GS activity was determined in the hippocampus and FC. Specific uptake of radiolabeled L-glutamate was studied using synaptosome-enriched fractions that were freshly prepared from both brain areas. RESULTS: We observed that the activity of GS increased in the hippocampus of PH rats, as compared to control animals, and decreased in the FC. A significant decrease in glutamate uptake was found in both brain areas, and was more marked in the hippocampus. The decrease in glutamate uptake might have been caused by a deficient transport function, significantly and persistent increase in this excitatory neurotransmitter activity. CONCLUSION: The presence of moderate ammonia blood levels may add to the toxicity of excitotoxic glutamate in the brain, which causes alterations in brain function. Portal vein stricture that causes portal hypertension modifies the normal function in some brain regions. PMID:19533812

  4. Elevated venous glutamate levels in (pre)catabolic conditions result at least partly from a decreased glutamate transport activity.

    PubMed

    Hack, V; Stütz, O; Kinscherf, R; Schykowski, M; Kellerer, M; Holm, E; Dröge, W

    1996-06-01

    Abnormally high postabsorptive venous plasma glutamate levels have been reported for several diseases that are associated with a loss of body cell mass including cancer, human/simian immunodeficiency virus infection, and amyotrophic lateral sclerosis. Studies on exchange rates in well-nourished cancer patients now show that high venous plasma glutamate levels may serve as a bona fide indicator for a decreased uptake of glutamate by the peripheral muscle tissue in the postabsorptive period and may be indicative for a precachectic state. High glutamate levels are also moderately correlated with a decreased uptake of glucose and ketone bodies. Relatively high venous glutamate levels have also been found in non-insulin-dependent diabetes mellitus and to some extent also in the cubital vein of normal elderly subjects, i.e., in conditions commonly associated with a decreased glucose tolerance and progressive loss of body cell mass.

  5. Structural and biochemical characterization of the folyl-poly-γ-l-glutamate hydrolyzing activity of human glutamate carboxypeptidase II.

    PubMed

    Navrátil, Michal; Ptáček, Jakub; Šácha, Pavel; Starková, Jana; Lubkowski, Jacek; Bařinka, Cyril; Konvalinka, Jan

    2014-07-01

    In addition to its well-characterized role in the central nervous system, human glutamate carboxypeptidase II (GCPII; Uniprot ID Q04609) acts as a folate hydrolase in the small intestine, participating in the absorption of dietary polyglutamylated folates (folyl-n-γ-l-glutamic acid), which are the provitamin form of folic acid (also known as vitamin B9 ). Despite the role of GCPII as a folate hydrolase, nothing is known about the processing of polyglutamylated folates by GCPII at the structural or enzymological level. Moreover, many epidemiologic studies on the relationship of the naturally occurring His475Tyr polymorphism to folic acid status suggest that this polymorphism may be associated with several pathologies linked to impaired folate metabolism. In the present study, we report: (a) a series X-ray structures of complexes between a catalytically inactive GCPII mutant (Glu424Ala) and a panel of naturally occurring polyglutamylated folates; (b) the X-ray structure of the His475Tyr variant at a resolution of 1.83 Å; (c) the study of the recently identified arene-binding site of GCPII through mutagenesis (Arg463Leu, Arg511Leu and Trp541Ala), inhibitor binding and enzyme kinetics with polyglutamylated folates as substrates; and (d) a comparison of the thermal stabilities and folate-hydrolyzing activities of GCPII wild-type and His475Tyr variants. As a result, the crystallographic data reveal considerable details about the binding mode of polyglutamylated folates to GCPII, especially the engagement of the arene binding site in recognizing the folic acid moiety. Additionally, the combined structural and kinetic data suggest that GCPII wild-type and His475Tyr variant are functionally identical. PMID:24863754

  6. Traumatic Brain Injury Increases Cortical Glutamate Network Activity by Compromising GABAergic Control

    PubMed Central

    Cantu, David; Walker, Kendall; Andresen, Lauren; Taylor-Weiner, Amaro; Hampton, David; Tesco, Giuseppina; Dulla, Chris G.

    2015-01-01

    Traumatic brain injury (TBI) is a major risk factor for developing pharmaco-resistant epilepsy. Although disruptions in brain circuitry are associated with TBI, the precise mechanisms by which brain injury leads to epileptiform network activity is unknown. Using controlled cortical impact (CCI) as a model of TBI, we examined how cortical excitability and glutamatergic signaling was altered following injury. We optically mapped cortical glutamate signaling using FRET-based glutamate biosensors, while simultaneously recording cortical field potentials in acute brain slices 2–4 weeks following CCI. Cortical electrical stimulation evoked polyphasic, epileptiform field potentials and disrupted the input–output relationship in deep layers of CCI-injured cortex. High-speed glutamate biosensor imaging showed that glutamate signaling was significantly increased in the injured cortex. Elevated glutamate responses correlated with epileptiform activity, were highest directly adjacent to the injury, and spread via deep cortical layers. Immunoreactivity for markers of GABAergic interneurons were significantly decreased throughout CCI cortex. Lastly, spontaneous inhibitory postsynaptic current frequency decreased and spontaneous excitatory postsynaptic current increased after CCI injury. Our results suggest that specific cortical neuronal microcircuits may initiate and facilitate the spread of epileptiform activity following TBI. Increased glutamatergic signaling due to loss of GABAergic control may provide a mechanism by which TBI can give rise to post-traumatic epilepsy. PMID:24610117

  7. Voltage-dependent drug blockade of L-glutamate activated channels of the crayfish.

    PubMed Central

    Dekin, M S; Edwards, C

    1983-01-01

    The actions of d-tubocurarine (d-TC) and local anaesthetics on the L-glutamate activated channel at the voltage-clamped crayfish neuromuscular junction were studied. The effect of d-TC and local anaesthetics on the dose-response relationship between ionophoretically applied L-glutamate and synaptic current suggested that both acted as non-competitive inhibitors. The amount of inhibition was voltage dependent, and increased as the membrane potential was hyperpolarized. This voltage-dependent block was also manifest in a flattening of the I-V relationship between L-glutamate induced current and membrane potential in the presence of d-TC. However, the reversal potential for the L-glutamate activated channel was not affected; it was about +7 mV in both the presence and absence of d-TC. The neurally evoked excitatory post-synaptic current (e.p.s.c.) was depressed in the presence of these drugs and this effect was also voltage dependent. The time course of the e.p.s.c. was affected, but less so than expected if the L-glutamate activated channel were identical to the channel opened by acetylcholine at the vertebrate neuromuscular junction. Possible reasons for this discrepancy are discussed. PMID:6312026

  8. Solubilization, partial purification, and reconstitution of glutamate- and N-methyl-D-aspartate-activated cation channels from brain synaptic membranes

    SciTech Connect

    Ly, A.M.; Michaelis, E.K. )

    1991-04-30

    L-Glutamate-activated cation channel proteins from rat brain synaptic membranes were solubilized, partially purified, and reconstituted into liposomes. Optimal conditions for solubilization and reconstitution included treatment of the membranes with nonionic detergents in the presence of neutral phospholipids plus glycerol. Quench-flow procedures were developed to characterize the rapid kinetics of ion flux induced by receptor agonists. ({sup 14}C)Methylamine, a cation that permeates through the open channel of both vertebrate and invertebrate glutamate receptors, was used to measure the activity of glutamate receptor-ion channel complexes in reconstituted liposomes. L-Glutamate caused an increase in the rate of ({sup 14}C)methylamine influx into liposomes reconstituted with either solubilized membrane proteins or partially purified glutamate-binding proteins. Of the major glutamate receptor agonists, only N-methyl-D-aspartate activated cation fluxes in liposomes reconstituted with glutamate-binding proteins. In liposomes reconstituted with glutamate-binding proteins, N-methyl-D-aspartate- or glutamate-induced influx of NA{sup +} led to a transient increase in the influx of the lipid-permeable anion probe S{sup 14}CN{sup {minus}}. These results indicate the functional reconstitution of N-methyl-D-aspartate-sensitive glutamate receptors and the role of the {approximately}69-kDa protein in the function of these ion channels.

  9. Hepatic alcohol dehydrogenase activity in alcoholic subjects with and without liver disease.

    PubMed Central

    Vidal, F; Perez, J; Morancho, J; Pinto, B; Richart, C

    1990-01-01

    Alcohol dehydrogenase activity was measured in samples of liver tissue from a group of alcoholic and non-alcoholic subjects to determine whether decreased liver alcohol dehydrogenase activity is a consequence of ethanol consumption or liver damage. The alcoholic patients were classified further into the following groups: control subjects with no liver disease (group 1), subjects with non-cirrhotic liver disease (group 2), and subjects with cirrhotic liver disease (group 3). The non-alcoholic subjects were also divided, using the same criteria, into groups 4, 5, and 6, respectively. The analysis of the results showed no significant differences when mean alcohol dehydrogenase activities of alcoholic and non-alcoholic patients with similar degrees of liver pathology were compared (groups 1 v 4, 2 v 5, and 3 v 6. Alcohol dehydrogenase activity was, however, severely reduced in patients with liver disease compared with control subjects. Our findings suggest that alcohol consumption does not modify hepatic alcohol dehydrogenase activity. The reduction in specific alcohol dehydrogenase activity in alcoholic liver disease is a consequence of liver damage. PMID:2379876

  10. Effect of phenylpyruvate on pyruvate dehydrogenase activity in rat brain mitochondria

    PubMed Central

    Land, John M.; Clark, John B.

    1973-01-01

    1. The effects of phenylpyruvate, a metabolite produced in phenylketonuria, on the pyruvate dehydrogenase-complex activity were investigated in rat brain mitochondria. 2. Pyruvate dehydrogenase activity was measured by two methods, one measuring the release of 14CO2 from [1-14C]pyruvate and the other measuring the acetyl-CoA formed by means of the coupling enzyme, pigeon liver arylamine acetyltransferase (EC 2.3.1.5). In neither case was there significant inhibition of the pyruvate dehydrogenase complex by phenylpyruvate at concentrations below 2mm. 3. However, phenylpyruvate acted as a classical competitive inhibitor of the coupling enzyme arylamine acetyltransferase, with a Ki of 100μm. 4. It was concluded that the inhibition of pyruvate dehydrogenase by phenylpyruvate is unlikely to be a primary enzyme defect in phenylketonuria. PMID:16742815

  11. Resveratrol Prevents Retinal Dysfunction by Regulating Glutamate Transporters, Glutamine Synthetase Expression and Activity in Diabetic Retina.

    PubMed

    Zeng, Kaihong; Yang, Na; Wang, Duozi; Li, Suping; Ming, Jian; Wang, Jing; Yu, Xuemei; Song, Yi; Zhou, Xue; Yang, Yongtao

    2016-05-01

    This study investigated the effects of resveratrol (RSV) on retinal functions, glutamate transporters (GLAST) and glutamine synthetase (GS) expression in diabetic rats retina, and on glutamate uptake, GS activity, GLAST and GS expression in high glucose-cultured Müller cells. The electroretinogram was used to evaluate retinal functions. Müller cells cultures were prepared from 5- to 7-day-old Sprague-Dawley rats. The expression of GLAST and GS was examined by qRT-PCR, ELISA and western-blotting. Glutamate uptake was measured as (3)H-glutamate contents of the lysates. GS activity was assessed by a spectrophotometric assay. 1- to 7-month RSV administrations (5 and 10 mg/kg/day) significantly alleviated hyperglycemia and weight loss in diabetic rats. RSV administrations also significantly attenuated diabetes-induced decreases in amplitude of a-wave in rod response, decreases in amplitude of a-, and b-wave in cone and rod response and decreases in amplitude of OP2 in oscillatory potentials. 1- to 7-month RSV treatments also significantly inhibited diabetes-induced delay in OP2 implicit times in scotopic 3.0 OPS test. The down-regulated mRNA and protein expression of GLAST and GS in diabetic rats retina was prevented by RSV administrations. In high glucose-treated cultures, Müller cells' glutamate uptake, GS activity, GLAST and GS expression were decreased significantly compared with normal control cultures. RSV (10, 20, and 30 mmol/l) significantly inhibited the HG-induced decreases in glutamate uptake, GS activity, GLAST and GS expression (at least P < 0.05). These beneficial results suggest that RSV may be considered as a therapeutic option to prevent from diabetic retinopathy.

  12. Age-dependent variations of lactate dehydrogenase and creatine kinase activities in water buffalo calf serum.

    PubMed

    Avallone, L; Lombardi, P; Florio, S; d'Angelo, A; Bogin, E

    1996-12-01

    The electrophoretic patterns of the serum enzymes lactate dehydrogenase and creatine kinase from water buffalo calves are described. Differences in total activities as well as their relative distribution were seen at ages ranging from 1 to 10 weeks. While total lactate dehydrogenase activity increased by over 100%, total creatine kinase increased by almost 400%. The relative activities of lactate dehydrogenase 1 and 5 decreased with age. Lactate dehydrogenase 2 and 3 increased and lactate dehydrogenase 4 did not change. In relation to creatine kinase, the prevalent isoenzyme was creatine kinase-MM, but it's relative activity gradually decreased in comparison to the other two isoenzymes (creatine kinase-MB and creatine kinase-BB). Creatine kinase-BB was completely absent until the 3rd week of age. The percentage modifications of creatine kinase isoenzymes were correlated to age. The results suggest that isoenzymatic separation and characterization of lactate dehydrogenase and creatine kinase in relation to the various tissues can significantly contribute to the diagnosis of diseases which are linked to tissue damage.

  13. Α-amino-β-fluorocyclopropanecarboxylic acids as a new tool for drug development: synthesis of glutamic acid analogs and agonist activity towards metabotropic glutamate receptor 4.

    PubMed

    Lemonnier, Gérald; Lion, Cédric; Quirion, Jean-Charles; Pin, Jean-Philippe; Goudet, Cyril; Jubault, Philippe

    2012-08-01

    Herein we describe the diastereoselective synthesis of glutamic acid analogs and the evaluation of their agonist activity towards metabotropic glutamate receptor subtype 4 (mGluR4). These analogs are based on a monofluorinated cyclopropane core substituted with an α-aminoacid function. The potential of this new building block as a tool for the development of a novel class of drugs is demonstrated with racemic analog 11a that displayed the best agonist activity with an EC50 of 340 nM.

  14. Methylmalonic semialdehyde dehydrogenase deficiency: demonstration of defective valine and beta-alanine metabolism and reduced malonic semialdehyde dehydrogenase activity in cultured fibroblasts

    SciTech Connect

    Gray, R.G.; Pollitt, R.J.; Webley, J.

    1987-08-01

    Intact cultured fibroblasts from a child with a new metabolic disorder, thought to be due to a deficiency of methylmalonic semialdehyde dehydrogenase, produced labeled CO/sub 2/ normally from (1-/sup 14/C)valine but not from (2-/sup 14/C)valine. CO/sub 2/ production from labeled beta-alanine was also much reduced, confirming the suspicion that malonic semialdehyde dehydrogenase is also deficient in this condition. An assay for malonic semialdehyde dehydrogenase in cell homogenates showed low activity but it was impossible to assess the degree of reduction.

  15. Re-engineering the discrimination between the oxidized coenzymes NAD+ and NADP+ in clostridial glutamate dehydrogenase and a thorough reappraisal of the coenzyme specificity of the wild-type enzyme.

    PubMed

    Capone, Marina; Scanlon, David; Griffin, Joanna; Engel, Paul C

    2011-07-01

    Clostridial glutamate dehydrogenase mutants, designed to accommodate the 2'-phosphate of disfavoured NADPH, showed the expected large specificity shifts with NAD(P)H. Puzzlingly, similar assays with oxidized cofactors initially revealed little improvement with NADP(+) , although rates with NAD(+) were markedly diminished. This article reveals that the enzyme's discrimination in favour of NAD(+) and against NADP(+) had been greatly underestimated and has indeed been abated by a factor of > 16,000 by the mutagenesis. Initially, stopped-flow studies of the wild-type enzyme showed a burst increase of A(340) with NADP(+) but not NAD(+), with amplitude depending on the concentration of the coenzyme, rather than enzyme. Amplitude also varied with the commercial source of the NADP(+). FPLC, HPLC and mass spectrometry identified NAD(+) contamination ranging from 0.04 to 0.37% in different commercial samples. It is now clear that apparent rates of NADP(+) utilization mainly reflected the reduction of contaminating NAD(+), creating an entirely false view of the initial coenzyme specificity and also of the effects of mutagenesis. Purification of the NADP(+) eliminated the burst. With freshly purified NADP(+), the NAD(+) : NADP(+) activity ratio under standard conditions, previously estimated as 300 : 1, is 11,000. The catalytic efficiency ratio is even higher at 80,000. Retested with pure cofactor, mutants showed marked specificity shifts in the expected direction, for example, 16 200 fold change in catalytic efficiency ratio for the mutant F238S/P262S, confirming that the key structural determinants of specificity have been successfully identified. Of wider significance, these results underline that, without purification, even the best commercial coenzyme preparations are inadequate for such studies.

  16. Effects of 3-aminoglutarate, a "silent" false transmitter for glutamate neurons, on synaptic transmission and epileptiform activity.

    PubMed

    Wu, Zheng; Foster, Alan C; Staubli, Ursula; Wu, Xia; Sun, Chicheng; Tang, Xin; Li, Yong-Xin; Chen, Gong

    2015-10-01

    Pharmacological tools that interact with the mechanisms that regulate vesicular filling and release of the neurotransmitter L-glutamate would be of enormous value. In this study, we provide physiological evidence that the glutamate analog, 3-aminoglutarate (3-AG), acts as a false transmitter to reduce presynaptic glutamate release. 3-AG inhibits glutamate-mediated neurotransmission both in primary neuronal cultures and in brain slices with more intact neural circuits. When assayed with the low affinity glutamate receptor antagonist γ-DGG, we demonstrate that 3-AG significantly reduces the synaptic cleft glutamate concentration, suggesting that 3-AG may act as a false transmitter to compete with glutamate during vesicle filling. Furthermore, using three different epileptic models (Mg(2+)-free, 4-AP, and high K(+)), we demonstrate that 3-AG is capable of suppressing epileptiform activity both before and after its induction. Our studies, along with those of the companion paper by Foster et al. (2015) indicate that 3-AG is a "silent" false transmitter for glutamate neurons that is a useful pharmacological tool to probe the mechanisms governing vesicular storage and release of glutamate under both physiological and pathophysiological conditions. 3-AG may have potential therapeutic value in conditions where the glutamate neurotransmitter system is pathologically overactive.

  17. Exposure to high glutamate concentration activates aerobic glycolysis but inhibits ATP-linked respiration in cultured cortical astrocytes.

    PubMed

    Shen, Yao; Tian, Yueyang; Shi, Xiaojie; Yang, Jianbo; Ouyang, Li; Gao, Jieqiong; Lu, Jianxin

    2014-08-01

    Astrocytes play a key role in removing the synaptically released glutamate from the extracellular space and maintaining the glutamate below neurotoxic level in the brain. However, high concentration of glutamate leads to toxicity in astrocytes, and the underlying mechanisms are unclear. The purpose of this study was to investigate whether energy metabolism disorder, especially impairment of mitochondrial respiration, is involved in the glutamate-induced gliotoxicity. Exposure to 10-mM glutamate for 48 h stimulated glycolysis and respiration in astrocytes. However, the increased oxygen consumption was used for proton leak and non-mitochondrial respiration, but not for oxidative phosphorylation and ATP generation. When the exposure time extended to 72 h, glycolysis was still activated for ATP generation, but the mitochondrial ATP-linked respiration of astrocytes was reduced. The glutamate-induced astrocyte damage can be mimicked by the non-metabolized substrate d-aspartate but reversed by the non-selective glutamate transporter inhibitor TBOA. In addition, the glutamate toxicity can be partially reversed by vitamin E. These findings demonstrate that changes of bioenergetic profile occur in cultured cortical astrocytes exposed to high concentration of glutamate and highlight the role of mitochondria respiration in glutamate-induced gliotoxicity in cortical astrocytes.

  18. Valine but not leucine or isoleucine supports neurotransmitter glutamate synthesis during synaptic activity in cultured cerebellar neurons.

    PubMed

    Bak, Lasse K; Johansen, Maja L; Schousboe, Arne; Waagepetersen, Helle S

    2012-09-01

    Synthesis of neuronal glutamate from α-ketoglutarate for neurotransmission necessitates an amino group nitrogen donor; however, it is not clear which amino acid(s) serves this role. Thus, the ability of the three branched-chain amino acids (BCAAs), leucine, isoleucine, and valine, to act as amino group nitrogen donors for synthesis of vesicular neurotransmitter glutamate was investigated in cultured mouse cerebellar (primarily glutamatergic) neurons. The cultures were superfused in the presence of (15) N-labeled BCAAs, and synaptic activity was induced by pulses of N-methyl-D-aspartate (300 μM), which results in release of vesicular glutamate. At the end of the superfusion experiment, the vesicular pool of glutamate was released by treatment with α-latrotoxin (3 nM, 5 min). This experimental paradigm allows a separate analysis of the cytoplasmic and vesicular pools of glutamate. Amount and extent of (15) N labeling of intracellular amino acids plus vesicular glutamate were analyzed employing HPLC and LC-MS analysis. Only when [(15) N]valine served as precursor did the labeling of both cytoplasmic and vesicular glutamate increase after synaptic activity. In addition, only [(15) N]valine was able to maintain the amount of vesicular glutamate during synaptic activity. This indicates that, among the BCAAs, only valine supports the increased need for synthesis of vesicular glutamate.

  19. Glutamate activation of Oct-2 in cultured chick Bergmann glia cells: involvement of NFkappaB.

    PubMed

    Méndez, J Alfredo; López-Bayghen, Esther; Ortega, Arturo

    2005-07-01

    Glutamate, the major excitatory neurotransmitter in the central nervous system, is critically involved in gene expression regulation at the transcriptional and translational levels. Its activity through ionotropic as well as metabotropic receptors modifies the protein repertoire in neurons and glial cells. In avian cerebellar Bergmann glia cells, glutamate receptors trigger a diverse array of signaling cascades that include activity-dependent transcription factors such as the activator protein-1, the cAMP response-element binding protein, and Oct-2. We analyze the upstream regulatory elements involved in Oct-2 activation. Our results demonstrate that Ca2+ influx, protein kinase C, phosphatidylinositol-3 kinase, Src, and nuclear factor (NF)kappaB are involved in this signaling pathway. Our findings link alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptor activation to a negative phase of chkbp gene regulation, controlled by NFkappaB.

  20. Interplay among platelet-activating factor, oxidative stress, and group I metabotropic glutamate receptors modulates neuronal survival.

    PubMed

    Zhu, Peimin; DeCoster, Mark A; Bazan, Nicolas G

    2004-08-15

    Platelet-activating factor (PAF) is a potent phospholipid messenger in the nervous system that participates in synaptic plasticity and in pathologic processes, including neurodegeneration. Oxidative stress plays important roles in neuronal cell death. To define the interaction between PAF and oxidative radicals in neuronal death, we studied the effects of PAF in the presence of oxidative radicals in primary neurons in culture. Exogenous PAF (50 microM) caused PAF receptor-independent injury to neurons. A nonneurotoxic PAF concentration (500 nM) potentiated neuronal death caused by hydrogen peroxide as determined by lactate dehydrogenase (LDH) assay, Hoechst staining, and TUNEL analysis, but it did not potentiate neuronal death caused by menadione, a superoxide donor, or by the nitric oxide donors 3-morpholino-sydnonimine (SIN-1) and sodium nitroprusside (SNP). This potentiation of the hydrogen peroxide effect was selectively blocked by a PAF membrane-receptor antagonist, BN52021 (5 microM). The neurotoxic effect of PAF and hydrogen peroxide was also completely blocked by ebselen and partially decreased by pretreatment with (S)-3,5-dihydroxyphenylglycine (DHPG), a group I metabotropic glutamate receptor (mGluR) agonist. This study suggests that PAF-receptor antagonists may be useful for neuroprotection. A similar effect might also be obtained with group I mGluR agonists, probably by way of a different underlying mechanism.

  1. Convergence of dopamine and glutamate signaling onto striatal ERK activation in response to drugs of abuse

    PubMed Central

    Cahill, Emma; Salery, Marine; Vanhoutte, Peter; Caboche, Jocelyne

    2014-01-01

    Despite their distinct targets, all addictive drugs commonly abused by humans evoke increases in dopamine (DA) concentration within the striatum. The main DA Guanine nucleotide binding protein couple receptors (GPCRs) expressed by medium-sized spiny neurons of the striatum are the D1R and D2R, which are positively and negatively coupled to cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) signaling, respectively. These two DA GPCRs are largely segregated into distinct neuronal populations, where they are co-expressed with glutamate receptors in dendritic spines. Direct and indirect interactions between DA GPCRs and glutamate receptors are the molecular basis by which DA modulates glutamate transmission and controls striatal plasticity and behavior induced by drugs of abuse. A major downstream target of striatal D1R is the extracellular signal-regulated kinase (ERK) kinase pathway. ERK activation by drugs of abuse behaves as a key integrator of D1R and glutamate NMDAR signaling. Once activated, ERK can trigger chromatin remodeling and induce gene expression that permits long-term cellular alterations and drug-induced morphological and behavioral changes. Besides the classical cAMP/PKA pathway, downstream of D1R, recent evidence implicates a cAMP-independent crosstalk mechanism by which the D1R potentiates NMDAR-mediated calcium influx and ERK activation. The mounting evidence of reciprocal modulation of DA and glutamate receptors adds further intricacy to striatal synaptic signaling and is liable to prove relevant for addictive drug-induced signaling, plasticity, and behavior. Herein, we review the evidence that built our understanding of the consequences of this synergistic signaling for the actions of drugs of abuse. PMID:24409148

  2. Gabapentin inhibits the activity of the rat excitatory glutamate transporter 3 expressed in Xenopus oocytes.

    PubMed

    Gil, Yang Sook; Kim, Jong Hak; Kim, Chi Hyo; Han, Jong In; Zuo, Zhiyi; Baik, Hee Jung

    2015-09-01

    Gabapentin, a derivative of γ-aminobutyric acid (GABA), is used to treat epilepsy and neuropathic pain. The pharmacological mechanisms for gabapentin effects are not completely elucidated. We investigated the effect of gabapentin on the activity of excitatory amino acid transporter 3 (EAAT3) that can regulate extracellular glutamate concentrations. EAAT3 was expressed in Xenopus oocytes. Membrane currents were recorded after application of l-glutamate in the presence or absence of different concentrations of gabapentin (1-300μM) by using a two-electrode voltage clamp. To determine the effect of gabapentin on Vmax and Km of EAAT3 for l-glutamate, l-glutamate at 3-300μM was used. To study the effects of protein kinase C (PKC) and phosphatidylinositol 3-kinase (PI3K) on gabapentin-induced changes in EAAT3 activity, oocytes were incubated with the PKC activator (Phorbol 12-myristate 13-acetate, PMA), the PKC inhibitors (chelerythrine or staurosporine), and the PI3K inhibitor wortmannin. Gabapentin decreased EAAT3 activity in a concentration-dependent manner and EAAT3 activity was significantly inhibited by 10-300μM gabapentin. Gabapentin significantly decreased Vmax without affecting Km. PMA increased EAAT3 activity; however, gabapentin attenuated the PMA-induced increase in EAAT3 activity. Pre-incubation of oocytes with chelerythrine, staurosporine, or wortmannin decreased basal EAAT3 activity, which was further reduced by gabapentin. We conclude that gabapentin decreases EAAT3 activity at clinically relevant and higher concentrations, in which PKC and PI3K may not be involved. The results suggest that EAAT3 might not be a target for the anticonvulsant action of gabapentin.

  3. Relationship of lactate dehydrogenase activity to body measurements of Angus x Charolais cows and calves

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Objectives were to examine 1) relationships between lactate dehydrogenase (LDH) activity and body measurements of grazing beef cows, and 2) the association between maternal LDH activity in late gestation and subsequent calf birth weight (BRW), hip height (HH) at weaning, and adjusted weaning weight ...

  4. Cloning of the rat pyruvate dehydrogenase kinase 4 gene promoter: activation of pyruvate dehydrogenase kinase 4 by the peroxisome proliferator-activated receptor gamma coactivator.

    PubMed

    Ma, Ke; Zhang, Yi; Elam, Marshall B; Cook, George A; Park, Edwards A

    2005-08-19

    The pyruvate dehydrogenase complex catalyzes the conversion of pyruvate to acetyl-CoA in mitochondria and is a key regulatory enzyme in the metabolism of glucose to acetyl-CoA. Phosphorylation of pyruvate dehydrogenase by the pyruvate dehydrogenase kinases (PDK) inhibits pyruvate dehydrogenase complex activity. There are four PDK isoforms, and expression of PDK4 and PDK2 genes is elevated in starvation and diabetes, allowing glucose to be conserved while fatty acid oxidation is increased. In these studies we have investigated the transcriptional mechanisms by which the expression of the PDK4 gene is increased. The peroxisome proliferator-activated receptor gamma coactivator (PGC-1alpha) stimulates the expression of genes involved in hepatic gluconeogenesis and mitochondrial fatty acid oxidation. We have found that PGC-1alpha will induce the expression of both the PDK2 and PDK4 genes in primary rat hepatocytes and ventricular myocytes. We cloned the promoter for the rat PDK4 gene. Hepatic nuclear factor 4 (HNF4), which activates many genes in the liver, will induce PDK4 expression. Although HNF4 and PGC-1alpha interact to stimulate several genes encoding gluconeogenic enzymes, the induction of PDK4 does not involve interactions of PGC-1alpha with HNF4. Using the chromatin immunoprecipitation assay, we have demonstrated that HNF4 and PGC-1alpha are associated with the PDK4 gene in vivo. Our data suggest that by inducing PDK genes PGC-1alpha will direct pyruvate away from metabolism into acetyl-CoA and toward the formation of oxaloacetate and into the gluconeogenic pathway. PMID:15967803

  5. Activation of the Glutamic Acid-Dependent Acid Resistance System in Escherichia coli BL21(DE3) Leads to Increase of the Fatty Acid Biotransformation Activity

    PubMed Central

    Woo, Ji-Min; Kim, Ji-Won; Song, Ji-Won; Blank, Lars M.; Park, Jin-Byung

    2016-01-01

    The biosynthesis of carboxylic acids including fatty acids from biomass is central in envisaged biorefinery concepts. The productivities are often, however, low due to product toxicity that hamper whole-cell biocatalyst performance. Here, we have investigated factors that influence the tolerance of Escherichia coli to medium chain carboxylic acid (i.e., n-heptanoic acid)-induced stress. The metabolic and genomic responses of E. coli BL21(DE3) and MG1655 grown in the presence of n-heptanoic acid indicated that the GadA/B-based glutamic acid-dependent acid resistance (GDAR) system might be critical for cellular tolerance. The GDAR system, which is responsible for scavenging intracellular protons by catalyzing decarboxylation of glutamic acid, was inactive in E. coli BL21(DE3). Activation of the GDAR system in this strain by overexpressing the rcsB and dsrA genes, of which the gene products are involved in the activation of GadE and RpoS, respectively, resulted in acid tolerance not only to HCl but also to n-heptanoic acid. Furthermore, activation of the GDAR system allowed the recombinant E. coli BL21(DE3) expressing the alcohol dehydrogenase of Micrococcus luteus and the Baeyer-Villiger monooxygenase of Pseudomonas putida to reach 60% greater product concentration in the biotransformation of ricinoleic acid (i.e., 12-hydroxyoctadec-9-enoic acid (1)) into n-heptanoic acid (5) and 11-hydroxyundec-9-enoic acid (4). This study may contribute to engineering E. coli-based biocatalysts for the production of carboxylic acids from renewable biomass. PMID:27681369

  6. Measuring the Impact of Microenvironmental Conditions on Mitochondrial Dehydrogenase Activity in Cultured Cells.

    PubMed

    Sun, Ramon C; Koong, Albert; Giaccia, Amato; Denko, Nicholas C

    2016-01-01

    Mitochondria are powerhouses of a cell, producing much of the cellular ATP. However, mitochondrial enzymes also participate in many cellular biosynthetic processes. They are responsible for helping to maintain NAD(P)/H and redox balance, supplying metabolic intermediates for cell growth, and regulating several types of programed cell death. Several mitochondrial enzymes have even been shown to participate in the oncogenic process such as isocitrate dehydrogenase, succinate dehydrogenase, and fumarate hydratase. Recent advances have identified significant metabolic changes in the mitochondria that are regulated by malignant transformation and environmental stimuli. Understanding the biological activity and regulation of mitochondrial enzymes can provide insight into how they participate in the process of oncogenic transformation and work to sustain malignant growth. This chapter describes a technique to measure mitochondrial dehydrogenase activities that is faster and more cost effective which can also be scaled up for high throughput. PMID:27325264

  7. Cost-effectiveness of a modified two-step algorithm using a combined glutamate dehydrogenase/toxin enzyme immunoassay and real-time PCR for the diagnosis of Clostridium difficile infection.

    PubMed

    Vasoo, Shawn; Stevens, Jane; Portillo, Lena; Barza, Ruby; Schejbal, Debra; Wu, May May; Chancey, Christina; Singh, Kamaljit

    2014-02-01

    The analytical performance and cost-effectiveness of the Wampole Toxin A/B EIA, the C. Diff. Quik Chek Complete (CdQCC) (a combined glutamate dehydrogenase antigen/toxin enzyme immunoassay), two RT-PCR assays (Progastro Cd and BD GeneOhm) and a modified two-step algorithm using the CdQCC reflexed to RT-PCR for indeterminate results were compared. The sensitivity of the Wampole Toxin A/B EIA, CdQCC (GDH antigen), BD GeneOhm and Progastro Cd RT-PCR were 85.4%, 95.8%, 100% and 93.8%, respectively. The algorithm provided rapid results for 86% of specimens and the remaining indeterminate results were resolved by RT-PCR, offering the best balance of sensitivity and cost savings per test (algorithm ∼US$13.50/test versus upfront RT-PCR ∼US$26.00/test).

  8. Origins of the high catalytic activity of human alcohol dehydrogenase 4 studied with horse liver A317C alcohol dehydrogenase.

    PubMed

    Herdendorf, Timothy J; Plapp, Bryce V

    2011-05-30

    The turnover numbers and other kinetic constants for human alcohol dehydrogenase (ADH) 4 ("stomach" isoenzyme) are substantially larger (10-100-fold) than those for human class I and horse liver alcohol dehydrogenases. Comparison of the primary amino acid sequences (69% identity) and tertiary structures of these enzymes led to the suggestion that residue 317, which makes a hydrogen bond with the nicotinamide amide nitrogen of the coenzyme, may account for these differences. Ala-317 in the class I enzymes is substituted with Cys in human ADH4, and locally different conformations of the peptide backbones could affect coenzyme binding. This hypothesis was tested by making the A317C substitution in horse liver ADH1E and comparisons to the wild-type ADH1E. The steady-state kinetic constants for the oxidation of benzyl alcohol and the reduction of benzaldehyde catalyzed by the A317C enzyme were very similar (up to about 2-fold differences) to those for the wild-type enzyme. Transient kinetics showed that the rate constants for binding of NAD(+) and NADH were also similar. Transient reaction data were fitted to the full Ordered Bi Bi mechanism and showed that the rate constants for hydride transfer decreased by about 2.8-fold with the A317C substitution. The structure of A317C ADH1E complexed with NAD(+) and 2,3,4,5,6-pentafluorobenzyl alcohol at 1.2 Å resolution is essentially identical to the structure of the wild-type enzyme, except near residue 317 where the additional sulfhydryl group displaces a water molecule that is present in the wild-type enzyme. ADH is adaptable and can tolerate internal substitutions, but the protein dynamics apparently are affected, as reflected in rates of hydride transfer. The A317C substitution is not solely responsible for the larger kinetic constants in human ADH4; thus, the differences in catalytic activity must arise from one or more of the other hundred substitutions in the enzyme.

  9. Studies on the active center of D- and L-lactate dehydrogenases using oxamate-diaminohexyl-Sepharose affinity chromatography.

    PubMed Central

    Tuengler, P; Stein, T N; Long, G L

    1980-01-01

    Vertebrate and invertebrate L-lactate dehydrogenases (L-lactate:NAD+ oxidoreductase, EC 1.1.1.27) are effectively bound to oxamate-diaminohexyl-Sepharose, whereas several D-lactate dehydrogenases (D-lactate:NAD+ oxidoreductase, EC 1.1.1.28) do not bind to the same Sepharose. One explanation for our findings is that the enzymes' substrate is oriented in a reversed manner in the active center of the D- and L-lactate dehydrogenases. PMID:6934514

  10. Amino ketone formation and aminopropanol-dehydrogenase activity in rat-liver preparations

    PubMed Central

    Turner, J. M.; Willetts, A. J.

    1967-01-01

    1. Rat tissue homogenates convert dl-1-aminopropan-2-ol into aminoacetone. Liver homogenates have relatively high aminopropanol-dehydrogenase activity compared with kidney, heart, spleen and muscle preparations. 2. Maximum activity of liver homogenates is exhibited at pH9·8. The Km for aminopropanol is approx. 15mm, calculated for a single enantiomorph, and the maximum activity is approx. 9mμmoles of aminoacetone formed/mg. wet wt. of liver/hr.at 37°. Aminoacetone is also formed from l-threonine, but less rapidly. An unidentified amino ketone is formed from dl-4-amino-3-hydroxybutyrate, the Km for which is approx. 200mm at pH9·8. 3. Aminopropanol-dehydrogenase activity in homogenates is inhibited non-competitively by dl-3-hydroxybutyrate, the Ki being approx. 200mm. EDTA and other chelating agents are weakly inhibitory, and whereas potassium chloride activates slightly at low concentrations, inhibition occurs at 50–100mm. 4. It is concluded that aminopropanol-dehydrogenase is located in mitochondria, and in contrast with l-threonine dehydrogenase can be readily solubilized from mitochondrial preparations by ultrasonic treatment. 5. Soluble extracts of disintegrated mitochondria exhibit maximum aminopropanol-dehydrogenase activity at pH9·1 At this pH, Km values for the amino alcohol and NAD+ are approx. 200 and 1·3mm respectively. Under optimum conditions the maximum velocity is approx. 70mμmoles of aminoacetone formed/mg. of protein/hr. at 37°. Chelating agents and thiol reagents appear to have little effect on enzyme activity, but potassium chloride inhibits at all concentrations tested up to 80mm. dl-3-Hydroxybutyrate is only slightly inhibitory. 6. Dehydrogenase activities for l-threonine and dl-4-amino-3-hydroxybutyrate appear to be distinct from that for aminopropanol. 7. Intraperitoneal injection of aminopropanol into rats leads to excretion of aminoacetone in the urine. Aminoacetone excretion proportional to the amount of the amino alcohol

  11. Synthesis and antiproliferative activity of glutamic acid-based dipeptides.

    PubMed

    Silveira-Dorta, Gastón; Martín, Víctor S; Padrón, José M

    2015-08-01

    A small and focused library of 22 dipeptides derived from N,N-dibenzylglutamic acid α- and γ-benzyl esters was prepared in a straightforward manner. The evaluation of the antiproliferative activity in the human solid tumor cell lines HBL-100 (breast), HeLa (cervix), SW1573 (non-small cell lung), T-47D (breast), and WiDr (colon) provided γ-glutamyl methionine (GI50 = 6.0-41 μM) and α-glutamyl proline (GI50 = 7.5-18 μM) as lead compounds. In particular, glutamyl serine and glutamyl proline dipeptides were more active in the resistant cancer cell line WiDr than the conventional anticancer drugs cisplatin and etoposide. Glutamyl tryptophan dipeptides did not affect cell growth of HBL-100, while in T-47D cells, proliferation was inhibited. This result might be attributed to the inhibition of the ATB(0,+) transporter.

  12. Synthesis and antiproliferative activity of glutamic acid-based dipeptides.

    PubMed

    Silveira-Dorta, Gastón; Martín, Víctor S; Padrón, José M

    2015-08-01

    A small and focused library of 22 dipeptides derived from N,N-dibenzylglutamic acid α- and γ-benzyl esters was prepared in a straightforward manner. The evaluation of the antiproliferative activity in the human solid tumor cell lines HBL-100 (breast), HeLa (cervix), SW1573 (non-small cell lung), T-47D (breast), and WiDr (colon) provided γ-glutamyl methionine (GI50 = 6.0-41 μM) and α-glutamyl proline (GI50 = 7.5-18 μM) as lead compounds. In particular, glutamyl serine and glutamyl proline dipeptides were more active in the resistant cancer cell line WiDr than the conventional anticancer drugs cisplatin and etoposide. Glutamyl tryptophan dipeptides did not affect cell growth of HBL-100, while in T-47D cells, proliferation was inhibited. This result might be attributed to the inhibition of the ATB(0,+) transporter. PMID:25900811

  13. CPG2 Recruits Endophilin B2 to the Cytoskeleton for Activity-Dependent Endocytosis of Synaptic Glutamate Receptors.

    PubMed

    Loebrich, Sven; Benoit, Marc Robert; Konopka, Jaclyn Aleksandra; Cottrell, Jeffrey Richard; Gibson, Joanne; Nedivi, Elly

    2016-02-01

    Internalization of glutamate receptors at the postsynaptic membrane via clathrin-mediated endocytosis (CME) is a key mechanism for regulating synaptic strength. A role for the F-actin cytoskeleton in CME is well established, and recently, PKA-dependent association of candidate plasticity gene 2 (CPG2) with the spine-cytoskeleton has been shown to mediate synaptic glutamate receptor internalization. Yet, how the endocytic machinery is physically coupled to the actin cytoskeleton to facilitate glutamate receptor internalization has not been demonstrated. Moreover, there has been no distinction of endocytic-machinery components that are specific to activity-dependent versus constitutive glutamate receptor internalization. Here, we show that CPG2, through a direct physical interaction, recruits endophilin B2 (EndoB2) to F-actin, thus anchoring the endocytic machinery to the spine cytoskeleton and facilitating glutamate receptor internalization. Regulation of CPG2 binding to the actin cytoskeleton by protein kinase A directly impacts recruitment of EndoB2 and clathrin. Specific disruption of EndoB2 or the CPG2-EndoB2 interaction impairs activity-dependent, but not constitutive, internalization of both NMDA- and AMPA-type glutamate receptors. These results demonstrate that, through direct interactions with F-actin and EndoB2, CPG2 physically bridges the spine cytoskeleton and the endocytic machinery, and this tripartite association is critical specifically for activity-dependent CME of synaptic glutamate receptors. PMID:26776730

  14. Modulation of pineal melatonin synthesis by glutamate involves paracrine interactions between pinealocytes and astrocytes through NF-κB activation.

    PubMed

    Villela, Darine; Atherino, Victoria Fairbanks; Lima, Larissa de Sá; Moutinho, Anderson Augusto; do Amaral, Fernanda Gaspar; Peres, Rafael; Martins de Lima, Thais; Torrão, Andréa da Silva; Cipolla-Neto, José; Scavone, Cristóforo; Afeche, Solange Castro

    2013-01-01

    The glutamatergic modulation of melatonin synthesis is well known, along with the importance of astrocytes in mediating glutamatergic signaling in the central nervous system. Pinealocytes and astrocytes are the main cell types in the pineal gland. The objective of this work was to investigate the interactions between astrocytes and pinealocytes as a part of the glutamate inhibitory effect on melatonin synthesis. Rat pinealocytes isolated or in coculture with astrocytes were incubated with glutamate in the presence of norepinephrine, and the melatonin content, was quantified. The expression of glutamate receptors, the intracellular calcium content and the NF- κ B activation were analyzed in astrocytes and pinealocytes. TNF- α 's possible mediation of the effect of glutamate was also investigated. The results showed that glutamate's inhibitory effect on melatonin synthesis involves interactions between astrocytes and pinealocytes, possibly through the release of TNF- α . Moreover, the activation of the astrocytic NF- κ B seems to be a necessary step. In astrocytes and pinealocytes, AMPA, NMDA, and group I metabotropic glutamate receptors were observed, as well as the intracellular calcium elevation. In conclusion, there is evidence that the modulation of melatonin synthesis by glutamate involves paracrine interactions between pinealocytes and astrocytes through the activation of the astrocytic NF- κ B transcription factor and possibly by subsequent TNF- α release.

  15. Glutamate protects against Ca(2+) paradox-induced injury and inhibits calpain activity in isolated rat hearts.

    PubMed

    Zhang, Jian-Ying; Kong, Ling-Heng; Lai, Dong; Jin, Zhen-Xiao; Gu, Xiao-Ming; Zhou, Jing-Jun

    2016-10-01

    This study determined the effects of glutamate on the Ca(2+) paradoxical heart, which is a model for Ca(2+) overload-induced injury during myocardial ischaemia and reperfusion, and evaluated its effect on a known mediator of injury, calpain. An isolated rat heart was retrogradely perfused in a Langendorff apparatus. Ca(2+) paradox was elicited via perfusion with a Ca(2+) -free Krebs-Henseleit (KH) solution for 3 minutes followed by Ca(2+) -containing normal KH solution for 30 minutes. The Ca(2+) paradoxical heart exhibited almost no viable tissue on triphenyltetrazolium chloride staining and markedly increased LDH release, caspase-3 activity, cytosolic cytochrome c content, and apoptotic index. These hearts also displayed significantly increased LVEDP and a disappearance of LVDP. Glutamate (5 and 20 mmol/L) significantly alleviated Ca(2+) paradox-induced injury. In contrast, 20 mmol/L mannitol had no effect on Ca(2+) paradox. Ca(2+) paradox significantly increased the extent of the translocation of μ-calpain to the sarcolemmal membrane and the proteolysis of α-fodrin, which suggests calpain activation. Glutamate also blocked these effects. A non-selective inhibitor of glutamate transporters, dl-TBOA (10 μmol/L), had no effect on control hearts, but it reversed glutamate-induced cardioprotection and reduction in calpain activity. Glutamate treatment significantly increased intracellular glutamate content in the Ca(2+) paradoxical heart, which was also blocked by dl-TBOA. We conclude that glutamate protects the heart against Ca(2+) overload-induced injury via glutamate transporters, and the inhibition of calpain activity is involved in this process.

  16. Oligomers of Amyloid β Prevent Physiological Activation of the Cellular Prion Protein-Metabotropic Glutamate Receptor 5 Complex by Glutamate in Alzheimer Disease.

    PubMed

    Haas, Laura T; Strittmatter, Stephen M

    2016-08-12

    The dysfunction and loss of synapses in Alzheimer disease are central to dementia symptoms. We have recently demonstrated that pathological Amyloid β oligomer (Aβo) regulates the association between intracellular protein mediators and the synaptic receptor complex composed of cellular prion protein (PrP(C)) and metabotropic glutamate receptor 5 (mGluR5). Here we sought to determine whether Aβo alters the physiological signaling of the PrP(C)-mGluR5 complex upon glutamate activation. We provide evidence that acute exposure to Aβo as well as chronic expression of familial Alzheimer disease mutant transgenes in model mice prevents protein-protein interaction changes of the complex induced by the glutamate analog 3,5-dihydroxyphenylglycine. We further show that 3,5-dihydroxyphenylglycine triggers the phosphorylation and activation of protein-tyrosine kinase 2-β (PTK2B, also referred to as Pyk2) and of calcium/calmodulin-dependent protein kinase II in wild-type brain slices but not in Alzheimer disease transgenic brain slices or wild-type slices incubated with Aβo. This study further distinguishes two separate Aβo-dependent signaling cascades, one dependent on extracellular Ca(2+) and Fyn kinase activation and the other dependent on the release of Ca(2+) from intracellular stores. Thus, Aβo triggers multiple distinct PrP(C)-mGluR5-dependent events implicated in neurodegeneration and dementia. We propose that targeting the PrP(C)-mGluR5 complex will reverse aberrant Aβo-triggered states of the complex to allow physiological fluctuations of glutamate signaling.

  17. The activity of class I, II, III and IV of alcohol dehydrogenase (ADH) isoenzymes and aldehyde dehydrogenase (ALDH) in brain cancer.

    PubMed

    Laniewska-Dunaj, Magdalena; Jelski, Wojciech; Orywal, Karolina; Kochanowicz, Jan; Rutkowski, Robert; Szmitkowski, Maciej

    2013-07-01

    The brain being highly sensitive to the action of alcohol is potentially susceptible to its carcinogenic effects. Alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) are the main enzymes involved in ethanol metabolism, which leads to the generation of carcinogenic acetaldehyde. Human brain tissue contains various ADH isoenzymes and possess also ALDH activity. The purpose of this study was to compare the capacity for ethanol metabolism measured by ADH isoenzymes and ALDH activity in cancer tissues and healthy brain cells. The samples were taken from 62 brain cancer patients (36 glioblastoma, 26 meningioma). For the measurement of the activity of class I and II ADH isoenzymes and ALDH activity, the fluorometric methods were used. The total ADH activity and activity of class III and IV isoenzymes were measured by the photometric method. The total activity of ADH, and activity of class I ADH were significantly higher in cancer cells than in healthy tissues. The other tested classes of ADH and ALDH did not show statistically significant differences of activity in cancer and in normal cells. Analysis of the enzymes activity did not show significant differences depending on the location of the tumor. The differences in the activity of total alcohol dehydrogenase, and class I isoenzyme between cancer tissues and healthy brain cells might be a factor for metabolic changes and disturbances in low mature cancer cells and additionally might be a reason for higher level of acetaldehyde which can intensify the carcinogenesis.

  18. Mechanisms underlying regulation of the expression and activities of the mammalian pyruvate dehydrogenase kinases.

    PubMed

    Sugden, Mary C; Holness, Mark J

    2006-07-01

    The mechanisms that control mammalian pyruvate dehydrogenase complex (PDC) activity include its phosphorylation (inactivation) by a family of pyruvate dehydrogenase kinases (PDKs 1 - 4). Here we review new developments in the regulation of the activities and expression of the PDKs, in particular PDK2 and PDK4, in relation to glucose and lipid homeostasis. This review describes recent advances relating to the acute and long-term modes of regulation of the PDKs, with particular emphasis on the regulatory roles of nuclear receptors including peroxisome proliferator-activated receptor (PPAR) alpha and Liver X receptor (LXR), PPAR gamma coactivator alpha (PGC-1alpha) and insulin, and the impact of changes in PDK activity and expression in glucose and lipid homeostasis. Since PDK4 may assist in lipid clearance when there is an imbalance between lipid delivery and oxidation, it may represent an attractive target for interventions aimed at rectifying abnormal lipid as well as glucose homeostasis in disease states. PMID:17132539

  19. Postsynaptic actin regulates active zone spacing and glutamate receptor apposition at the Drosophila neuromuscular junction.

    PubMed

    Blunk, Aline D; Akbergenova, Yulia; Cho, Richard W; Lee, Jihye; Walldorf, Uwe; Xu, Ke; Zhong, Guisheng; Zhuang, Xiaowei; Littleton, J Troy

    2014-07-01

    Synaptic communication requires precise alignment of presynaptic active zones with postsynaptic receptors to enable rapid and efficient neurotransmitter release. How transsynaptic signaling between connected partners organizes this synaptic apparatus is poorly understood. To further define the mechanisms that mediate synapse assembly, we carried out a chemical mutagenesis screen in Drosophila to identify mutants defective in the alignment of active zones with postsynaptic glutamate receptor fields at the larval neuromuscular junction. From this screen we identified a mutation in Actin 57B that disrupted synaptic morphology and presynaptic active zone organization. Actin 57B, one of six actin genes in Drosophila, is expressed within the postsynaptic bodywall musculature. The isolated allele, act(E84K), harbors a point mutation in a highly conserved glutamate residue in subdomain 1 that binds members of the Calponin Homology protein family, including spectrin. Homozygous act(E84K) mutants show impaired alignment and spacing of presynaptic active zones, as well as defects in apposition of active zones to postsynaptic glutamate receptor fields. act(E84K) mutants have disrupted postsynaptic actin networks surrounding presynaptic boutons, with the formation of aberrant actin swirls previously observed following disruption of postsynaptic spectrin. Consistent with a disruption of the postsynaptic actin cytoskeleton, spectrin, adducin and the PSD-95 homolog Discs-Large are all mislocalized in act(E84K) mutants. Genetic interactions between act(E84K) and neurexin mutants suggest that the postsynaptic actin cytoskeleton may function together with the Neurexin-Neuroligin transsynaptic signaling complex to mediate normal synapse development and presynaptic active zone organization.

  20. Cinnabarinic acid, an endogenous metabolite of the kynurenine pathway, activates type 4 metabotropic glutamate receptors.

    PubMed

    Fazio, F; Lionetto, L; Molinaro, G; Bertrand, H O; Acher, F; Ngomba, R T; Notartomaso, S; Curini, M; Rosati, O; Scarselli, P; Di Marco, R; Battaglia, G; Bruno, V; Simmaco, M; Pin, J P; Nicoletti, F; Goudet, C

    2012-05-01

    Cinnabarinic acid is an endogenous metabolite of the kynurenine pathway that meets the structural requirements to interact with glutamate receptors. We found that cinnabarinic acid acts as a partial agonist of type 4 metabotropic glutamate (mGlu4) receptors, with no activity at other mGlu receptor subtypes. We also tested the activity of cinnabarinic acid on native mGlu4 receptors by examining 1) the inhibition of cAMP formation in cultured cerebellar granule cells; 2) protection against excitotoxic neuronal death in mixed cultures of cortical cells; and 3) protection against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine toxicity in mice after local infusion into the external globus pallidus. In all these models, cinnabarinic acid behaved similarly to conventional mGlu4 receptor agonists, and, at least in cultured neurons, the action of low concentrations of cinnabarinic acid was largely attenuated by genetic deletion of mGlu4 receptors. However, high concentrations of cinnabarinic acid were still active in the absence of mGlu4 receptors, suggesting that the compound may have off-target effects. Mutagenesis and molecular modeling experiments showed that cinnabarinic acid acts as an orthosteric agonist interacting with residues of the glutamate binding pocket of mGlu4. Accordingly, cinnabarinic acid did not activate truncated mGlu4 receptors lacking the N-terminal Venus-flytrap domain, as opposed to the mGlu4 receptor enhancer, N-phenyl-7-(hydroxyimino)cyclopropa[b]chromen-1a-carboxamide (PHCCC). Finally, we could detect endogenous cinnabarinic acid in brain tissue and peripheral organs by high-performance liquid chromatography-tandem mass spectrometry analysis. Levels increased substantially during inflammation induced by lipopolysaccharide. We conclude that cinnabarinic acid is a novel endogenous orthosteric agonist of mGlu4 receptors endowed with neuroprotective activity. PMID:22311707

  1. Multichannel Simultaneous Determination of Activities of Lactate Dehydrogenase

    SciTech Connect

    Ma, L.

    2000-09-12

    It is very important to find the best conditions for some enzymes to do the best catalysis in current pharmaceutical industries. Based on the results above, we could say that this set-up could be widely used in finding the optimal condition for best enzyme activity of a certain enzyme. Instead of looking for the best condition for enzyme activity by doing many similar reactions repeatedly, we can complete this assignment with just one run if we could apply enough conditions.

  2. Reduced mitochondrial malate dehydrogenase activity has a strong effect on photorespiratory metabolism as revealed by 13C labelling

    PubMed Central

    Lindén, Pernilla; Keech, Olivier; Stenlund, Hans; Gardeström, Per; Moritz, Thomas

    2016-01-01

    Mitochondrial malate dehydrogenase (mMDH) catalyses the interconversion of malate and oxaloacetate (OAA) in the tricarboxylic acid (TCA) cycle. Its activity is important for redox control of the mitochondrial matrix, through which it may participate in regulation of TCA cycle turnover. In Arabidopsis, there are two isoforms of mMDH. Here, we investigated to which extent the lack of the major isoform, mMDH1 accounting for about 60% of the activity, affected leaf metabolism. In air, rosettes of mmdh1 plants were only slightly smaller than wild type plants although the fresh weight was decreased by about 50%. In low CO2 the difference was much bigger, with mutant plants accumulating only 14% of fresh weight as compared to wild type. To investigate the metabolic background to the differences in growth, we developed a 13CO2 labelling method, using a custom-built chamber that enabled simultaneous treatment of sets of plants under controlled conditions. The metabolic profiles were analysed by gas- and liquid- chromatography coupled to mass spectrometry to investigate the metabolic adjustments between wild type and mmdh1. The genotypes responded similarly to high CO2 treatment both with respect to metabolite pools and 13C incorporation during a 2-h treatment. However, under low CO2 several metabolites differed between the two genotypes and, interestingly most of these were closely associated with photorespiration. We found that while the glycine/serine ratio increased, a concomitant altered glutamine/glutamate/α-ketoglutarate relation occurred. Taken together, our results indicate that adequate mMDH activity is essential to shuttle reductants out from the mitochondria to support the photorespiratory flux, and strengthen the idea that photorespiration is tightly intertwined with peripheral metabolic reactions. PMID:26889011

  3. Activation of glutamate transport evokes rapid glutamine release from perisynaptic astrocytes

    PubMed Central

    Uwechue, Nneka M; Marx, Mari-Carmen; Chevy, Quentin; Billups, Brian

    2012-01-01

    Stimulation of astrocytes by neuronal activity and the subsequent release of neuromodulators is thought to be an important regulator of synaptic communication. In this study we show that astrocytes juxtaposed to the glutamatergic calyx of Held synapse in the rat medial nucleus of the trapezoid body (MNTB) are stimulated by the activation of glutamate transporters and consequently release glutamine on a very rapid timescale. MNTB principal neurones express electrogenic system A glutamine transporters, and were exploited as glutamine sensors in this study. By simultaneous whole-cell voltage clamping astrocytes and neighbouring MNTB neurones in brainstem slices, we show that application of the excitatory amino acid transporter (EAAT) substrate d-aspartate stimulates astrocytes to rapidly release glutamine, which is detected by nearby MNTB neurones. This release is significantly reduced by the toxins l-methionine sulfoximine and fluoroacetate, which reduce glutamine concentrations specifically in glial cells. Similarly, glutamine release was also inhibited by localised inactivation of EAATs in individual astrocytes, using internal dl-threo-β-benzyloxyaspartic acid (TBOA) or dissipating the driving force by modifying the patch-pipette solution. These results demonstrate that astrocytes adjacent to glutamatergic synapses can release glutamine in a temporally precise, controlled manner in response to glial glutamate transporter activation. Since glutamine can be used by neurones as a precursor for glutamate and GABA synthesis, this represents a potential feedback mechanism by which astrocytes can respond to synaptic activation and react in a way that sustains or enhances further communication. This would therefore represent an additional manifestation of the tripartite relationship between synapses and astrocytes. PMID:22411007

  4. Fructose 1,6-diphosphate-activated L-lactate dehydrogenase from Streptococcus lactis: kinetic properties and factors affecting activation.

    PubMed Central

    Crow, V L; Pritchard, G G

    1977-01-01

    The L-(+)-lactate dehydrogenase (L-lactate:NAD+ oxidoreductase, EC 1.1.1.27) of Streptococcus lactis C10, like that of other streptococci, was activated by fructose 1,6-diphosphate (FDP). The enzyme showed some activity in the absence of FDP, with a pH optimum of 8.2; FDP decreased the Km for both pyruvate and reduced nicotinamide adenine dinucleotide (NADH) and shifted the pH optimum to 6.9. Enzyme activity showed a hyperbolic response to both NADH and pyruvate in all the buffers tried except phosphate buffer, in which the response to increasing NADH was sigmoidal. The FDP concentration required for half-maximal velocity (FDP0.5V) was markedly influenced by the nature of the assay buffer used. Thus the FDP0.5V was 0.002 mM in 90 mM triethanolamine buffer, 0.2 mM in 90 mM tris(hydroxymethyl)aminomethanemaleate buffer, and 4.4 mM in 90 mM phosphate buffer. Phosphate inhibition of FDP binding is not a general property of streptococcal lactate dehydrogenase, since the FDP0.5V value for S. faecalis 8043 lactate dehydrogenase was not increased by phosphate. The S. faecalis and S. lactis lactate dehydrogenases also differed in that Mn2+ enhanced FDP binding in S. faecalis but had no effect on the S. lactis dehydrogenase. The FDP concentration (12 to 15 mM) found in S. lactis cells during logarithmic growth on a high-carbohydrate (3% lactose) medium would be adequate to give almost complete activation of the lactate dehydrogenase even if the high FDP0.5V value found in 90 mM phosphate were similar to the FDP requirement in vivo. PMID:17595

  5. The dihydrolipoamide dehydrogenase of Aeromonas caviae ST exhibits NADH-dependent tellurite reductase activity.

    PubMed

    Castro, Miguel E; Molina, Roberto; Díaz, Waldo; Pichuantes, Sergio E; Vásquez, Claudio C

    2008-10-10

    Potassium tellurite (K(2)TeO(3)) is extremely toxic for most forms of life and only a limited number of organisms are naturally resistant to the toxic effects of this compound. Crude extracts prepared from the environmental isolate Aeromonas caviae ST catalize the in vitro reduction of TeO32- in a NADH-dependent reaction. Upon fractionation by ionic exchange column chromatography three major polypeptides identified as the E1, E2, and E3 components of the pyruvate dehydrogenase (PDH) complex were identified in fractions exhibiting tellurite-reducing activity. Tellurite reductase and pyruvate dehydrogenase activities co-eluted from a Sephadex gel filtration column. To determine which component(s) of the PDH complex has tellurite reductase activity, the A. caviae ST structural genes encoding for E1 (aceE), E2 (aceF), and E3 (lpdA) were independently cloned and expressed in Escherichia coli and their gene products purified. Results indicated that tellurite reductase activity lies almost exclusively in the E3 component, dihydrolipoamide dehydrogenase. The E3 component of the PDH complex from E. coli, Zymomonas mobilis, Streptococcus pneumoniae, and Geobacillus stearothermophilus also showed NADH-dependent tellurite reductase in vitro suggesting that this enzymatic activity is widely distributed among microorganisms. PMID:18675788

  6. Stability and activity of alcohol dehydrogenases in W/O-microemulsions: enantioselective reduction including cofactor regeneration.

    PubMed

    Orlich, B; Berger, H; Lade, M; Schomäcker, R

    2000-12-20

    Microemulsions provide an interesting alternative to classical methods for the conversion of less water-soluble substrates by alcohol dehydrogenase, but until now stability and activity were too low for economically useful processes. The activity and stability of the enzymes are dependent on the microemulsion composition, mostly the water and the surfactant concentration. Therefore, it is necessary to know the exact phase behavior of a given microemulsion reaction system and the corresponding enzyme behavior therein. Because of their economic and ecologic suitability polyethoxylated fatty alcohols were investigated concerning their phase behavior and their compatibility with enzymes in ternary mixtures. The phase behavior of Marlipal O13-60 (C13EO6 in industrial quality)/cyclohexane/water and its effect on the activity and stability of alcohol dehydrogenase from Yeast (YADH) and horse liver (HLADH) and the carbonyl reductase from Candida parapsilosis (CPCR) is presented in this study. Beside the macroscopic phase behavior of the reaction system, the viscosity of the system indicates structural changes of aggregates in the microemulsion. The changes of the enzyme activities with the composition are discussed on the basis of transitions from reverse micelles to swollen reverse micelles and finally, the transition to the phase separation. The formate dehydrogenase from Candida boidinii was used for the NADH-regeneration during reduction reactions. While the formate dehydrogenase did not show any kinetic effect on the microemulsion composition, the other enzymes show significant changes of activity and stability varying the water or surfactant concentration of the microemulsion. Under certain conditions, stability could be maintained with HLADH for several weeks. Successful experiments with semi-batch processes including cofactor regeneration and product separation were performed.

  7. Succinate dehydrogenase activity and soma size of motoneurons innervating different portions of the rat tibialis anterior

    NASA Technical Reports Server (NTRS)

    Ishihara, A.; Roy, R. R.; Edgerton, V. R.

    1995-01-01

    The spatial distribution, soma size and oxidative enzyme activity of gamma and alpha motoneurons innervating muscle fibres in the deep (away from the surface of the muscle) and superficial (close to the surface of the muscle) portions of the tibialis anterior in normal rats were determined. The deep portion had a higher percentage of high oxidative fibres than the superficial portion of the muscle. Motoneurons were labelled by retrograde neuronal transport of fluorescent tracers: Fast Blue and Nuclear Yellow were injected into the deep portion and Nuclear Yellow into the superficial portion of the muscle. Therefore, motoneurons innervating the deep portion were identified by both a blue fluorescent cytoplasm and a golden-yellow fluorescent nucleus, while motoneurons innervating the superficial portion were identified by only a golden-yellow fluorescent nucleus. After staining for succinate dehydrogenase activity on the same section used for the identification of the motoneurons, soma size and succinate dehydrogenase activity of the motoneurons were measured. The gamma and alpha motoneurons innervating both the deep and superficial portions were located primarily at L4 and were intermingled within the same region of the dorsolateral portion of the ventral horn in the spinal cord. Mean soma size was similar for either gamma or alpha motoneurons in the two portions of the muscle. The alpha motoneurons innervating the superficial portion had a lower mean succinate dehydrogenase activity than those innervating the deep portion of the muscle. An inverse relationship between soma size and succinate dehydrogenase activity of alpha, but not gamma, motoneurons innervating both the deep and superficial portions was observed. Based on three-dimensional reconstructions within the spinal cord, there were no apparent differences in the spatial distribution of the motoneurons, either gamma or alpha, associated with the deep and superficial compartments of the muscle. The data

  8. Removal of CO dehydrogenase from Pseudomonas carboxydovorans cytoplasmic membranes, rebinding of CO dehydrogenase to depleted membranes, and restoration of respiratory activities.

    PubMed Central

    Jacobitz, S; Meyer, O

    1989-01-01

    In Pseudomonas carboxydovorans, CO dehydrogenase and hydrogenase were found in association with the cytoplasmic membrane in a weakly bound and a tightly bound pool. The pools could be experimentally distinguished on the basis of resistance to removal by washes in low-ionic-strength buffer. The tightly bound pool of the enzymes could be differentially solubilized under conditions leaving the electron transport system intact and with the nondenaturing zwitterionic detergent 3-(3-cholamidopropyl) dimethylammonio 1-propane-sulfonic acid (CHAPS) and the nonionic detergent dodecyl beta-D-maltoside. In vitro reconstitution of depleted membranes with the corresponding supernatants containing CO dehydrogenase led to binding of the enzyme and to reactivation of respiratory activities with CO. The reconstitution reaction required cations with effectiveness which increased with increasing ionic charge: monovalent (Li+), divalent (Mg2+, Mn2+), or trivalent (Cr3+, La3+). Reconstitution of depleted membranes with CO dehydrogenase was specific for CO-grown bacteria. Cytoplasmic membranes from H2- or heterotrophically grown Pseudomonas carboxydovorans had no affinity for CO dehydrogenase at all, indicating the absence of the physiological electron acceptor of the enzyme, which presumably is cytochrome b561, or another membrane anchor. PMID:2808305

  9. Determination of Dehydrogenase Activities Involved in D-Glucose Oxidation in Gluconobacter and Acetobacter Strains

    PubMed Central

    Sainz, Florencia; Jesús Torija, María; Matsutani, Minenosuke; Kataoka, Naoya; Yakushi, Toshiharu; Matsushita, Kazunobu; Mas, Albert

    2016-01-01

    Acetic acid bacteria (AAB) are known for rapid and incomplete oxidation of an extensively variety of alcohols and carbohydrates, resulting in the accumulation of organic acids as the final products. These oxidative fermentations in AAB are catalyzed by PQQ- or FAD- dependent membrane-bound dehydrogenases. In the present study, the enzyme activity of the membrane-bound dehydrogenases [membrane-bound PQQ-glucose dehydrogenase (mGDH), D-gluconate dehydrogenase (GADH) and membrane-bound glycerol dehydrogenase (GLDH)] involved in the oxidation of D-glucose and D-gluconic acid (GA) was determined in six strains of three different species of AAB (three natural and three type strains). Moreover, the effect of these activities on the production of related metabolites [GA, 2-keto-D-gluconic acid (2KGA) and 5-keto-D-gluconic acid (5KGA)] was analyzed. The natural strains belonging to Gluconobacter showed a high mGDH activity and low activity in GADH and GLDH, whereas the Acetobacter malorum strain presented low activity in the three enzymes. Nevertheless, no correlation was observed between the activity of these enzymes and the concentration of the corresponding metabolites. In fact, all the tested strains were able to oxidize D-glucose to GA, being maximal at the late exponential phase of the AAB growth (24 h), which coincided with D-glucose exhaustion and the maximum mGDH activity. Instead, only some of the tested strains were capable of producing 2KGA and/or 5KGA. In the case of Gluconobacter oxydans strains, no 2KGA production was detected which is related to the absence of GADH activity after 24 h, while in the remaining strains, detection of GADH activity after 24 h resulted in a high accumulation of 2KGA. Therefore, it is possible to choose the best strain depending on the desired product composition. Moreover, the sequences of these genes were used to construct phylogenetic trees. According to the sequence of gcd, gene coding for mGDH, Acetobacter and Komagataeibacter

  10. Determination of Dehydrogenase Activities Involved in D-Glucose Oxidation in Gluconobacter and Acetobacter Strains.

    PubMed

    Sainz, Florencia; Jesús Torija, María; Matsutani, Minenosuke; Kataoka, Naoya; Yakushi, Toshiharu; Matsushita, Kazunobu; Mas, Albert

    2016-01-01

    Acetic acid bacteria (AAB) are known for rapid and incomplete oxidation of an extensively variety of alcohols and carbohydrates, resulting in the accumulation of organic acids as the final products. These oxidative fermentations in AAB are catalyzed by PQQ- or FAD- dependent membrane-bound dehydrogenases. In the present study, the enzyme activity of the membrane-bound dehydrogenases [membrane-bound PQQ-glucose dehydrogenase (mGDH), D-gluconate dehydrogenase (GADH) and membrane-bound glycerol dehydrogenase (GLDH)] involved in the oxidation of D-glucose and D-gluconic acid (GA) was determined in six strains of three different species of AAB (three natural and three type strains). Moreover, the effect of these activities on the production of related metabolites [GA, 2-keto-D-gluconic acid (2KGA) and 5-keto-D-gluconic acid (5KGA)] was analyzed. The natural strains belonging to Gluconobacter showed a high mGDH activity and low activity in GADH and GLDH, whereas the Acetobacter malorum strain presented low activity in the three enzymes. Nevertheless, no correlation was observed between the activity of these enzymes and the concentration of the corresponding metabolites. In fact, all the tested strains were able to oxidize D-glucose to GA, being maximal at the late exponential phase of the AAB growth (24 h), which coincided with D-glucose exhaustion and the maximum mGDH activity. Instead, only some of the tested strains were capable of producing 2KGA and/or 5KGA. In the case of Gluconobacter oxydans strains, no 2KGA production was detected which is related to the absence of GADH activity after 24 h, while in the remaining strains, detection of GADH activity after 24 h resulted in a high accumulation of 2KGA. Therefore, it is possible to choose the best strain depending on the desired product composition. Moreover, the sequences of these genes were used to construct phylogenetic trees. According to the sequence of gcd, gene coding for mGDH, Acetobacter and Komagataeibacter

  11. Determination of Dehydrogenase Activities Involved in D-Glucose Oxidation in Gluconobacter and Acetobacter Strains.

    PubMed

    Sainz, Florencia; Jesús Torija, María; Matsutani, Minenosuke; Kataoka, Naoya; Yakushi, Toshiharu; Matsushita, Kazunobu; Mas, Albert

    2016-01-01

    Acetic acid bacteria (AAB) are known for rapid and incomplete oxidation of an extensively variety of alcohols and carbohydrates, resulting in the accumulation of organic acids as the final products. These oxidative fermentations in AAB are catalyzed by PQQ- or FAD- dependent membrane-bound dehydrogenases. In the present study, the enzyme activity of the membrane-bound dehydrogenases [membrane-bound PQQ-glucose dehydrogenase (mGDH), D-gluconate dehydrogenase (GADH) and membrane-bound glycerol dehydrogenase (GLDH)] involved in the oxidation of D-glucose and D-gluconic acid (GA) was determined in six strains of three different species of AAB (three natural and three type strains). Moreover, the effect of these activities on the production of related metabolites [GA, 2-keto-D-gluconic acid (2KGA) and 5-keto-D-gluconic acid (5KGA)] was analyzed. The natural strains belonging to Gluconobacter showed a high mGDH activity and low activity in GADH and GLDH, whereas the Acetobacter malorum strain presented low activity in the three enzymes. Nevertheless, no correlation was observed between the activity of these enzymes and the concentration of the corresponding metabolites. In fact, all the tested strains were able to oxidize D-glucose to GA, being maximal at the late exponential phase of the AAB growth (24 h), which coincided with D-glucose exhaustion and the maximum mGDH activity. Instead, only some of the tested strains were capable of producing 2KGA and/or 5KGA. In the case of Gluconobacter oxydans strains, no 2KGA production was detected which is related to the absence of GADH activity after 24 h, while in the remaining strains, detection of GADH activity after 24 h resulted in a high accumulation of 2KGA. Therefore, it is possible to choose the best strain depending on the desired product composition. Moreover, the sequences of these genes were used to construct phylogenetic trees. According to the sequence of gcd, gene coding for mGDH, Acetobacter and Komagataeibacter

  12. Determination of Dehydrogenase Activities Involved in D-Glucose Oxidation in Gluconobacter and Acetobacter Strains

    PubMed Central

    Sainz, Florencia; Jesús Torija, María; Matsutani, Minenosuke; Kataoka, Naoya; Yakushi, Toshiharu; Matsushita, Kazunobu; Mas, Albert

    2016-01-01

    Acetic acid bacteria (AAB) are known for rapid and incomplete oxidation of an extensively variety of alcohols and carbohydrates, resulting in the accumulation of organic acids as the final products. These oxidative fermentations in AAB are catalyzed by PQQ- or FAD- dependent membrane-bound dehydrogenases. In the present study, the enzyme activity of the membrane-bound dehydrogenases [membrane-bound PQQ-glucose dehydrogenase (mGDH), D-gluconate dehydrogenase (GADH) and membrane-bound glycerol dehydrogenase (GLDH)] involved in the oxidation of D-glucose and D-gluconic acid (GA) was determined in six strains of three different species of AAB (three natural and three type strains). Moreover, the effect of these activities on the production of related metabolites [GA, 2-keto-D-gluconic acid (2KGA) and 5-keto-D-gluconic acid (5KGA)] was analyzed. The natural strains belonging to Gluconobacter showed a high mGDH activity and low activity in GADH and GLDH, whereas the Acetobacter malorum strain presented low activity in the three enzymes. Nevertheless, no correlation was observed between the activity of these enzymes and the concentration of the corresponding metabolites. In fact, all the tested strains were able to oxidize D-glucose to GA, being maximal at the late exponential phase of the AAB growth (24 h), which coincided with D-glucose exhaustion and the maximum mGDH activity. Instead, only some of the tested strains were capable of producing 2KGA and/or 5KGA. In the case of Gluconobacter oxydans strains, no 2KGA production was detected which is related to the absence of GADH activity after 24 h, while in the remaining strains, detection of GADH activity after 24 h resulted in a high accumulation of 2KGA. Therefore, it is possible to choose the best strain depending on the desired product composition. Moreover, the sequences of these genes were used to construct phylogenetic trees. According to the sequence of gcd, gene coding for mGDH, Acetobacter and Komagataeibacter

  13. [Enhancing glutamate decarboxylase activity by site-directed mutagenesis: an insight from Ramachandran plot].

    PubMed

    Ke, Piyu; Huang, Jun; Hu, Sheng; Zhao, Weirui; Lü, Changjiang; Yu, Kai; Lei, Yinlin; Wang, Jinbo; Mei, Lehe

    2016-01-01

    Glutamate decarboxylase (GAD) can catalyze the decarboxylation of glutamate into γ-aminobutyrate (GABA) and is the only enzyme of GABA biosynthesis. Improving GAD activity and thermostability will be helpful for the highly efficient biosynthesis of GABA. According to the Ramachandran plot information of GAD 1407 three-dimensional structure from Lactobacillus brevis CGMCC No. 1306, we identified the unstable site K413 as the mutation target, constructed the mutant GAD by site-directed mutagenesis and measured the thermostability and activity of the wide type and mutant GAD. Mutant K413A led to a remarkably slower inactivation rate, and its half-life at 50 °C reached 105 min which was 2.1-fold higher than the wild type GAD1407. Moreover, mutant K413I exhibited 1.6-fold higher activity in comparison with the wide type GAD1407, although it had little improvement in thermostability of GAD. Ramachandran plot can be considered as a potential approach to increase GAD thermostability and activity. PMID:27443004

  14. [Enhancing glutamate decarboxylase activity by site-directed mutagenesis: an insight from Ramachandran plot].

    PubMed

    Ke, Piyu; Huang, Jun; Hu, Sheng; Zhao, Weirui; Lü, Changjiang; Yu, Kai; Lei, Yinlin; Wang, Jinbo; Mei, Lehe

    2016-01-01

    Glutamate decarboxylase (GAD) can catalyze the decarboxylation of glutamate into γ-aminobutyrate (GABA) and is the only enzyme of GABA biosynthesis. Improving GAD activity and thermostability will be helpful for the highly efficient biosynthesis of GABA. According to the Ramachandran plot information of GAD 1407 three-dimensional structure from Lactobacillus brevis CGMCC No. 1306, we identified the unstable site K413 as the mutation target, constructed the mutant GAD by site-directed mutagenesis and measured the thermostability and activity of the wide type and mutant GAD. Mutant K413A led to a remarkably slower inactivation rate, and its half-life at 50 °C reached 105 min which was 2.1-fold higher than the wild type GAD1407. Moreover, mutant K413I exhibited 1.6-fold higher activity in comparison with the wide type GAD1407, although it had little improvement in thermostability of GAD. Ramachandran plot can be considered as a potential approach to increase GAD thermostability and activity.

  15. Pyruvate dehydrogenase complex activity controls metabolic and malignant phenotype in cancer cells.

    PubMed

    McFate, Thomas; Mohyeldin, Ahmed; Lu, Huasheng; Thakar, Jay; Henriques, Jeremy; Halim, Nader D; Wu, Hong; Schell, Michael J; Tsang, Tsz Mon; Teahan, Orla; Zhou, Shaoyu; Califano, Joseph A; Jeoung, Nam Ho; Harris, Robert A; Verma, Ajay

    2008-08-15

    High lactate generation and low glucose oxidation, despite normal oxygen conditions, are commonly seen in cancer cells and tumors. Historically known as the Warburg effect, this altered metabolic phenotype has long been correlated with malignant progression and poor clinical outcome. However, the mechanistic relationship between altered glucose metabolism and malignancy remains poorly understood. Here we show that inhibition of pyruvate dehydrogenase complex (PDC) activity contributes to the Warburg metabolic and malignant phenotype in human head and neck squamous cell carcinoma. PDC inhibition occurs via enhanced expression of pyruvate dehydrogenase kinase-1 (PDK-1), which results in inhibitory phosphorylation of the pyruvate dehydrogenase alpha (PDHalpha) subunit. We also demonstrate that PDC inhibition in cancer cells is associated with normoxic stabilization of the malignancy-promoting transcription factor hypoxia-inducible factor-1alpha (HIF-1alpha) by glycolytic metabolites. Knockdown of PDK-1 via short hairpin RNA lowers PDHalpha phosphorylation, restores PDC activity, reverts the Warburg metabolic phenotype, decreases normoxic HIF-1alpha expression, lowers hypoxic cell survival, decreases invasiveness, and inhibits tumor growth. PDK-1 is an HIF-1-regulated gene, and these data suggest that the buildup of glycolytic metabolites, resulting from high PDK-1 expression, may in turn promote HIF-1 activation, thus sustaining a feed-forward loop for malignant progression. In addition to providing anabolic support for cancer cells, altered fuel metabolism thus supports a malignant phenotype. Correction of metabolic abnormalities offers unique opportunities for cancer treatment and may potentially synergize with other cancer therapies. PMID:18541534

  16. A high-throughput colorimetric assay to measure the activity of glutamate decarboxylase.

    PubMed

    Yu, Kai; Hu, Sheng; Huang, Jun; Mei, Le-He

    2011-08-10

    A pH-sensitive colorimetric assay has been established to quantitatively measure glutamate decarboxylase (GAD) activity in bacterial cell extracts using a microplate format. GAD catalyzes the irreversible α-decarboxylation of L-glutamate to γ-aminobutyrate. The assay is based on the color change of bromocresol green due to an increase in pH as protons are consumed during the enzyme-catalyzed reaction. Bromocresol green was chosen as the indicator because it has a similar pK(a) to the acetate buffer used. The corresponding absorbance change at 620 nm was recorded with a microplate reader as the reaction proceeded. A difference in the enzyme preparation pH and optimal pH for GAD activity of 2.5 did not prevent this method from successfully allowing the determination of reaction kinetic parameters and the detection of improvements in enzymatic activity with a low coefficient of variance. Our assay is simple, rapid, requires minimal sample concentration and can be carried out in robotic high-throughput devices used as standard in directed evolution experiments. In addition, it is also applicable to other reactions that involve a change in pH.

  17. Evolutionary origins of retinoid active short-chain dehydrogenases/reductases of SDR16C family

    PubMed Central

    Belyaeva, Olga V.; Chang, Chenbei; Berlett, Michael C; Kedishvili, Natalia Y.

    2014-01-01

    Vertebrate enzymes that belong to the 16C family of short-chain dehydrogenases/reductases (SDR16C) were shown to play an essential role in the control of retinoic acid (RA) levels during development. To trace the evolution of enzymatic function of SDR16C family, and to examine the origins of the pathway for RA biosynthesis from vitamin A, we identified putative SDR16C enzymes through the extensive search of available genome sequencing data in a subset of species representing major metazoan phyla. The phylogenetic analysis revealed that enzymes from protostome, non-chordate deuterostome and invertebrate chordate species are found in three clades of SDR16C family containing retinoid active enzymes, which are retinol dehydrogenase 10 (RDH10), retinol dehydrogenases E2 (RDHE2) and RDHE2-similar, and dehydrogenase reductase (SDR family) member 3 (DHRS3). For the initial functional analysis, we cloned RDH10- and RDHE2-related enzymes from the early developmental stages of a non-chordate deuterostome, green sea urchin Lytechinus variegatus, and an invertebrate chordate, sea squirt Ciona intestinalis. In situ hybridization revealed that these proteins are expressed in a pattern relevant to development, while assays performed on proteins expressed in mammalian cell culture showed that they possess retinol-oxidizing activity as their vertebrate homologs. The existence of invertebrate homologs of DHRS3 was inferred from the analysis of phylogeny and cofactor-binding residues characteristic of preference for NADP(H). The presence of invertebrate homologs in the DHRS3 group of SDR16C is interesting in light of the complex mutually activating interaction, which we have recently described for human RDH10 and DHRS3 enzymes. Further functional analysis of these homologs will establish whether this interaction evolved to control retinoid homeostasis only in vertebrates, or is also conserved in pre-vertebrates. PMID:25451586

  18. Mechanistic insights into ferredoxin-NADP(H) reductase catalysis involving the conserved glutamate in the active site.

    PubMed

    Dumit, Verónica I; Essigke, Timm; Cortez, Néstor; Ullmann, G Matthias

    2010-04-01

    Plant-type ferredoxin-NADP(H) reductases (FNRs) are flavoenzymes harboring one molecule of noncovalently bound flavin adenine dinucleotide that catalyze reversible reactions between obligatory one-electron carriers and obligatory two-electron carriers. A glutamate next to the C-terminus is strictly conserved in FNR and has been proposed to function as proton donor/acceptor during catalysis. However, experimental studies of this proposed function led to contradicting conclusions about the role of this glutamate in the catalytic mechanism. In the present work, we study the titration behavior of the glutamate in the active site of FNR using theoretical methods. Protonation probabilities for maize FNR were computed for the reaction intermediates of the catalytic cycle by Poisson-Boltzmann electrostatic calculations and Metropolis Monte Carlo titration. The titration behavior of the highly conserved glutamate was found to vary depending on the bound substrates NADP(H) and ferredoxin and also on the redox states of these substrates and the flavin adenine dinucleotide. Our results support the involvement of the glutamate in the FNR catalytic mechanism not only as a proton donor but also as a key residue for stabilizing and destabilizing reaction intermediates. On the basis of our findings, we propose a model rationalizing the function of the glutamate in the reaction cycle, which allows reinterpretation of previous experimental results.

  19. Escherichia coli d-Malate Dehydrogenase, a Generalist Enzyme Active in the Leucine Biosynthesis Pathway*

    PubMed Central

    Vorobieva, Anastassia A.; Khan, Mohammad Shahneawz; Soumillion, Patrice

    2014-01-01

    The enzymes of the β-decarboxylating dehydrogenase superfamily catalyze the oxidative decarboxylation of d-malate-based substrates with various specificities. Here, we show that, in addition to its natural function affording bacterial growth on d-malate as a carbon source, the d-malate dehydrogenase of Escherichia coli (EcDmlA) naturally expressed from its chromosomal gene is capable of complementing leucine auxotrophy in a leuB− strain lacking the paralogous isopropylmalate dehydrogenase enzyme. To our knowledge, this is the first example of an enzyme that contributes with a physiologically relevant level of activity to two distinct pathways of the core metabolism while expressed from its chromosomal locus. EcDmlA features relatively high catalytic activity on at least three different substrates (l(+)-tartrate, d-malate, and 3-isopropylmalate). Because of these properties both in vivo and in vitro, EcDmlA may be defined as a generalist enzyme. Phylogenetic analysis highlights an ancient origin of DmlA, indicating that the enzyme has maintained its generalist character throughout evolution. We discuss the implication of these findings for protein evolution. PMID:25160617

  20. KCa2 channels activation prevents [Ca2+]i deregulation and reduces neuronal death following glutamate toxicity and cerebral ischemia

    PubMed Central

    Dolga, A M; Terpolilli, N; Kepura, F; Nijholt, I M; Knaus, H-G; D'Orsi, B; Prehn, J H M; Eisel, U L M; Plant, T; Plesnila, N; Culmsee, C

    2011-01-01

    Exacerbated activation of glutamate receptor-coupled calcium channels and subsequent increase in intracellular calcium ([Ca2+]i) are established hallmarks of neuronal cell death in acute and chronic neurological diseases. Here we show that pathological [Ca2+]i deregulation occurring after glutamate receptor stimulation is effectively modulated by small conductance calcium-activated potassium (KCa2) channels. We found that neuronal excitotoxicity was associated with a rapid downregulation of KCa2.2 channels within 3 h after the onset of glutamate exposure. Activation of KCa2 channels preserved KCa2 expression and significantly reduced pathological increases in [Ca2+]i providing robust neuroprotection in vitro and in vivo. These data suggest a critical role for KCa2 channels in excitotoxic neuronal cell death and propose their activation as potential therapeutic strategy for the treatment of acute and chronic neurodegenerative disorders. PMID:21509037

  1. Inhibition of mammillary body neurons by direct activation of Group II metabotropic glutamate receptors

    PubMed Central

    Lee, Charles C.

    2016-01-01

    The mammillary body is an important neural component of limbic circuitry implicated in learning and memory. Excitatory and inhibitory inputs, primarily mediated by glutamate and gamma-amino butyric acid (GABA), respectively, converge and integrate in this region, before sending information to the thalamus. One potentially overlooked mechanism for inhibition of mammillary body neurons is through direct activation of Group II metabotropic glutamate receptors (mGluRs). Here, whole-cell patch clamp recordings of in vitro slice preparations containing the mammillary body nuclei of the mouse were employed to record responses to bath application of pharmacological agents to isolate the direct effect of activating Group II mGluRs. Application of the Group II mGluR specific agonist, APDC, resulted in a hyperpolarization of the membrane potential in mammillary body neurons, likely resulting from the opening of a potassium conductance. These data suggest that glutamatergic inputs to the mammillary body may be attenuated via Group II mGluRs and implicates a functional role for these receptors in memory-related circuits and broadly throughout the central nervous system. PMID:27390777

  2. Asymmetry of the active site loop conformation between subunits of glutamate-1-semialdehyde aminomutase in solution.

    PubMed

    Campanini, Barbara; Bettati, Stefano; di Salvo, Martino Luigi; Mozzarelli, Andrea; Contestabile, Roberto

    2013-01-01

    Glutamate-1-semialdehyde aminomutase (GSAM) is a dimeric, pyridoxal 5'-phosphate (PLP)- dependent enzyme catalysing in plants and some bacteria the isomerization of L-glutamate-1-semialdehyde to 5-aminolevulinate, a common precursor of chlorophyll, haem, coenzyme B12, and other tetrapyrrolic compounds. During the catalytic cycle, the coenzyme undergoes conversion from pyridoxamine 5'-phosphate (PMP) to PLP. The entrance of the catalytic site is protected by a loop that is believed to switch from an open to a closed conformation during catalysis. Crystallographic studies indicated that the structure of the mobile loop is related to the form of the cofactor bound to the active site, allowing for asymmetry within the dimer. Since no information on structural and functional asymmetry of the enzyme in solution is available in the literature, we investigated the active site accessibility by determining the cofactor fluorescence quenching of PMP- and PLP-GSAM forms. PLP-GSAM is partially quenched by potassium iodide, suggesting that at least one catalytic site is accessible to the anionic quencher and therefore confirming the asymmetry observed in the crystal structure. Iodide induces release of the cofactor from PMP-GSAM, apparently from only one catalytic site, therefore suggesting an asymmetry also in this form of the enzyme in solution, in contrast with the crystallographic data.

  3. [Imbalance of system of glutamin - glutamic acid in the placenta and amniotic fluid at placental insufficiency].

    PubMed

    Pogorelova, T N; Gunko, V O; Linde, V A

    2014-01-01

    Metabolism of glutamine and glutamic acid has been investigated in the placenta and amniotic fluid under conditions of placental insufficiency. The development of placental insufficiency is characterized by the increased content of glutamic acid and a decrease of glutamine in both placenta and amniotic fluid. These changes changes were accompanied by changes in the activity of enzymes involved in the metabolism of these amino acids. There was a decrease in glutamate dehydrogenase activity and an increase in glutaminase activity with the simultaneous decrease of glutamine synthetase activity. The compensatory decrease in the activity of glutamine keto acid aminotransferase did not prevent a decrease in the glutamine level. The impairments in the system glutamic acid-glutamine were more pronounced during the development of premature labor.

  4. [The pentose phosphate pathway and NADP-dependent glycerol-3-phosphate dehydrogenase activity in some tissues of albino rat].

    PubMed

    Glushankov, E P; Epifanova, Iu E; Kolotilova, A I

    1976-10-01

    The NADP-dependent glycerol-3-phosphate dehydrogenase activity in liver, heart and skeletal muscle of rat was studied. The activity is found when glyceraldehyde-3-phosphate or ribose-5-phosphate in the presence of ATP are taken as substrates. The data obtained confirm that NADP-dependent glycerol-3-phosphate dehydrogenase exists in skeletal muscle and demonstrate that it is found in heart muscle as well.

  5. Naphthazarin protects against glutamate-induced neuronal death via activation of the Nrf2/ARE pathway

    SciTech Connect

    Son, Tae Gen; Kawamoto, Elisa M.; Yu, Qian-Sheng; Greig, Nigel H.; Mattson, Mark P.; Camandola, Simonetta

    2013-04-19

    Highlights: •Naphthazarin activates the Nrf2/ARE pathway. •Naphthazarin induces Nrf2-driven genes in neurons and astrocytes. •Naphthazarin protects neurons against excitotoxicity. -- Abstract: Nuclear factor E2-related factor 2 (Nrf2)/antioxidant response element (ARE) pathway is an important cellular stress response pathway involved in neuroprotection. We previously screened several natural phytochemicals and identified plumbagin as a novel activator of the Nrf2/ARE pathway that can protect neurons against ischemic injury. Here we extended our studies to natural and synthetic derivatives of plumbagin. We found that 5,8-dimethoxy-1,4-naphthoquinone (naphthazarin) is a potent activator of the Nrf2/ARE pathway, up-regulates the expression of Nrf2-driven genes in primary neuronal and glial cultures, and protects neurons against glutamate-induced excitotoxicity.

  6. Complete Deficiency of Leukocyte Glucose-6-Phosphate Dehydrogenase with Defective Bactericidal Activity

    PubMed Central

    Cooper, M. Robert; DeChatelet, Lawrence R.; McCall, Charles E.; La Via, Mariano F.; Spurr, Charles L.; Baehner, Robert L.

    1972-01-01

    A 52 yr old Caucasian female (F. E.) had hemolytic anemia, a leukemoid reaction, and fatal sepsis due to Escherichia coli. Her leukocytes ingested bacteria normally but did not kill catalase positive Staphylococcus aureus, Escherichia coli, and Serratia marcescens. An H2O2-producing bacterium, Streptococcus faecalis, was killed normally. Granule myeloperoxidase, acid and alkaline phosphatase, and beta glucuronidase activities were normal, and these enzymes shifted normally to the phagocyte vacuole (light and electron microscopy). Intravacuolar reduction of nitroblue tetrazolium did not occur. Moreover, only minimal quantities of H2O2 were generated, and the hexose monophosphate shunt (HMPS) was not stimulated during phagocytosis. These observations suggested the diagnosis of chronic granulomatous disease. However, in contrast to control and chronic granulomatous disease leukocytes, glucose-6-phosphate dehydrogenase activity was completely absent in F. E. leukocytes whereas NADH oxidase and NADPH oxidase activities were both normal. Unlike chronic granulomatous disease, methylene blue did not stimulate the hexose monophosphate shunt in F. E. cells. Thus, F. E. and chronic granulomatous disease leukocytes appear to share certain metabolic and bactericidal defects, but the metabolic basis of the abnormality differs. Chronic granulomatous disease cells lack oxidase activity which produces H2O2; F. E. cells had normal levels of oxidase activity but failed to produce NADPH due to complete glucose-6-phosphate dehydrogenase deficiency. These data indicate that a complete absence of leukocyte glucose-6-phosphate dehydrogenase with defective hexose monophosphate shunt activity is associated with low H2O2 production and inadequate bactericidal activity, and further suggest an important role for NADPH in the production of H2O2 in human granulocytes. Images PMID:4401271

  7. Fluorescence imaging of glutamate release in neurons

    SciTech Connect

    Wang, Ziqiang; Yeung, Edward S.

    1999-12-01

    A noninvasive detection scheme based on glutamate dehydrogenase (GDH) enzymatic assay combined with microscopy was developed to measure the glutamate release in cultured cells from the central nervous system (CNS). The enzyme reaction is very specific and sensitive. The detection limit with charge-coupled device (CCD) imaging is down to {mu}M levels of glutamate with reasonable response time ({approx}30 s). The standard glutamate test shows a linear response over 3 orders of magnitude, from {mu}M to 0.1 mM range. The in vitro monitoring of glutamate release from cultured neuron cells demonstrated excellent spatial and temporal resolution. (c) 1999 Society for Applied Spectroscopy.

  8. Mitochondrial complex I, aconitase, and succinate dehydrogenase during hypoxia-reoxygenation: modulation of enzyme activities by MnSOD.

    PubMed

    Powell, Charles S; Jackson, Robert M

    2003-07-01

    Both NADH dehydrogenase (complex I) and aconitase are inactivated partially in vitro by superoxide (O2-.) and other oxidants that cause loss of iron from enzyme cubane (4Fe-4S) centers. We tested whether hypoxia-reoxygenation (H-R) by itself would decrease lung epithelial cell NADH dehydrogenase, aconitase, and succinate dehydrogenase (SDH) activities and whether transfection with adenoviral vectors expressing MnSOD (Ad.MnSOD) would inhibit oxidative enzyme inactivation and thus confirm a mechanism involving O2-. Human lung carcinoma cells with alveolar epithelial cell characteristics (A549 cells) were exposed to <1% O2-5% CO2 (hypoxia) for 24 h followed by air-5% CO2 for 24 h (reoxygenation). NADH dehydrogenase activity was assayed in submitochondrial particles; aconitase and SDH activities were measured in cell lysates. H-R significantly decreased NADH dehydrogenase, aconitase, and SDH activities. Ad.MnSOD increased mitochondrial MnSOD substantially and prevented the inhibitory effects of H-R on enzyme activities. Addition of alpha-ketoglutarate plus aspartate, but not succinate, to medium prevented cytotoxicity due to 2,3-dimethoxy-1,4-naphthoquinone. After hypoxia, cells displayed significantly increased dihydrorhodamine fluorescence, indicating increased mitochondrial oxidant production. Inhibition of NADH dehydrogenase, aconitase, and SDH activities during reoxygenation are due to excess O2-. produced in mitochondria, because enzyme inactivation can be prevented by overexpression of MnSOD. PMID:12665464

  9. Neuronal activity mediated regulation of glutamate transporter GLT‐1 surface diffusion in rat astrocytes in dissociated and slice cultures

    PubMed Central

    Al Awabdh, Sana; Gupta‐Agarwal, Swati; Sheehan, David F.; Muir, James; Norkett, Rosalind; Twelvetrees, Alison E.; Griffin, Lewis D.

    2016-01-01

    The astrocytic GLT‐1 (or EAAT2) is the major glutamate transporter for clearing synaptic glutamate. While the diffusion dynamics of neurotransmitter receptors at the neuronal surface are well understood, far less is known regarding the surface trafficking of transporters in subcellular domains of the astrocyte membrane. Here, we have used live‐cell imaging to study the mechanisms regulating GLT‐1 surface diffusion in astrocytes in dissociated and brain slice cultures. Using GFP‐time lapse imaging, we show that GLT‐1 forms stable clusters that are dispersed rapidly and reversibly upon glutamate treatment in a transporter activity‐dependent manner. Fluorescence recovery after photobleaching and single particle tracking using quantum dots revealed that clustered GLT‐1 is more stable than diffuse GLT‐1 and that glutamate increases GLT‐1 surface diffusion in the astrocyte membrane. Interestingly, the two main GLT‐1 isoforms expressed in the brain, GLT‐1a and GLT‐1b, are both found to be stabilized opposed to synapses under basal conditions, with GLT‐1b more so. GLT‐1 surface mobility is increased in proximity to activated synapses and alterations of neuronal activity can bidirectionally modulate the dynamics of both GLT‐1 isoforms. Altogether, these data reveal that astrocytic GLT‐1 surface mobility, via its transport activity, is modulated during neuronal firing, which may be a key process for shaping glutamate clearance and glutamatergic synaptic transmission. GLIA 2016;64:1252–1264 PMID:27189737

  10. A bifunctional enzyme from Rhodococcus erythropolis exhibiting secondary alcohol dehydrogenase-catalase activities.

    PubMed

    Martinez-Rojas, Enriqueta; Kurt, Tutku; Schmidt, Udo; Meyer, Vera; Garbe, Leif-Alexander

    2014-11-01

    Alcohol dehydrogenases have long been recognized as potential biocatalyst for production of chiral fine and bulk chemicals. They are relevant for industry in enantiospecific production of chiral compounds. In this study, we identified and purified a nicotinamide adenine dinucleotide (NAD)-dependent secondary alcohol dehydrogenase (SdcA) from Rhodococcus erythropolis oxidizing γ-lactols into γ-lactones. SdcA showed broad substrate specificity on γ-lactols; secondary aliphatic alcohols with 8 and 10 carbon atoms were also substrates and oxidized with (2S)-stereospecificity. The enzyme exhibited moderate stability with a half-life of 5 h at 40 °C and 20 days at 4 °C. Mass spectrometric identification revealed high sequence coverage of SdcA amino acid sequence to a highly conserved catalase from R. erythropolis. The corresponding encoding gene was isolated from genomic DNA and subsequently overexpressed in Escherichia coli BL21 DE3 cells. In addition, the recombinant SdcA was purified and characterized in order to confirm that the secondary alcohol dehydrogenase and catalase activity correspond to the same enzyme.

  11. Second-generation sulfonamide inhibitors of D-glutamic acid-adding enzyme: activity optimisation with conformationally rigid analogues of D-glutamic acid.

    PubMed

    Sosič, Izidor; Barreteau, Hélène; Simčič, Mihael; Sink, Roman; Cesar, Jožko; Zega, Anamarija; Grdadolnik, Simona Golič; Contreras-Martel, Carlos; Dessen, Andréa; Amoroso, Ana; Joris, Bernard; Blanot, Didier; Gobec, Stanislav

    2011-07-01

    D-Glutamic acid-adding enzyme (MurD) catalyses the essential addition of d-glutamic acid to the cytoplasmic peptidoglycan precursor UDP-N-acetylmuramoyl-l-alanine, and as such it represents an important antibacterial drug-discovery target enzyme. Based on a series of naphthalene-N-sulfonyl-d-Glu derivatives synthesised recently, we synthesised two series of new, optimised sulfonamide inhibitors of MurD that incorporate rigidified mimetics of d-Glu. The compounds that contained either constrained d-Glu or related rigid d-Glu mimetics showed significantly better inhibitory activities than the parent compounds, thereby confirming the advantage of molecular rigidisation in the design of MurD inhibitors. The binding modes of the best inhibitors were examined with high-resolution NMR spectroscopy and X-ray crystallography. We have solved a new crystal structure of the complex of MurD with an inhibitor bearing a 4-aminocyclohexane-1,3-dicarboxyl moiety. These data provide an additional step towards the development of sulfonamide inhibitors with potential antibacterial activities. PMID:21524830

  12. Glutamate-activated chloride channels: Unique fipronil targets present in insects but not in mammals

    PubMed Central

    Narahashi, Toshio; Zhao, Xilong; Ikeda, Tomoko; Salgado, Vincent L.; Yeh, Jay Z.

    2009-01-01

    Selectivity to insects over mammals is one of the important characteristics for a chemical to become a useful insecticide. Fipronil was found to block cockroach GABA receptors more potently than rat GABAA receptors. Furthermore, glutamate-activated chloride channels (GluCls), which are present in cockroaches but not in mammals, were very sensitive to the blocking action of fipronil. The IC50s of fipronil block were 30 nM in cockroach GABA receptors and 1600 nM in rat GABAA receptors. Moreover, GluCls of cockroach neurons had low IC50s for fipronil. Two types of glutamate-induced chloride current were obswerved: desensitizing and non-desensitizing, with fipronil IC50s of 800 and 10 nM, respectively. We have developed methods to separately record these two types of GluCls. The non-desensitizing and desensitizing currents were selectively inhibited by trypsin and polyvinylpyrrolidone, respectively. In conclusion, in addition to GABA receptors, GluCls play a crucial role in selectivity of fipronil to insects over mammals. GluCls form the basis for development of selective and safe insecticides. PMID:20563240

  13. Alleviating pain hypersensitivity through activation of type 4 metabotropic glutamate receptor.

    PubMed

    Vilar, Bruno; Busserolles, Jérôme; Ling, Bing; Laffray, Sophie; Ulmann, Lauriane; Malhaire, Fanny; Chapuy, Eric; Aissouni, Youssef; Etienne, Monique; Bourinet, Emmanuel; Acher, Francine; Pin, Jean-Philippe; Eschalier, Alain; Goudet, Cyril

    2013-11-27

    Hyperactivity of the glutamatergic system is involved in the development of central sensitization in the pain neuraxis, associated with allodynia and hyperalgesia observed in patients with chronic pain. Herein we study the ability of type 4 metabotropic glutamate receptors (mGlu4) to regulate spinal glutamate signaling and alleviate chronic pain. We show that mGlu4 are located both on unmyelinated C-fibers and spinal neurons terminals in the inner lamina II of the spinal cord where they inhibit glutamatergic transmission through coupling to Cav2.2 channels. Genetic deletion of mGlu4 in mice alters sensitivity to strong noxious mechanical compression and accelerates the onset of the nociceptive behavior in the inflammatory phase of the formalin test. However, responses to punctate mechanical stimulation and nocifensive responses to thermal noxious stimuli are not modified. Accordingly, pharmacological activation of mGlu4 inhibits mechanical hypersensitivity in animal models of inflammatory or neuropathic pain while leaving acute mechanical perception unchanged in naive animals. Together, these results reveal that mGlu4 is a promising new target for the treatment of chronic pain. PMID:24285900

  14. Activation of metabotropic glutamate receptors regulates ribosomes of cochlear nucleus neurons.

    PubMed

    Carzoli, Kathryn L; Hyson, Richard L

    2014-01-01

    The brain stem auditory system of the chick is an advantageous model for examining changes that occur as a result of deafness. Elimination of acoustic input through cochlear ablation results in the eventual death of approximately 30% of neurons in the chick cochlear nucleus, nucleus magnocellularis (NM). One early change following deafness is an alteration in NM ribosomes, evidenced both by a decrease in protein synthesis and reduction in antigenicity for Y10B, a monoclonal antibody that recognizes a ribosomal epitope. Previous studies have shown that mGluR activation is necessary to maintain Y10B antigenicity and NM viability. What is still unclear, however, is whether or not mGluR activation is sufficient to prevent deafness-induced changes in these neurons, or if other activity-dependent factors are also necessary. The current study investigated the ability of mGluR activation to regulate cochlear nucleus ribosomes in the absence of auditory nerve input. In vitro methods were employed to periodically pressure eject glutamate or mGluR agonists over neurons on one side of a slice preparation leaving the opposite side of the same slice untreated. Immunohistochemistry was then performed using Y10B in order to assess ribosomal changes. Application of glutamate and both group I and II selective mGluR agonists effectively rescued ribosomal antigenicity on the treated side of the slice in comparison to ribosomes on the untreated side. These findings suggest that administration of mGluR agonists is sufficient to reduce the early interruption of normal ribosomal integrity that is typically seen following loss of auditory nerve activity.

  15. Differential effects of acute and chronic fructose administration on pyruvate dehydrogenase activity and lipogenesis

    SciTech Connect

    Wilson, L.

    1988-01-01

    These studies were undertaken to distinguish between the acute and chronic effects of fructose administration. In vivo, liver lipogenesis, as measured by {sup 3}H{sub 2}O incorporation, was greater in rats fed 60% fructose than in their glucose fed controls. Both fructose feeding, and fructose feeding plus intraperitoneal fructose injection increased the activities of 6-phosphogluconate dehydrogenase and malic enzyme. Liver PDH activity was increased by fructose feeding, and was increased even more by fructose feeding and injection of fructose, but this was not associated with any changes in hepatic ATP concentrations.

  16. Clostridium difficile testing algorithms using glutamate dehydrogenase antigen and C. difficile toxin enzyme immunoassays with C. difficile nucleic acid amplification testing increase diagnostic yield in a tertiary pediatric population.

    PubMed

    Ota, Kaede V; McGowan, Karin L

    2012-04-01

    We evaluated the performance of the rapid C. diff Quik Chek Complete's glutamate dehydrogenase antigen (GDH) and toxin A/B (CDT) tests in two algorithmic approaches for a tertiary pediatric population: algorithm 1 entailed initial testing with GDH/CDT followed by loop-mediated isothermal amplification (LAMP), and algorithm 2 entailed GDH/CDT followed by cytotoxicity neutralization assay (CCNA) for adjudication of discrepant GDH-positive/CDT-negative results. A true positive (TP) was defined as positivity by CCNA or positivity by LAMP plus another test (GDH, CDT, or the Premier C. difficile toxin A and B enzyme immunoassay [P-EIA]). A total of 141 specimens from 141 patients yielded 27 TPs and 19% prevalence. Sensitivity, specificity, positive predictive value, and negative predictive value were 56%, 100%, 100%, and 90% for P-EIA and 81%, 100%, 100%, and 96% for both algorithm 1 and algorithm 2. In summary, GDH-based algorithms detected C. difficile infections with superior sensitivity compared to P-EIA. The algorithms allowed immediate reporting of half of all TPs, but LAMP or CCNA was required to confirm the presence or absence of toxigenic C. difficile in GDH-positive/CDT-negative specimens.

  17. Evaluation of the C.Diff Quik Chek Complete Assay, a new glutamate dehydrogenase and A/B toxin combination lateral flow assay for use in rapid, simple diagnosis of clostridium difficile disease.

    PubMed

    Sharp, Susan E; Ruden, Lila O; Pohl, Julie C; Hatcher, Patricia A; Jayne, Linda M; Ivie, W Michael

    2010-06-01

    The diagnosis of Clostridium difficile infection continues to be a challenge for many clinical microbiology laboratories. A new lateral flow assay, the C.Diff Quik Chek Complete assay, which tests for the presence of both glutamate dehydrogenase (GDH) and C. difficile toxins A and B, was evaluated for its ability to diagnose C. difficile disease. The results of this assay were compared to those of both PCR and toxigenic culture. The results showed that this assay allows 88% of specimens to be accurately screened as either positive (both tests positive) or negative (both tests negative) for the presence of toxigenic C. difficile in less than 30 min and with minimal hands-on time. Use of a random-access PCR for the analysis of specimens with discrepant results (one test positive and the other negative) allows the easy, rapid, and highly sensitive (100%; 95% confidence interval [CI], 89.6 to 100%) and specific (99.6%; 95% CI, 97.3 to 99.9%) diagnosis of C. difficile disease. The use of this algorithm would save institutional costs, curtail unnecessary isolation days, reduce the nosocomial transmission of disease, and increase the quality of care for patients.

  18. Purification and Partial Kinetic and Physical Characterization of Two Chloroplast-Localized NADP-Specific Glutamate Dehydrogenase Isoenzymes and Their Preferential Accumulation in Chlorella sorokiniana Cells Cultured at Low or High Ammonium Levels 1

    PubMed Central

    Bascomb, Newell F.; Schmidt, Robert R.

    1987-01-01

    Two ammonium-inducible, chloroplast-localized NADP-specific glutamate dehydrogenase isoenzymes were purified to homogeneity from Chlorella sorokiniana. These isoenzymes were homopolymers of either α- or β-subunits with molecular weights of 55,500 or 53,000, respectively. The α-isoenzyme was preferentially induced at low ammonium concentrations (2 millimolar or lower), whereas only the β-isoenzyme accumulated after cells were fully induced (120 minutes) at high ammonium concentrations (29 millimolar). Purification of isoenzymes was achieved by (NH4)2SO4 fractionation, gel-filtration, anion-exchange fast protein liquid chromatography, and affinity chromatography. The α- and β-isoenzymes were separated by their differential binding to Type 4 nicotinamide adenine dinucleotide phosphate-Sepharose. Both isoenzymes bound to an antibody affinity column to which purified antibody (prepared against β-isoenzyme) was covalently attached. Peptide mapping of the subunits showed them to have a high degree of sequence homology. Both subunits were synthesized in vitro from precursor protein(s) with a molecular weight of 58,500. Although the subunits have similar chemical, physical, and antigenic properties, their holoenzymes have strikingly different ammonium Km values. The ammonium Km of the β-isoenzyme remained constant at approximately 75 millimolar, whereas this Km of the α-isoenzyme ranged from 0.02 to 3.5 millimolar, depending upon nicotinamide adenine dinucleotide phosphate concentration. Images Fig. 1 Fig. 2 Fig. 7 Fig. 8 PMID:16665219

  19. TRPA1 activation by lidocaine in nerve terminals results in glutamate release increase

    SciTech Connect

    Piao, L.-H.; Fujita, Tsugumi; Jiang, C.-Y.; Liu Tao; Yue, H.-Y.; Nakatsuka, Terumasa; Kumamoto, Eiichi

    2009-02-20

    We examined the effects of local anesthetics lidocaine and procaine on glutamatergic spontaneous excitatory transmission in substantia gelatinosa (SG) neurons in adult rat spinal cord slices with whole-cell patch-clamp techniques. Bath-applied lidocaine (1-5 mM) dose-dependently and reversibly increased the frequency but not the amplitude of spontaneous excitatory postsynaptic current (sEPSC) in SG neurons. Lidocaine activity was unaffected by the Na{sup +}-channel blocker, tetrodotoxin, and the TRPV1 antagonist, capsazepine, but was inhibited by the TRP antagonist, ruthenium red. In the same neuron, the TRPA1 agonist, allyl isothiocyanate, and lidocaine both increased sEPSC frequency. In contrast, procaine did not produce presynaptic enhancement. These results indicate that lidocaine activates TRPA1 in nerve terminals presynaptic to SG neurons to increase the spontaneous release of L-glutamate.

  20. Gene algD coding for GDPmannose dehydrogenase is transcriptionally activated in mucoid Pseudomonas aeruginosa.

    PubMed Central

    Deretic, V; Gill, J F; Chakrabarty, A M

    1987-01-01

    Transcriptional regulation of alginate biosynthesis by Pseudomonas aeruginosa was studied. A DNA region complementing the alg-5 mutation within the alginate gene cluster was found by RNA-DNA dot blot and Northern hybridization to be transcriptionally activated in mucoid P. aeruginosa. This region was subcloned as a 3.2-kilobase BglII-ClaI DNA fragment on the broad-host-range controlled transcription vector pMMB24, and gene products were analyzed by expression from the tac promoter. A 48-kilodalton polypeptide was detected in extracts of P. aeruginosa and 35S-labeled Escherichia coli maxicells. By using the same expression system, GDPmannose dehydrogenase activity was detected in both P. aeruginosa and E. coli. Thus, gene algD coding for this enzyme was found to be present in the transcriptionally active DNA area. Insertion of the xylE gene within the BglII-ClaI fragment disrupted the induction of the 48-kilodalton polypeptide, GDPmannose dehydrogenase activity, and alg-5 complementing ability. With the algD-xylE transcription fusion, activation of algD gene expression was shown to occur in mucoid P. aeruginosa of different origins. In addition, regulation of the algD promoter activity was demonstrated to be mediated by a diffusible factor. Images PMID:3025179

  1. Corneal aldehyde dehydrogenase and glutathione S-transferase activity after excimer laser keratectomy in guinea pigs

    PubMed Central

    Bilgihan, K.; Bilgihan, A.; Turkozkan, N.

    1998-01-01

    BACKGROUND—The free radical balance of the eye may be changed by excimer laser keratectomy. Previous studies have demonstrated that excimer laser keratectomy increases the corneal temperature, decreases the superoxide dismutase activity of the aqueous, and induces lipid peroxidation in the superficial corneal stroma. Aldehyde dehydrogenase (ALDH) and glutathione S-transferase (GST) are known to play an important role in corneal metabolism, particularly in detoxification of aldehydes, which are generated from free radical reactions.
METHODS—In three groups of guinea pigs mechanical corneal de-epithelialisation was performed in group I, superficial corneal photoablation in group II, and deep corneal photoablation in group III, and the corneal ALDH and GST activities measured after 48 hours.
RESULTS—The mean ALDH and GST activities of group I and II showed no differences compared with the controls (p>0.05). The corneal ALDH activities were found to be significantly decreased (p<0.05) and GST activities increased (p<0.05) in group III.
CONCLUSION—These results suggest that excimer laser treatment of high myopia may change the ALDH and GST activities, metabolism, and free radical balance of the cornea.

 Keywords: excimer laser keratectomy; aldehyde dehydrogenase; glutathione S-transferase PMID:9602629

  2. Pituitary Adenylate cyclase-activating polypeptide orchestrates neuronal regulation of the astrocytic glutamate-releasing mechanism system xc (.).

    PubMed

    Kong, Linghai; Albano, Rebecca; Madayag, Aric; Raddatz, Nicholas; Mantsch, John R; Choi, SuJean; Lobner, Doug; Baker, David A

    2016-05-01

    Glutamate signaling is achieved by an elaborate network involving neurons and astrocytes. Hence, it is critical to better understand how neurons and astrocytes interact to coordinate the cellular regulation of glutamate signaling. In these studies, we used rat cortical cell cultures to examine whether neurons or releasable neuronal factors were capable of regulating system xc (-) (Sxc), a glutamate-releasing mechanism that is expressed primarily by astrocytes and has been shown to regulate synaptic transmission. We found that astrocytes cultured with neurons or exposed to neuronal-conditioned media displayed significantly higher levels of Sxc activity. Next, we demonstrated that the pituitary adenylate cyclase-activating polypeptide (PACAP) may be a neuronal factor capable of regulating astrocytes. In support, we found that PACAP expression was restricted to neurons, and that PACAP receptors were expressed in astrocytes. Interestingly, blockade of PACAP receptors in cultures comprised of astrocytes and neurons significantly decreased Sxc activity to the level observed in purified astrocytes, whereas application of PACAP to purified astrocytes increased Sxc activity to the level observed in cultures comprised of neurons and astrocytes. Collectively, these data reveal that neurons coordinate the actions of glutamate-related mechanisms expressed by astrocytes, such as Sxc, a process that likely involves PACAP. A critical gap in modeling excitatory signaling is how distinct components of the glutamate system expressed by neurons and astrocytes are coordinated. In these studies, we found that system xc (-) (Sxc), a glutamate release mechanism expressed by astrocytes, is regulated by releasable neuronal factors including PACAP. This represents a novel form of neuron-astrocyte communication, and highlights the possibility that pathological changes involving astrocytic Sxc may stem from altered neuronal activity.

  3. Overexpression of α-synuclein simultaneously increases glutamate NMDA receptor phosphorylation and reduces glucocerebrosidase activity.

    PubMed

    Yang, Junfeng; Hertz, Ellen; Zhang, Xiaoqun; Leinartaité, Lina; Lundius, Ebba Gregorsson; Li, Jie; Svenningsson, Per

    2016-01-12

    Progressive accumulation of α-synuclein (α-syn)-containing protein aggregates throughout the nervous system is a pathological hallmark of Parkinson's disease (PD). The mechanisms whereby α-syn exerts neurodegeneration remain to be fully understood. Here we show that overexpression of α-syn in transgenic mice leads to increased phosphorylation of glutamate NMDA receptor (NMDAR) subunits NR1 and NR2B in substantia nigra and striatum as well as reduced glucocerebrosidase (GCase) levels. Similarly, molecular studies performed in mouse N2A cells stably overexpressing human α-syn ((α-syn)N2A) showed that phosphorylation states of the same NMDAR subunits were increased, whereas GCase levels and lysosomal GCase activity were reduced. (α-syn)N2A cells showed an increased sensitivity to neurotoxicity towards 6-hydroxydopamine and NMDA. However, wildtype N2A, but not (α-syn)N2A cells, showed a further reduction in viability when co-incubated with 6-hydroxydopamine and the lysosomal inhibitors NH4Cl and leupeptin, suggesting that α-syn per se perturbs lysosomal functions. NMDA treatment reduced lysosomal GCase activity to the same extent in (α-syn)N2A cells as in wildtype N2A cells, indicating that the α-syn-dependent difference in NMDA neurotoxicity is unrelated to an altered GCase activity. Nevertheless, these data provide molecular evidence that overexpression of α-syn simultaneously induces two potential neurotoxic hits by increasing glutamate NMDA receptor phosphorylation, consistent with increased NMDA receptors functionality, and reducing GCase activity. PMID:26610904

  4. Glutamate Stimulates Local Protein Synthesis in the Axons of Rat Cortical Neurons by Activating α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid (AMPA) Receptors and Metabotropic Glutamate Receptors.

    PubMed

    Hsu, Wei-Lun; Chung, Hui-Wen; Wu, Chih-Yueh; Wu, Huei-Ing; Lee, Yu-Tao; Chen, En-Chan; Fang, Weilun; Chang, Yen-Chung

    2015-08-21

    Glutamate is the principal excitatory neurotransmitter in the mammalian CNS. By analyzing the metabolic incorporation of azidohomoalanine, a methionine analogue, in newly synthesized proteins, we find that glutamate treatments up-regulate protein translation not only in intact rat cortical neurons in culture but also in the axons emitting from cortical neurons before making synapses with target cells. The process by which glutamate stimulates local translation in axons begins with the binding of glutamate to the ionotropic AMPA receptors and metabotropic glutamate receptor 1 and members of group 2 metabotropic glutamate receptors on the plasma membrane. Subsequently, the activated mammalian target of rapamycin (mTOR) signaling pathway and the rise in Ca(2+), resulting from Ca(2+) influxes through calcium-permeable AMPA receptors, voltage-gated Ca(2+) channels, and transient receptor potential canonical channels, in axons stimulate the local translation machinery. For comparison, the enhancement effects of brain-derived neurotrophic factor (BDNF) on the local protein synthesis in cortical axons were also studied. The results indicate that Ca(2+) influxes via transient receptor potential canonical channels and activated the mTOR pathway in axons also mediate BDNF stimulation to local protein synthesis. However, glutamate- and BDNF-induced enhancements of translation in axons exhibit different kinetics. Moreover, Ca(2+) and mTOR signaling appear to play roles carrying different weights, respectively, in transducing glutamate- and BDNF-induced enhancements of axonal translation. Thus, our results indicate that exposure to transient increases of glutamate and more lasting increases of BDNF would stimulate local protein synthesis in migrating axons en route to their targets in the developing brain.

  5. Experimentally induced diabetes causes glial activation, glutamate toxicity and cellular damage leading to changes in motor function

    PubMed Central

    Nagayach, Aarti; Patro, Nisha; Patro, Ishan

    2014-01-01

    Behavioral impairments are the most empirical consequence of diabetes mellitus documented in both humans and animal models, but the underlying causes are still poorly understood. As the cerebellum plays a major role in coordination and execution of the motor functions, we investigated the possible involvement of glial activation, cellular degeneration and glutamate transportation in the cerebellum of rats, rendered diabetic by a single injection of streptozotocin (STZ; 45 mg/kg body weight; intraperitoneally). Motor function alterations were studied using Rotarod test (motor coordination) and grip strength (muscle activity) at 2nd, 4th, 6th, 8th, 10th, and 12th week post-diabetic confirmation. Scenario of glial (astroglia and microglia) activation, cell death and glutamate transportation was gaged using immunohistochemistry, histological study and image analysis. Cellular degeneration was clearly demarcated in the diabetic cerebellum. Glial cells were showing sequential and marked activation following diabetes in terms of both morphology and cell number. Bergmann glial cells were hypertrophied and distorted. Active caspase-3 positive apoptotic cells were profoundly present in all three cerebellar layers. Reduced co-labeling of GLT-1 and GFAP revealed the altered glutamate transportation in cerebellum following diabetes. These results, exclusively derived from histology, immunohistochemistry and cellular quantification, provide first insight over the associative reciprocity between the glial activation, cellular degeneration and reduced glutamate transportation, which presumably lead to the behavioral alterations following STZ-induced diabetes. PMID:25400546

  6. Novel mutations in dihydrolipoamide dehydrogenase deficiency in two cousins with borderline-normal PDH complex activity.

    PubMed

    Cameron, Jessie M; Levandovskiy, Valeriy; Mackay, Neviana; Raiman, Julian; Renaud, Deborah L; Clarke, Joe T R; Feigenbaum, Annette; Elpeleg, Orly; Robinson, Brian H

    2006-07-15

    We have diagnosed dihydrolipoamide dehydrogenase (DLD) deficiency in two male second cousins, who presented with markedly different clinical phenotypes. Patient 1 had a recurrent encephalopathy, and patient 2 had microcephaly and lactic acidosis. Their presentation is unusual, in that the DLD subunit deficiency had little effect on pyruvate dehydrogenase complex activity, but caused a severe reduction in the activities of other enzymes that utilize this subunit. We have identified two mutations in the DLD gene in each patient. The second cousins have one novel mutation in common resulting in a substitution of isoleucine for threonine (I47T), which has not been previously reported in the literature. Patient 1 has a second mutation that has been reported to be common in the Ashkenazi Jewish population, G229C. Patient 2 has a second mutation, E375K, which has also been previously reported in the literature. Enzyme kinetic measurements on patient fibroblasts show that under certain conditions, one heteroallelic mutation may have a higher K(m). This may account for the differing clinical phenotypes. These findings have important repercussions for other patients with similar clinical phenotypes, as DLD activity is not normally measured in cases with normal PDHc activity.

  7. Novel biohybrids of layered double hydroxide and lactate dehydrogenase enzyme: Synthesis, characterization and catalytic activity studies

    NASA Astrophysics Data System (ADS)

    Djebbi, Mohamed Amine; Braiek, Mohamed; Hidouri, Slah; Namour, Philippe; Jaffrezic-Renault, Nicole; Ben Haj Amara, Abdesslem

    2016-02-01

    The present work introduces new biohybrid materials involving layered double hydroxides (LDH) and biomolecule such as enzyme to produce bioinorganic system. Lactate dehydrogenase (Lac Deh) has been chosen as a model enzyme, being immobilized onto MgAl and ZnAl LDH materials via direct ion-exchange (adsorption) and co-precipitation methods. The immobilization efficiency was largely dependent upon the immobilization methods. A comparative study shows that the co-precipitation method favors the immobilization of great and tunable amount of enzyme. The structural behavior, chemical bonding composition and morphology of the resulting biohybrids were determined by X-ray diffraction (XRD) study, Fourier transform infrared (FTIR) spectroscopy and transmission electron microscopy (TEM), respectively. The free and immobilized enzyme activity and kinetic parameters were also reported using UV-Visible spectroscopy. However, the modified LDH materials showed a decrease in crystallinity as compared to the unmodified LDH. The change in activity of the immobilized lactate dehydrogenase was considered to be due, to the reduced accessibility of substrate molecules to the active sites of the enzyme and the partial conformational change of the Lac Deh molecules as a result of the immobilization way. Finally, it was proven that there is a correlation between structure/microstructure and enzyme activity dependent on the immobilization process.

  8. Lactic dehydrogenase isozyme patterns and alpha-hydroxybutyrate dehydrogenase activities in serum from newborns, patients with ovarian cancer or myocardial infarction.

    PubMed

    Kikuchi, Y; Kita, T; Furuya, K; Kato, K

    1988-11-01

    Lactic dehydrogenase (LDH) and alpha-hydroxybutyrate dehydrogenase (HBD) and LDH isozyme patterns were studied in serum from newborns and patients with ovarian cancer or myocardial infarction. LDH and HBD activities from newborns and patients with ovarian cancer or myocardial infarction were significantly increased, compared with those from patients with benign ovarian tumor. These increases were accompanied with a decrease of LDH-H and an increase of LDH-M in serum from newborns and patients with ovarian cancer, while an increase of LDH-H in serum from patients with myocardial infarction was dominant. However, the raised HBD activities in serum from patients with benign ovarian tumor did not affect the LDH isozyme patterns. From analysis of linear regression, a negative correlation between LDH-1 or -2 and HBD activity in serum from patients with ovarian cancer was observed while there was a positive correlation between LDH-4 and HBD activity. Similar patterns in serum from newborns were observed. On the other hand, a positive correlation between LDH-1 and HBD activity and a negative correlation between LDH-4 and HBD activity were found in serum from patients with myocardial infarction.

  9. Induction of an Olfactory Memory by the Activation of a Metabotropic Glutamate Receptor

    NASA Astrophysics Data System (ADS)

    Kaba, Hideto; Hayashi, Yasunori; Higuchi, Takashi; Nakanishi, Shigetada

    1994-07-01

    Female mice form an olfactory memory of male pheromones at mating; exposure to the pheromones of a strange male after that mating will block pregnancy. The formation of this memory is mediated by the accessory olfactory system, in which an increase in norepinephrine after mating reduces inhibitory transmission of γ-aminobutyric acid from the granule cells to the mitral cells. This study shows that the activation of mGluR2, a metabotropic glutamate receptor that suppresses the γ-aminobutyric acid inhibition of the mitral cells, permits the formation of a specific olfactory memory without the occurrence of mating by infusion of mGluR2 agonists into the female's accessory olfactory bulb. This memory faithfully reflects the memory formed at mating.

  10. Endogenous activation of metabotropic glutamate receptors supports the proliferation and survival of neural progenitor cells.

    PubMed

    Di Giorgi-Gerevini, V; Melchiorri, D; Battaglia, G; Ricci-Vitiani, L; Ciceroni, C; Busceti, C L; Biagioni, F; Iacovelli, L; Canudas, A M; Parati, E; De Maria, R; Nicoletti, F

    2005-08-01

    The use of neural progenitor cells (NPCs) is limited by the incomplete knowledge of the extracellular signals regulating their proliferation and survival. We report that cultured mouse NPCs express functional mGlu3 and mGlu5 metabotropic glutamate receptors. Pharmacological blockade of both receptors reduced NPC proliferation and survival, whereas activation of mGlu5 receptors substantially enhanced cell proliferation. Adult mice lacking mGlu5 receptors or treated with mGlu5 or mGlu3 receptor antagonists showed a dramatic reduction in the number of dividing neuroprogenitors present in the subventricular zone and in the dentate gyrus of the hippocampus. These data disclose a novel function of mGlu receptors and offer new potential strategies for the optimization of cell replacement therapy in neurodegenerative disorders. PMID:15947794

  11. [Activity of liver mitochondrial NAD+-dependent dehydrogenases of the krebs cycle in rats with acetaminophen-induced hepatitis developed under conditions of alimentary protein deficiency].

    PubMed

    Voloshchuk, O N; Kopylchuk, G P

    2016-01-01

    Activity of isocitrate dehydrogenase, α-ketoglutarate dehydrogenase, malate dehydrogenase, and the NAD(+)/NADН ratio were studied in the liver mitochondrial fraction of rats with toxic hepatitis induced by acetaminophen under conditions of alimentary protein deprivation. Acetaminophen-induced hepatitis was characterized by a decrease of isocitrate dehydrogenase, α-ketoglutarate dehydrogenase and malate dehydrogenase activities, while the mitochondrial NAD(+)/NADН ratio remained at the control level. Modeling of acetaminophen-induced hepatitis in rats with alimentary protein caused a more pronounced decrease in the activity of NAD(+)-dependent dehydrogenases studied and a 2.2-fold increase of the mitochondrial NAD(+)/NADН ratio. This suggests that alimentary protein deprivation potentiated drug-induced liver damage.

  12. [Activity of liver mitochondrial NAD+-dependent dehydrogenases of the krebs cycle in rats with acetaminophen-induced hepatitis developed under conditions of alimentary protein deficiency].

    PubMed

    Voloshchuk, O N; Kopylchuk, G P

    2016-01-01

    Activity of isocitrate dehydrogenase, α-ketoglutarate dehydrogenase, malate dehydrogenase, and the NAD(+)/NADН ratio were studied in the liver mitochondrial fraction of rats with toxic hepatitis induced by acetaminophen under conditions of alimentary protein deprivation. Acetaminophen-induced hepatitis was characterized by a decrease of isocitrate dehydrogenase, α-ketoglutarate dehydrogenase and malate dehydrogenase activities, while the mitochondrial NAD(+)/NADН ratio remained at the control level. Modeling of acetaminophen-induced hepatitis in rats with alimentary protein caused a more pronounced decrease in the activity of NAD(+)-dependent dehydrogenases studied and a 2.2-fold increase of the mitochondrial NAD(+)/NADН ratio. This suggests that alimentary protein deprivation potentiated drug-induced liver damage. PMID:27143375

  13. [Effects of Light Near-Infrared Radiation on Rats Assessed by Succinate Dehydrogenase Activity in Lymphocytes on Blood Smears].

    PubMed

    Khunderyakova, N V; Zakharchenko, A V; Zakharchenko, M V; Muller, H; Fedotcheva, I; Kondrashova, M N

    2015-01-01

    Biological effects of light near infrared radiation (850 nm), with modulation acoustic frequency of 101 Hz, was studied. The study was conducted on rats, the effect was recorded by succinate dehydrogenase activity in lymphocytes on the blood smear after administration of the activating dose of adrenaline, which simulates the state of the organism in the early stages of the pathogenic effects (stress). A pronounced regulating effect of infrared radiation on the activity of succinate dehydrogenase in animals activated by adrenaline was shown. Infrared radiation has a normalizing effect reducing the degree of inhibition or activation of the enzyme induced by adrenaline and had no effect on the control animals. Thus, by modulating the activity of succinate dehydrogenase infrared radiation regulates energy production in the mitochondria supported by the most powerful oxidation substrate--succinic acid, which is especially pronounced under stress. PMID:26841503

  14. [Effects of Light Near-Infrared Radiation on Rats Assessed by Succinate Dehydrogenase Activity in Lymphocytes on Blood Smears].

    PubMed

    Khunderyakova, N V; Zakharchenko, A V; Zakharchenko, M V; Muller, H; Fedotcheva, I; Kondrashova, M N

    2015-01-01

    Biological effects of light near infrared radiation (850 nm), with modulation acoustic frequency of 101 Hz, was studied. The study was conducted on rats, the effect was recorded by succinate dehydrogenase activity in lymphocytes on the blood smear after administration of the activating dose of adrenaline, which simulates the state of the organism in the early stages of the pathogenic effects (stress). A pronounced regulating effect of infrared radiation on the activity of succinate dehydrogenase in animals activated by adrenaline was shown. Infrared radiation has a normalizing effect reducing the degree of inhibition or activation of the enzyme induced by adrenaline and had no effect on the control animals. Thus, by modulating the activity of succinate dehydrogenase infrared radiation regulates energy production in the mitochondria supported by the most powerful oxidation substrate--succinic acid, which is especially pronounced under stress.

  15. Single channel currents of different amplitude activated by glutamate in a tonic (slow) crayfish muscle.

    PubMed

    Finger, W; Pareto, A

    1987-09-11

    Single channel currents were recorded by means of the patch-clamp technique from a tonic (slow) crayfish muscle in the presence of 5 mM glutamate. The experiments were carried out with 'Gigaohm-seals' in the 'cell-attached' mode at 15-17 degrees C. Five classes of single channel currents with different mean amplitudes were resolved: i1 = -0.75 +/- 0.43 (S.D.) pA, i2 = -1.4 +/- 0.4 pA, i3 = -3.5 +/- 0.63 pA, i4 = -8.5 +/- 0.92 pA and i5 approximately equal to 2 X i4, i2, i3 and i4 were recorded at resting membrane potential, Eo approximately equal to -80 mV (pipette potential Vp = 0), while i1 and i5 were recorded at 40 mV hyperpolarized to Eo (Vp = +40 mV). The current most frequently seen was i4 which is the excitatory glutamate-activated single channel current recorded previously by Franke et al. The membrane reversal potentials and channel conductances for i2 and i4 were estimated to be +60 mV (Eo + 140 mV), 13 pS for i2 and +40 mV (Eo + 120 mV), 80 pS for i4. It was assumed that up to 40 i1 currents could superpose in a single patch to generate a DC current of up to -30 pA with current fluctuations the intensity of which increased with the DC current amplitude. Often variable combinations of i1 to i4 currents could be recorded simultaneously in a single patch. In particular, simultaneous activity of i1, i4; i2, i4 and i3, i4 currents was observed in different single patches.

  16. Thinking outside the cleft to understand synaptic activity: contribution of the cystine-glutamate antiporter (System xc-) to normal and pathological glutamatergic signaling.

    PubMed

    Bridges, Richard; Lutgen, Victoria; Lobner, Doug; Baker, David A

    2012-07-01

    System x(c)(-) represents an intriguing target in attempts to understand the pathological states of the central nervous system. Also called a cystine-glutamate antiporter, system x(c)(-) typically functions by exchanging one molecule of extracellular cystine for one molecule of intracellular glutamate. Nonvesicular glutamate released during cystine-glutamate exchange activates extrasynaptic glutamate receptors in a manner that shapes synaptic activity and plasticity. These findings contribute to the intriguing possibility that extracellular glutamate is regulated by a complex network of release and reuptake mechanisms, many of which are unique to glutamate and rarely depicted in models of excitatory signaling. Because system x(c)(-) is often expressed on non-neuronal cells, the study of cystine-glutamate exchange may advance the emerging viewpoint that glia are active contributors to information processing in the brain. It is noteworthy that system x(c)(-) is at the interface between excitatory signaling and oxidative stress, because the uptake of cystine that results from cystine-glutamate exchange is critical in maintaining the levels of glutathione, a critical antioxidant. As a result of these dual functions, system x(c)(-) has been implicated in a wide array of central nervous system diseases ranging from addiction to neurodegenerative disorders to schizophrenia. In the current review, we briefly discuss the major cellular components that regulate glutamate homeostasis, including glutamate release by system x(c)(-). This is followed by an in-depth discussion of system x(c)(-) as it relates to glutamate release, cystine transport, and glutathione synthesis. Finally, the role of system x(c)(-) is surveyed across a number of psychiatric and neurodegenerative disorders.

  17. Activation of Group II Metabotropic Glutamate Receptors Induces Depotentiation in Amygdala Slices and Reduces Fear-Potentiated Startle in Rats

    ERIC Educational Resources Information Center

    Lin, Chia-Ho; Lee, Chia-Ching; Huang, Ya-Chun; Wang, Su-Jane; Gean, Po-Wu

    2005-01-01

    There is a close correlation between long-term potentiation (LTP) in the synapses of lateral amygdala (LA) and fear conditioning in animals. We predict that reversal of LTP (depotentiation) in this area of the brain may ameliorate conditioned fear. Activation of group II metabotropic glutamate receptors (mGluR II) with DCG-IV induces…

  18. Alcohol and polyol dehydrogenases are both divided into two protein types, and structural properties cross-relate the different enzyme activities within each type.

    PubMed Central

    Jörnvall, H; Persson, M; Jeffery, J

    1981-01-01

    Sorbitol dehydrogenase from sheep liver shows similarities to mammalian and yeast alcohol dehydrogenases. Comparisons based on peptides from segments of sorbitol dehydrogenase reveal that homologous regions with 38% identity include two ligands to the active site zinc atom in liver alcohol dehydrogenase, as well as further important residues. Similarities in in other regions are less extensive, exactly as they are between different alcohol dehydrogenases. In all aspects, sorbitol dehydrogenase appears as a typical member of the alcohol dehydrogenase family. On the other hand, alcohol dehydrogenase from Drosophila, which has a shorter subunit, is not closely related to either of these enzymes, except for a region that probably corresponds to the first part of the coenzyme binding domain in many dehydrogenases. Instead, Drosophila alcohol dehydrogenase in its supposed catalytic region shows similarities toward Klebsiella ribitol dehydrogenase, which also has a small subunit. It may be concluded that both alcohol and polyol dehydrogenases show two types of protein subunit, reflecting an early subdivision of polypeptide types into "long" and "short" subunits rather than into different enzymatic specificities or quaternary structures. The relationships explain known properties of all these enzymes and provide insight into functional mechanisms and evolutionary interpretations. PMID:7027257

  19. Augmented cystine-glutamate exchange by pituitary adenylate cyclase-activating polypeptide signaling via the VPAC1 receptor.

    PubMed

    Resch, Jon M; Albano, Rebecca; Liu, XiaoQian; Hjelmhaug, Julie; Lobner, Doug; Baker, David A; Choi, SuJean

    2014-07-28

    In the central nervous system, cystine import in exchange for glutamate through system xc(-) is critical for the production of the antioxidant glutathione by astrocytes, as well as the maintenance of extracellular glutamate. Therefore, regulation of system xc(-) activity affects multiple aspects of cellular physiology and may contribute to disease states. Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuronally-derived peptide that has already been demonstrated to modulate multiple aspects of glutamate signaling suggesting PACAP may also target activity of cystine-glutamate exchange via system xc(-) . In the current study, 24-hour treatment of primary cortical cultures containing neurons and glia with PACAP concentration-dependently increased system xc(-) function as measured by radiolabeled cystine uptake. Furthermore, the increase in cystine uptake was completely abolished by the system xc(-) inhibitor, (S)-4-carboxyphenylglycine (CPG), attributing increases in cystine uptake specifically to system xc(-) activity. Time course and quantitative PCR results indicate that PACAP signaling may increase cystine-glutamate exchange by increasing expression of xCT, the catalytic subunit of system xc(-) . Furthermore, the potentiation of system xc(-) activity by PACAP occurs via a PKA-dependent pathway that is not mediated by the PAC1R, but rather the shared vasoactive intestinal polypeptide receptor VPAC1R. Finally, assessment of neuronal, astrocytic, and microglial-enriched cultures demonstrated that only astrocyte-enriched cultures exhibit enhanced cystine uptake following both PACAP and VIP treatment. These data introduce a novel mechanism by which both PACAP and VIP regulate system xc(-) activity. Synapse, 2014. © 2014 Wiley Periodicals, Inc.

  20. Genetics Home Reference: pyruvate dehydrogenase deficiency

    MedlinePlus

    ... control the activity of the complex: pyruvate dehydrogenase phosphatase turns on (activates) the complex, while pyruvate dehydrogenase ... binding protein (the PDHX gene), and pyruvate dehydrogenase phosphatase (the PDP1 gene) have been identified in people ...

  1. Activity of the lactate-alanine shuttle is independent of glutamate-glutamine cycle activity in cerebellar neuronal-astrocytic cultures.

    PubMed

    Bak, Lasse K; Sickmann, Helle M; Schousboe, Arne; Waagepetersen, Helle S

    The glutamate-glutamine cycle describes the neuronal release of glutamate into the synaptic cleft, astrocytic uptake, and conversion into glutamine, followed by release for use as a neuronal glutamate precursor. This only explains the fate of the carbon atoms, however, and not that of the ammonia. Recently, a role for alanine has been proposed in transfer of ammonia between glutamatergic neurons and astrocytes, denoted the lactate-alanine shuttle (Waagepetersen et al. [ 2000] J. Neurochem. 75:471-479). The role of alanine in this context has been studied further using cerebellar neuronal cultures and corresponding neuronal-astrocytic cocultures. A superfusion paradigm was used to induce repetitively vesicular glutamate release by N-methyl-D-aspartate (NMDA) in the neurons, allowing the relative activity dependency of the lactate-alanine shuttle to be assessed. [(15)N]Alanine (0.2 mM), [2-(15)N]/[5-(15)N]glutamine (0.25 mM), and [(15)N]ammonia (0.3 mM) were used as precursors and cell extracts were analyzed by mass spectrometry. Labeling from [(15)N]alanine in glutamine, aspartate, and glutamate in cerebellar cocultures was independent of depolarization of the neurons. Employing glutamine with the amino group labeled ([2-(15)N]glutamine) as the precursor, an activity-dependent increase in the labeling of both glutamate and aspartate (but not alanine) was observed in the cerebellar neurons. When the amide group of glutamine was labeled ([5-(15)N]glutamine), no labeling could be detected in the analyzed metabolites. Altogether, the results of this study support the existence of the lactate-alanine shuttle and the associated glutamate-glutamine cycle. No direct coupling of the two shuttles was observed, however, and only the glutamate-glutamine cycle seemed activity dependent.

  2. Glutamate and glutathione interplay in a motor neuronal model of amyotrophic lateral sclerosis reveals altered energy metabolism.

    PubMed

    D'Alessandro, Giuseppina; Calcagno, Eleonora; Tartari, Silvia; Rizzardini, Milena; Invernizzi, Roberto William; Cantoni, Lavinia

    2011-08-01

    Impairment of mitochondrial function might contribute to oxidative stress associated with neurodegeneration in amyotrophic lateral sclerosis (ALS). Glutamate levels in tissues of ALS patients are sometimes altered. In neurons, mitochondrial metabolism of exogenous glutamine is mainly responsible for the net synthesis of glutamate, which is a neurotransmitter, but it is also necessary for the synthesis of glutathione, the main endogenous antioxidant. We investigated glutathione synthesis and glutamine/glutamate metabolism in a motor neuronal model of familial ALS. In standard culture conditions (with glutamine) or restricting glutamine or cystine, the level of glutathione was always lower in the cell line expressing the mutant (G93A) human Cu, Zn superoxide dismutase (G93ASOD1) than in the line expressing wild-type SOD1. With glutamine the difference in glutathione was associated with a lower glutamate and impairment of the glutamine/glutamate metabolism as evidenced by lower glutaminase and cytosolic malate dehydrogenase activity. d-β-hydroxybutyrate, as an alternative to glutamine as energy substrate in addition to glucose, reversed the decreases of cytosolic malate dehydrogenase activity and glutamate and glutathione. However, in the G93ASOD1 cell line, in all culture conditions the expression of pyruvate dehydrogenase kinase l protein, which down-regulates pyruvate dehydrogenase activity, was induced, together with an increase in lactate release in the medium. These findings suggest that the glutathione decrease associated with mutant SOD1 expression is due to mitochondrial dysfunction caused by the reduction of the flow of glucose-derived pyruvate through the TCA cycle; it implies altered glutamate metabolism and depends on the different mitochondrial energy substrates. PMID:21530659

  3. Post-synaptic calcium influx at the giant synapse of the squid during activation by glutamate.

    PubMed Central

    Eusebi, F; Miledi, R; Parker, I; Stinnakre, J

    1985-01-01

    Changes in free calcium were monitored in the post-synaptic axon of the giant synapse of the squid, using the calcium indicators aequorin and Arsenazo III. The peak size of the calcium-dependent optical signals recorded from aequorin and Arsenazo III both showed a linear relation with the amount of calcium injected ionophoretically into the axon, but the Arsenazo signal had a slower time course than the aequorin. Ionophoretic application of glutamate to the post-synaptic axon depolarized the axon and caused a rise in intracellular free calcium. Aequorin signals were detected in natural sea water, and their size increased when the calcium concentration in the sea water was raised. Arsenazo signals could be detected only in high-calcium (55 mM) sea water. Intracellular calcium signals were detected also during bath application of several glutamate analogues, including kainate, ibotenate, and aspartate. The peak amplitude of the intracellular calcium signal, monitored with both indicators, increased with increasing ionophoretic glutamate dose, and varied linearly with the integral of the glutamate-induced membrane depolarization. No calcium signals were detected when depolarizations, similar to those produced by glutamate, were induced by current injection in the absence of glutamate. We conclude that glutamate increases the calcium permeability of the post-synaptic membrane, independently of the glutamate-induced depolarization. The glutamate-induced depolarization and the rise in intracellular free calcium increased roughly linearly as the membrane potential was made more negative. Extrapolation of these data indicated that the glutamate depolarization would reduce to zero at about -30 mV, while the calcium signals would be suppressed at about +50 mV. PMID:2869144

  4. Decreased succinate dehydrogenase activity of gamma and alpha motoneurons in mouse spinal cords following 13 weeks of exposure to microgravity.

    PubMed

    Ishihara, Akihiko; Nagatomo, Fumiko; Fujino, Hidemi; Kondo, Hiroyo; Ohira, Yoshinobu

    2013-10-01

    Cell body size and succinate dehydrogenase activity of motoneurons in the dorsolateral region of the ventral horn in the lumbar and cervical segments of the mouse spinal cord were assessed after long-term exposure to microgravity and compared with those of ground-based controls. Mice were housed in a mouse drawer system on the International Space Station for 13 weeks. The mice were transported to the International Space Station by the Space Shuttle Discovery and returned to Earth by the Space Shuttle Atlantis. No changes in the cell body size of motoneurons were observed in either segment after exposure to microgravity, but succinate dehydrogenase activity of small-sized (<300 μm(2)) gamma and medium-sized (300-700 μm(2)) alpha motoneurons, which have higher succinate dehydrogenase activity than large-sized (>700 μm(2)) alpha motoneurons, in both segments was lower than that of ground-based controls. We concluded that exposure to microgravity for longer than 3 months induced decreased succinate dehydrogenase activity of both gamma and slow-type alpha motoneurons. In particular, the decreased succinate dehydrogenase activity of gamma motoneurons was observed only after long-term exposure to microgravity. PMID:23943522

  5. Cytoplasm-to-myonucleus ratios and succinate dehydrogenase activities in adult rat slow and fast muscle fibers

    NASA Technical Reports Server (NTRS)

    Tseng, B. S.; Kasper, C. E.; Edgerton, V. R.

    1994-01-01

    The relationship between myonuclear number, cellular size, succinate dehydrogenase activity, and myosin type was examined in single fiber segments (n = 54; 9 +/- 3 mm long) mechanically dissected from soleus and plantaris muscles of adult rats. One end of each fiber segment was stained for DNA before quantitative photometric analysis of succinate dehydrogenase activity; the other end was double immunolabeled with fast and slow myosin heavy chain monoclonal antibodies. Mean +/- S.D. cytoplasmic volume/myonucleus ratio was higher in fast and slow plantaris fibers (112 +/- 69 vs. 34 +/- 21 x 10(3) microns3) than fast and slow soleus fibers (40 +/- 20 vs. 30 +/- 14 x 10(3) microns3), respectively. Slow fibers always had small volumes/myonucleus, regardless of fiber diameter, succinate dehydrogenase activity, or muscle of origin. In contrast, smaller diameter (< 70 microns) fast soleus and plantaris fibers with high succinate dehydrogenase activity appeared to have low volumes/myonucleus while larger diameter (> 70 microns) fast fibers with low succinate dehydrogenase activity always had large volume/myonucleus. Slow soleus fibers had significantly greater numbers of myonuclei/mm than did either fast soleus or fast plantaris fibers (116 +/- 51 vs. 55 +/- 22 and 44 +/- 23), respectively. These data suggest that the myonuclear domain is more limited in slow than fast fibers and in the fibers with a high, compared to a low, oxidative metabolic capability.

  6. Decreased succinate dehydrogenase activity of gamma and alpha motoneurons in mouse spinal cords following 13 weeks of exposure to microgravity.

    PubMed

    Ishihara, Akihiko; Nagatomo, Fumiko; Fujino, Hidemi; Kondo, Hiroyo; Ohira, Yoshinobu

    2013-10-01

    Cell body size and succinate dehydrogenase activity of motoneurons in the dorsolateral region of the ventral horn in the lumbar and cervical segments of the mouse spinal cord were assessed after long-term exposure to microgravity and compared with those of ground-based controls. Mice were housed in a mouse drawer system on the International Space Station for 13 weeks. The mice were transported to the International Space Station by the Space Shuttle Discovery and returned to Earth by the Space Shuttle Atlantis. No changes in the cell body size of motoneurons were observed in either segment after exposure to microgravity, but succinate dehydrogenase activity of small-sized (<300 μm(2)) gamma and medium-sized (300-700 μm(2)) alpha motoneurons, which have higher succinate dehydrogenase activity than large-sized (>700 μm(2)) alpha motoneurons, in both segments was lower than that of ground-based controls. We concluded that exposure to microgravity for longer than 3 months induced decreased succinate dehydrogenase activity of both gamma and slow-type alpha motoneurons. In particular, the decreased succinate dehydrogenase activity of gamma motoneurons was observed only after long-term exposure to microgravity.

  7. The neuroprotective action of pyrroloquinoline quinone against glutamate-induced apoptosis in hippocampal neurons is mediated through the activation of PI3K/Akt pathway

    SciTech Connect

    Zhang Qi; Shen Mi; Ding Mei; Shen Dingding; Ding Fei

    2011-04-01

    Pyrroloquinoline quinone (PQQ), a cofactor in several enzyme-catalyzed redox reactions, possesses a potential capability of scavenging reactive oxygen species (ROS) and inhibiting cell apoptosis. In this study, we investigated the effects of PQQ on glutamate-induced cell death in primary cultured hippocampal neurons and the possible underlying mechanisms. We found that glutamate-induced apoptosis in cultured hippocampal neurons was significantly attenuated by the ensuing PQQ treatment, which also inhibited the glutamate-induced increase in Ca2+ influx, caspase-3 activity, and ROS production, and reversed the glutamate-induced decrease in Bcl-2/Bax ratio. The examination of signaling pathways revealed that PQQ treatment activated the phosphorylation of Akt and suppressed the glutamate-induced phosphorylation of c-Jun N-terminal protein kinase (JNK). And inhibition of phosphatidylinositol-3-kinase (PI3K)/Akt cascade by LY294002 and wortmannin significantly blocked the protective effects of PQQ, and alleviated the increase in Bcl-2/Bax ratio. Taken together, our results indicated that PQQ could protect primary cultured hippocampal neurons against glutamate-induced cell damage by scavenging ROS, reducing Ca2+ influx, and caspase-3 activity, and suggested that PQQ-activated PI3K/Akt signaling might be responsible for its neuroprotective action through modulation of glutamate-induced imbalance between Bcl-2 and Bax. - Research Highlights: >PQQ attenuated glutamate-induced cell apoptosis of cultured hippocampal neurons. >PQQ inhibited glutamate-induced Ca{sup 2+} influx and caspase-3 activity. >PQQ reduced glutamate-induced increase in ROS production. >PQQ affected phosphorylation of Akt and JNK signalings after glutamate injury. >PI3K/Akt was required for neuroprotection of PQQ by modulating Bcl-2/Bax ratio.

  8. Effect of MS-153, a glutamate transporter activator, on the conditioned rewarding effects of morphine, methamphetamine and cocaine in mice.

    PubMed

    Nakagawa, Takayuki; Fujio, Mayumi; Ozawa, Tohru; Minami, Masabumi; Satoh, Masamichi

    2005-01-30

    There is a body of evidence implying the involvement of the glutamatergic system in the conditioned rewarding effects of drugs of abuse. It is recognized that the release of extracellular glutamate from nerve terminals is counterbalanced by the functions of neuronal and glial glutamate transporters. In the present study, we investigated the effects of (R)-(-)-5-methyl-1-nicotinoyl-2-pyrazoline (MS-153), a glutamate transporter activator, on the induction of the conditioned place preference to morphine, methamphetamine and cocaine in mice. In the conditioned place preference paradigm, mice were conditioned with repeated subcutaneous injections of morphine (5 mg/kg), methamphetamine (2 mg/kg) or cocaine (8 mg/kg) in combination with or without MS-153 (3 and 10 mg/kg). Co-administration of MS-153 at a dose of 10 mg/kg, but not 3 mg/kg, significantly attenuated the induction of conditioned place preference to morphine, methamphetamine and cocaine. However, MS-153 itself produced neither conditioned place preference nor aversion. On the other hand, co-administration of MS-153 (10 mg/kg) did not alter the acute locomotor activation elicited by a single injection of morphine, methamphetamine and cocaine. These results suggest that MS-153, a glutamate transporter activator, has an inhibitory effect on the conditioned rewarding effects of morphine, methamphetamine and cocaine without affecting their acute locomotor responses.

  9. Dynamic changes in gamma-aminobutyric acid and glutamate decarboxylase activity in oats (Avena nuda L.) during steeping and germination.

    PubMed

    Xu, Jian Guo; Hu, Qing Ping; Duan, Jiang Lian; Tian, Cheng Rui

    2010-09-01

    Gamma-aminobutyric acid (GABA) is the principal inhibitory neurotransmitter in the central nervous system and provides beneficial effects for human and other animals health. To accumulate GABA, samples from two different naked oat cultivars, Baiyan II and Bayou I, were steeped and germinated in an incubator. The content of GABA and glutamic acid as well as the activity of the glutamate decarboxylase (GAD) in oats during steeping and germination were investigated with an amino acid automatic analyzer. Compared with raw groats, an increase in GABA content of oat groats during steeping and germination was continuously observed for two oat cultivars. The activity of GAD increased greatly at the end of steeping and the second stage of germination for Baiyan II and Bayou I, respectively. Glutamic acid content of treated oat groats was significantly lower than that in raw groats until the later period of germination. GABA was correlated (p<0.01) significantly and positively with the glutamic acid rather than GAD activity in the current study. The results indicates that steeping and germination process under highly controlled conditions can effectively accumulate the GABA in oat groats for Baiyan II and Bayou I, which would greatly facilitate production of nutraceuticals or food ingredients that enable consumers to gain greater access to the health benefits of oats. However, more assays need to be further performed with more oat cultivars.

  10. Topiramate protects against glutamate excitotoxicity via activating BDNF/TrkB-dependent ERK pathway in rodent hippocampal neurons.

    PubMed

    Mao, Xiao-Yuan; Cao, Yong-Gang; Ji, Zhong; Zhou, Hong-Hao; Liu, Zhao-Qian; Sun, Hong-Li

    2015-07-01

    Topiramate (TPM) was previously found to have neuroprotection against neuronal injury in epileptic and ischemic models. However, whether TPM protects against glutamate-induced excitotoxicity in hippocampal neurons is elusive. Our present work aimed to evaluate the protective effect of TPM against glutamate toxicity in hippocampal neurons and further figure out the potential molecular mechanisms. The in vitro glutamate excitotoxic model was prepared with 125μM glutamate for 20min. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) analysis and Hoechst 33342 staining were conducted to detect neuronal survival. The protein expressions of brain-derived neurotrophic factor (BDNF), TrkB, mitogen-activated protein kinase (MAPK) cascade (including extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and p38 MAPK), cyclic AMP response element binding protein (CREB), Bcl-2, Bax and β-actin were detected via Western blot assay. Our results demonstrated that TPM protected hippocampal neurons from glutamate toxicity. Meanwhile, the pretreatment of TPM for 10min significantly prevented the down-regulation of BDNF and the phosphorylation of TrkB. Furthermore, the elevation of phosphorylated EKR expression was significantly inhibited after blockade of TrkB by TrkB IgG, while no alterations of phosphorylated JNK and p38 MAPK were found in the cultured hippocampal neurons. Besides, it was also found that the enhanced phosphorylation of CREB was evidently reversed under excitotoxic conditions after treating with U0126 (the selective inhibitor of ERK). The protein level of Bcl-2 was also observed to be remarkably increased after TPM treatment. In conclusion, these findings implicate that TPM exerts neuroprotective effects against glutamate excitotoxicity in hippocampal neurons and its protection may be modulated through BDNF/TrkB-dependent ERK pathway.

  11. Preferential binding of allosteric modulators to active and inactive conformational states of metabotropic glutamate receptors

    PubMed Central

    Yanamala, Naveena; Tirupula, Kalyan C; Klein-Seetharaman, Judith

    2008-01-01

    Metabotropic glutamate receptors (mGluRs) are G protein coupled receptors that play important roles in synaptic plasticity and other neuro-physiological and pathological processes. Allosteric mGluR ligands are particularly promising drug targets because of their modulatory effects – enhancing or suppressing the response of mGluRs to glutamate. The mechanism by which this modulation occurs is not known. Here, we propose the hypothesis that positive and negative modulators will differentially stabilize the active and inactive conformations of the receptors, respectively. To test this hypothesis, we have generated computational models of the transmembrane regions of different mGluR subtypes in two different conformations. The inactive conformation was modeled using the crystal structure of the inactive, dark state of rhodopsin as template and the active conformation was created based on a recent model of the light-activated state of rhodopsin. Ligands for which the nature of their allosteric effects on mGluRs is experimentally known were docked to the modeled mGluR structures using ArgusLab and Autodock softwares. We find that the allosteric ligand binding pockets of mGluRs are overlapping with the retinal binding pocket of rhodopsin, and that ligands have strong preferences for the active and inactive states depending on their modulatory nature. In 8 out of 14 cases (57%), the negative modulators bound the inactive conformations with significant preference using both docking programs, and 6 out of 9 cases (67%), the positive modulators bound the active conformations. Considering results by the individual programs only, even higher correlations were observed: 12/14 (86%) and 8/9 (89%) for ArgusLab and 10/14 (71%) and 7/9 (78%) for AutoDock. These findings strongly support the hypothesis that mGluR allosteric modulation occurs via stabilization of different conformations analogous to those identified in rhodopsin where they are induced by photochemical isomerization

  12. Retour aux sources: defining the structural basis of glutamate receptor activation.

    PubMed

    Dawe, G Brent; Aurousseau, Mark R; Daniels, Bryan A; Bowie, Derek

    2015-01-01

    Ionotropic glutamate receptors (iGluRs) are the major excitatory neurotransmitter receptor in the vertebrate CNS and, as a result, their activation properties lie at the heart of much of the neuronal network activity observed in the developing and adult brain. iGluRs have also been implicated in many nervous system disorders associated with postnatal development (e.g. autism, schizophrenia), cerebral insult (e.g. stroke, epilepsy), and disorders of the ageing brain (e.g. Alzheimer's disease, Parkinsonism). In view of this, an emphasis has been placed on understanding how iGluRs activate and desensitize in functional and structural terms. Early structural models of iGluRs suggested that the strength of the agonist response was primarily governed by the degree of closure induced in the ligand-binding domain (LBD). However, recent studies have suggested a more nuanced role for the LBD with current evidence identifying the iGluR LBD interface as a "hotspot" regulating agonist behaviour. Such ideas remain to be consolidated with recently solved structures of full-length iGluRs to account for the global changes that underlie channel activation and desensitization.

  13. Stimulation of endocannabinoid formation in brain slice cultures through activation of group I metabotropic glutamate receptors.

    PubMed

    Jung, Kwang-Mook; Mangieri, Regina; Stapleton, Christopher; Kim, Janet; Fegley, Darren; Wallace, Matthew; Mackie, Ken; Piomelli, Daniele

    2005-11-01

    Activation of group I metabotropic glutamate (mGlu) receptors drives the endocannabinoid system to cause both short- and long-term changes of synaptic strength in the striatum, hippocampus, and other brain areas. Although there is strong electrophysiological evidence for a role of endocannabinoid release in mGlu receptor-dependent plasticity, the identity of the endocannabinoid transmitter mediating this phenomenon remains undefined. In this study, we show that activation of group I mGlu receptors triggers the biosynthesis of the endocannabinoid 2-arachidonoylglycerol (2-AG), but not anandamide, in primary cultures of corticostriatal and hippocampal slices prepared from early postnatal rat brain. Pharmacological studies suggest that 2-AG biosynthesis is initiated by activation of mGlu5 receptors, is catalyzed by phospholipase C (PLC) and 1,2-diacylglycerol lipase (DGL) activities, and is dependent on intracellular Ca2+ ions. Realtime polymerase chain reaction and immunostaining analyses indicate that DGL-beta is the predominant DGL isoform expressed in corticostriatal and hippocampal slices and that this enzyme is highly expressed in striatal neurons, where it is colocalized with PLC-beta1. The results suggest that 2-AG is a primary endocannabinoid mediator of mGlu receptor-dependent neuronal plasticity.

  14. Activity and stability of yeast alcohol dehydrogenase (YADH) entrapped in aerosol OT reverse micelles.

    PubMed

    Sarcar, S; Jain, T K; Maitra, A

    1992-02-20

    The activity and stability of yeast alcohol dehydrogenase (YADH) entrapped in aerosol OT reverse micellar droplets have been investigated spectrophotometrically. Various physical parameters, e.g., water pool size, w(0), pH, and temperature, were optimized for YADH in water/AOT/isooctane reverse micelles. It was found that the enzyme exhibits maximum activity at w(0) = 28 and pH 8.1. It was more active in reverse micelles than in aqueous buffers at a particular temperature and was denatured at about 307 degrees C in both the systems. At a particular temperature YADH entrapped in reverse micelles was less stable than when it was dissolved in aqueous buffer.

  15. Designing a highly active soluble PQQ-glucose dehydrogenase for efficient glucose biosensors and biofuel cells

    SciTech Connect

    Durand, Fabien; Stines-Chaumeil, Claire; Flexer, Victoria; Andre, Isabelle; Mano, Nicolas

    2010-11-26

    Research highlights: {yields} A new mutant of PQQ-GDH designed for glucose biosensors application. {yields} First mutant of PQQ-GDH with higher activity for D-glucose than the Wild type. {yields} Position N428 is a key point to increase the enzyme activity. {yields} Molecular modeling shows that the N428 C mutant displays a better interaction for PQQ than the WT. -- Abstract: We report for the first time a soluble PQQ-glucose dehydrogenase that is twice more active than the wild type for glucose oxidation and was obtained by combining site directed mutagenesis, modelling and steady-state kinetics. The observed enhancement is attributed to a better interaction between the cofactor and the enzyme leading to a better electron transfer. Electrochemical experiments also demonstrate the superiority of the new mutant for glucose oxidation and make it a promising enzyme for the development of high-performance glucose biosensors and biofuel cells.

  16. Plasma lactic dehydrogenase activities in men during bed rest with exercise training

    NASA Technical Reports Server (NTRS)

    Greenleaf, J. E.; Juhos, L. T.; Young, H. L.

    1985-01-01

    Peak oxygen uptake and the activity of lactic dehydrogenase (LDH-T) and its five isoenzymes were measured by spectrophotometer in seven men before, during, and after bed rest and exercise training. Exercise training consisted of isometric leg exercises of 250 kcal/hr for a period of one hour per day. It is found that LDH-T was reduced by 0.05 percent in all three regimens by day 10 of bed rest, and that the decrease occurred at different rates. The earliest reduction in LDH-T activity in the no-exercise regimen was associated with a decrease in peak oxygen uptake of 12.3 percent. It is concluded that isometric (aerobic) muscular strength training appear to maintain skeletal muscle integrity better during bed rest than isotonic exercise training. Reduced hydrostatic pressure during bed rest, however, ultimately counteracts the effects of both moderate isometric and isotonic exercise training, and may result in decreased LDH-T activity.

  17. [Lactate dehydrogenase and Krebs cycle enzyme activity in rat liver during the growth of transplanted and spontaneous tumors].

    PubMed

    Morozkina, T S

    1978-03-01

    Certain distinctions in the mouse and rat liver responses to transplanted and spontaneous tumours have been discovered at the initial periods of their growth. The most pronounced changes (the mosaic distribution of enzymatic activity in the lobe) are observed in the case of spontaneous tumours. Activities the Krebs cycle enzymes, especially of NAD-dependent enzymes are seen inhibited in the tumour-bearing liver at the terminal periods of growth of both spontaneous and transplanted tumours; lactate dehydrogenase activity increases (with the exception of mitochondrial lactate dehydrogenase in the rat liver with transplanted sarcomas). PMID:684845

  18. The Role of Glutamate Dehydrogenase (GDH) Testing Assay in the Diagnosis of Clostridium difficile Infections: A High Sensitive Screening Test and an Essential Step in the Proposed Laboratory Diagnosis Workflow for Developing Countries like China

    PubMed Central

    Cheng, Jing-Wei; Xiao, Meng; Kudinha, Timothy; Xu, Zhi-Peng; Sun, Lin-Ying; Hou, Xin; Zhang, Li; Fan, Xin; Kong, Fanrong; Xu, Ying-Chun

    2015-01-01

    The incidence and severity of Clostridium difficile infection (CDI) in North America and Europe has increased significantly since the 2000s. However, CDI is not widely recognized in China and other developing countries due to limited laboratory diagnostic capacity and low awareness. Most published studies on laboratory workflows for CDI diagnosis are from developed countries, and thus may not be suitable for most developing countries. Therefore, an alternative strategy for developing countries is needed. In this study, we evaluated the performance of the Glutamate Dehydrogenase (GDH) test and its associated workflow on 416 fecal specimens from suspected CDI cases. The assay exhibited excellent sensitivity (100.0%) and specificity (92.8%), compared to culture based method, and thus could be a good screening marker for C. difficile but not for indication of toxin production. The VIDAS CDAB assay, which can detect toxin A/B directly from fecal specimens, showed good specificity (99.7%) and positive predictive value (97.2%), but low sensitivity (45.0%) and negative predictive value (88.3%), compared with PCR-based toxin gene detection. Therefore, we propose a practical and efficient GDH test based workflow strategy for the laboratory diagnosis of CDI in developing countries like China. By applying this new workflow, the CDI laboratory diagnosis rate was notably improved in our center, yet the increasing cost was kept at a minimum level. Furthermore, to gain some insights into the genetic population structure of C. difficile isolates from our hospital, we performed MLST and PCR toxin gene typing. PMID:26659011

  19. The Role of Glutamate Dehydrogenase (GDH) Testing Assay in the Diagnosis of Clostridium difficile Infections: A High Sensitive Screening Test and an Essential Step in the Proposed Laboratory Diagnosis Workflow for Developing Countries like China.

    PubMed

    Cheng, Jing-Wei; Xiao, Meng; Kudinha, Timothy; Xu, Zhi-Peng; Sun, Lin-Ying; Hou, Xin; Zhang, Li; Fan, Xin; Kong, Fanrong; Xu, Ying-Chun

    2015-01-01

    The incidence and severity of Clostridium difficile infection (CDI) in North America and Europe has increased significantly since the 2000s. However, CDI is not widely recognized in China and other developing countries due to limited laboratory diagnostic capacity and low awareness. Most published studies on laboratory workflows for CDI diagnosis are from developed countries, and thus may not be suitable for most developing countries. Therefore, an alternative strategy for developing countries is needed. In this study, we evaluated the performance of the Glutamate Dehydrogenase (GDH) test and its associated workflow on 416 fecal specimens from suspected CDI cases. The assay exhibited excellent sensitivity (100.0%) and specificity (92.8%), compared to culture based method, and thus could be a good screening marker for C. difficile but not for indication of toxin production. The VIDAS CDAB assay, which can detect toxin A/B directly from fecal specimens, showed good specificity (99.7%) and positive predictive value (97.2%), but low sensitivity (45.0%) and negative predictive value (88.3%), compared with PCR-based toxin gene detection. Therefore, we propose a practical and efficient GDH test based workflow strategy for the laboratory diagnosis of CDI in developing countries like China. By applying this new workflow, the CDI laboratory diagnosis rate was notably improved in our center, yet the increasing cost was kept at a minimum level. Furthermore, to gain some insights into the genetic population structure of C. difficile isolates from our hospital, we performed MLST and PCR toxin gene typing. PMID:26659011

  20. Effect of different mulch materials on the soil dehydrogenase activity (DHA) in an organic pepper crop

    NASA Astrophysics Data System (ADS)

    Moreno, Marta M.; Peco, Jesús; Campos, Juan; Villena, Jaime; González, Sara; Moreno, Carmen

    2016-04-01

    The use biodegradable materials (biopolymers of different composition and papers) as an alternative to conventional mulches has increased considerably during the last years mainly for environmental reason. In order to assess the effect of these materials on the soil microbial activity during the season of a pepper crop organically grown in Central Spain, the soil dehydrogenase activity (DHA) was measured in laboratory. The mulch materials tested were: 1) black polyethylene (PE, 15 μm); black biopolymers (15 μm): 2) Mater-Bi® (corn starch based), 3) Sphere 4® (potato starch based), 4) Sphere 6® (potato starch based), 5) Bioflex® (polylactic acid based), 6) Ecovio® (polylactic acid based), 7) Mimgreen® (black paper, 85 g/m2). A randomized complete block design with four replications was adopted. The crop was drip irrigated following the water demand of each treatment. Soil samples (5-10 cm depth) under the different mulches were taken at different dates (at the beginning of the crop cycle and at different dates throughout the crop season). Additionally, samples of bare soil in a manual weeding and in an untreated control were taken. The results obtained show the negative effect of black PE on the DHA activity, mainly as result of the higher temperature reached under the mulch and the reduction in the gas interchange between the soil and the atmosphere. The values corresponding to the biodegradable materials were variable, although highlighting the low DHA activity observed under Bioflex®. In general, the uncovered treatments showed higher values than those reached under mulches, especially in the untreated control. Keywords: mulch, biodegradable, biopolymer, paper, dehydrogenase activity (DHA). Acknowledgements: the research was funded by Project RTA2011-00104-C04-03 from the INIA (Spanish Ministry of Economy and Competitiveness).

  1. Structure-activity relationship study of spider polyamine toxins as inhibitors of ionotropic glutamate receptors.

    PubMed

    Xiong, Xiao-Feng; Poulsen, Mette H; Hussein, Rama A; Nørager, Niels G; Strømgaard, Kristian

    2014-12-01

    The spider polyamine toxins Joro spider toxin-3 (JSTX-3) and Nephila polyamine toxins-1 and -8 (NPTX-1 and NPTX-8) are isolated from the venom of the orb-weaver spider Nephila clavata (Joro spider). They share a high degree of structural resemblance, their aromatic head groups being the only difference, and were recently found to be very potent open-channel blockers of ionotropic glutamate (iGlu) receptors. In this study we designed and synthesized a collection of 24 analogues of these toxins using a recently developed solid-phase synthetic methodology. Systematic variation in two regions of the toxins and subsequent evaluation of biological activity at AMPA and NMDA subtypes of iGlu receptors provided succinct information on structure-activity relationships. In particular, one set of analogues were found to display exquisite selectivity and potency for AMPA receptors relative to the natural products. Thus, this systematic SAR study has provided new pharmacological tools for studies of iGlu receptors.

  2. Potato tuber cytokinin oxidase/dehydrogenase genes: biochemical properties, activity, and expression during tuber dormancy progression.

    PubMed

    Suttle, Jeffrey C; Huckle, Linda L; Lu, Shunwen; Knauber, Donna C

    2014-03-15

    The enzymatic and biochemical properties of the proteins encoded by five potato cytokinin oxidase/dehydrogenase (CKX)-like genes functionally expressed in yeast and the effects of tuber dormancy progression on StCKX expression and cytokinin metabolism were examined in lateral buds isolated from field-grown tubers. All five putative StCKX genes encoded proteins with in vitro CKX activity. All five enzymes were maximally active at neutral to slightly alkaline pH with 2,6-dichloro-indophenol as the electron acceptor. In silico analyses indicated that four proteins were likely secreted. Substrate dependence of two of the most active enzymes varied; one exhibiting greater activity with isopentenyl-type cytokinins while the other was maximally active with cis-zeatin as a substrate. [(3)H]-isopentenyl-adenosine was readily metabolized by excised tuber buds to adenine/adenosine demonstrating that CKX was active in planta. There was no change in apparent in planta CKX activity during either natural or chemically forced dormancy progression. Similarly although expression of individual StCKX genes varied modestly during tuber dormancy, there was no clear correlation between StCKX gene expression and tuber dormancy status. Thus although CKX gene expression and enzyme activity are present in potato tuber buds throughout dormancy, they do not appear to play a significant role in the regulation of cytokinin content during tuber dormancy progression.

  3. Potato tuber cytokinin oxidase/dehydrogenase genes: biochemical properties, activity, and expression during tuber dormancy progression.

    PubMed

    Suttle, Jeffrey C; Huckle, Linda L; Lu, Shunwen; Knauber, Donna C

    2014-03-15

    The enzymatic and biochemical properties of the proteins encoded by five potato cytokinin oxidase/dehydrogenase (CKX)-like genes functionally expressed in yeast and the effects of tuber dormancy progression on StCKX expression and cytokinin metabolism were examined in lateral buds isolated from field-grown tubers. All five putative StCKX genes encoded proteins with in vitro CKX activity. All five enzymes were maximally active at neutral to slightly alkaline pH with 2,6-dichloro-indophenol as the electron acceptor. In silico analyses indicated that four proteins were likely secreted. Substrate dependence of two of the most active enzymes varied; one exhibiting greater activity with isopentenyl-type cytokinins while the other was maximally active with cis-zeatin as a substrate. [(3)H]-isopentenyl-adenosine was readily metabolized by excised tuber buds to adenine/adenosine demonstrating that CKX was active in planta. There was no change in apparent in planta CKX activity during either natural or chemically forced dormancy progression. Similarly although expression of individual StCKX genes varied modestly during tuber dormancy, there was no clear correlation between StCKX gene expression and tuber dormancy status. Thus although CKX gene expression and enzyme activity are present in potato tuber buds throughout dormancy, they do not appear to play a significant role in the regulation of cytokinin content during tuber dormancy progression. PMID:24594397

  4. Pyruvate dehydrogenase kinase isoform 2 activity stimulated by speeding up the rate of dissociation of ADP.

    PubMed

    Bao, Haiying; Kasten, Shane A; Yan, Xiaohua; Hiromasa, Yasuaki; Roche, Thomas E

    2004-10-26

    Pyruvate dehydrogenase kinase 2 (PDK2) activity is stimulated by NADH and NADH plus acetyl-CoA via the reduction and reductive acetylation of the lipoyl groups of the dihydrolipoyl acetyltransferase (E2) component. Elevated K(+) and Cl(-) were needed for significant stimulation. Stimulation substantially increased both k(cat) and the K(m) for ATP; the fractional stimulation increased with the level of ATP. With an E2 structure lacking the pyruvate dehydrogenase (E1) binding domain, stimulation of PDK2 was retained, the K(m) for E1 decreased, and the equilibrium dissociation constant for ATP increased but remained much lower than the K(m) for ATP. Stimulation of PDK2 activity greatly reduced the fraction of bound ADP. These results fit an ordered reaction mechanism with ATP binding before E1 and stimulation increasing the rate of dissociation of ADP. Conversion of all of the lipoyl groups in the E2 60mer to the oxidized form (E2(ox)) greatly reduced k(cat) and the K(m) of PDK2 for ATP. Retention over an extended period of time of a low portion of reduced lipoyl groups maintains E2 in a state that supported much higher PDK2 activity than short-term (5 min) reduction of a large portion of lipoyl groups of E2(ox), but reduction of E2(ox) produced a larger fold stimulation. Reduction and to a greater extent reductive acetylation increased PDK2 binding to E2; conversion to E2(ox) did not significantly hinder binding. We suggest that passing even limited reducing equivalents among lipoyl groups maintains E2 lipoyl domains in a conformation that aids kinase function. PMID:15491151

  5. Activities of citrate synthase and NAD+-linked and NADP+-linked isocitrate dehydrogenase in muscle from vertebrates and invertebrates.

    PubMed Central

    Alp, P R; Newsholme, E A; Zammit, V A

    1976-01-01

    1. The activities of citrate synthase, NAD+-linked and NADP+-linked isocitrate dehydrogenase were measured in muscles from a large number of animals, in order to provide some indication of the importance of the citric acid cycle in these muscles. According to the differences in enzyme activities, the muscles can be divided into three classes. First, in a number of both vertebrate and invertebrate muscles, the activities of all three enzymes are very low. It is suggested that either the muscles use energy at a very low rate or they rely largely on anaerobic glycolysis for higher rates of energy formation. Second, most insect flight muscles contain high activities of citrate synthase and NAD+-linked isocitrate dehydrogenase, but the activities of the NADP+-linked enzyme are very low. The high activities indicate the dependence of insect flight on energy generated via the citric acid cycle. The flight muscles of the beetles investigated contain high activities of both isocitrate dehydrogenases. Third, other muscles of both vertebrates and invertebrates contain high activities of citrate synthase and NADP+-liniked isocitrate dehydrogenase. Many, if not all, of these muscles are capable of sustained periods of mechanical activity (e.g. heart muscle, pectoral muscles of some birds). Consequently, to support this activity fuel must be supplied continually to the muscle via the circulatory system which, in most animals, also transports oxygen so that energy can be generated by complete oxidation of the fuel. It is suggested that the low activities of NAD+-linked isocitrate dehydrogenase in these muscles may be involved in oxidation of isocitrate in the cycle when the muscles are at rest. 2. A comparison of the maximal activities of the enzymes with the maximal flux through the cycle suggests that, in insect flight muscle, NAD+-linked isocitrate dehydrogenase catalyses a non-equilibrium reaction and citrate synthease catalyses a near-equilibrium reaction. In other muscles

  6. Increased activity of 6-phosphogluconate dehydrogenase and glucose-6-phosphate dehydrogenase in purified cell suspensions and single cells from the uterine cervix in cervical intraepithelial neoplasia.

    PubMed Central

    Jonas, S. K.; Benedetto, C.; Flatman, A.; Hammond, R. H.; Micheletti, L.; Riley, C.; Riley, P. A.; Spargo, D. J.; Zonca, M.; Slater, T. F.

    1992-01-01

    The activities of 6-phosphogluconate dehydrogenase and glucose-6-phosphate dehydrogenase have been measured in squamous epithelial cells of the uterine cervix from normal patients and cases of cervical intraepithelial neoplasia (CIN). A biochemical cycling method, which uses only simple equipment and is suited to routine use and to automation, was applied to cells separated by gradient centrifugation. In addition, cells were examined cytochemically, and the intensity of staining in the cytoplasm of single whole cells was measured using computerised microcytospectrophotometry. Twenty per cent of cells in samples from normal patients (n=61) showed staining intensities above an extinction of 0.15 at 540 nm, compared to 71% of cases of CIN 1 (n=14), 91% of cases of CIN 2 (n=11) and 67% of cases of CIN 3 (n=15). The cytochemical data do not allow definitive distinctions to be made between different grades of CIN whereas the biochemical assay applied to cell lysates shows convincing differences between normal samples and cases of CIN. There are no false negatives for CIN 3 (n=14) and CIN 2 (n=10) and 11% false negatives for CIN 1 (n=9) and 14% of false positives for normal cases (n=21). The results of this preliminary study with reference to automation are discussed [corrected]. Images Figure 1 PMID:1637668

  7. Evidence that the C-terminal domain of a type B PutA protein contributes to aldehyde dehydrogenase activity and substrate channeling.

    PubMed

    Luo, Min; Christgen, Shelbi; Sanyal, Nikhilesh; Arentson, Benjamin W; Becker, Donald F; Tanner, John J

    2014-09-01

    Proline utilization A (PutA) is a bifunctional enzyme that catalyzes the oxidation of proline to glutamate. Structures of type A PutAs have revealed the catalytic core consisting of proline dehydrogenase (PRODH) and Δ(1)-pyrroline-5-carboxylate dehydrogenase (P5CDH) modules connected by a substrate-channeling tunnel. Type B PutAs also have a C-terminal domain of unknown function (CTDUF) that is absent in type A PutAs. Small-angle X-ray scattering (SAXS), mutagenesis, and kinetics are used to determine the contributions of this domain to PutA structure and function. The 1127-residue Rhodobacter capsulatus PutA (RcPutA) is used as a representative CTDUF-containing type B PutA. The reaction progress curve for the coupled PRODH-P5CDH activity of RcPutA does not exhibit a time lag, implying a substrate channeling mechanism. RcPutA is monomeric in solution, which is unprecedented for PutAs. SAXS rigid body modeling with target-decoy validation is used to build a model of RcPutA. On the basis of homology to aldehyde dehydrogenases (ALDHs), the CTDUF is predicted to consist of a β-hairpin fused to a noncatalytic Rossmann fold domain. The predicted tertiary structural interactions of the CTDUF resemble the quaternary structural interactions in the type A PutA dimer interface. The model is tested by mutagenesis of the dimerization hairpin of a type A PutA and the CTDUF hairpin of RcPutA. Similar functional phenotypes are observed in the two sets of variants, supporting the hypothesis that the CTDUF mimics the type A PutA dimer interface. These results suggest annotation of the CTDUF as an ALDH superfamily domain that facilitates P5CDH activity and substrate channeling by stabilizing the aldehyde-binding site and sealing the substrate-channeling tunnel from the bulk medium. PMID:25137435

  8. Evidence That the C-Terminal Domain of a Type B PutA Protein Contributes to Aldehyde Dehydrogenase Activity and Substrate Channeling

    PubMed Central

    2015-01-01

    Proline utilization A (PutA) is a bifunctional enzyme that catalyzes the oxidation of proline to glutamate. Structures of type A PutAs have revealed the catalytic core consisting of proline dehydrogenase (PRODH) and Δ1-pyrroline-5-carboxylate dehydrogenase (P5CDH) modules connected by a substrate-channeling tunnel. Type B PutAs also have a C-terminal domain of unknown function (CTDUF) that is absent in type A PutAs. Small-angle X-ray scattering (SAXS), mutagenesis, and kinetics are used to determine the contributions of this domain to PutA structure and function. The 1127-residue Rhodobacter capsulatus PutA (RcPutA) is used as a representative CTDUF-containing type B PutA. The reaction progress curve for the coupled PRODH–P5CDH activity of RcPutA does not exhibit a time lag, implying a substrate channeling mechanism. RcPutA is monomeric in solution, which is unprecedented for PutAs. SAXS rigid body modeling with target–decoy validation is used to build a model of RcPutA. On the basis of homology to aldehyde dehydrogenases (ALDHs), the CTDUF is predicted to consist of a β-hairpin fused to a noncatalytic Rossmann fold domain. The predicted tertiary structural interactions of the CTDUF resemble the quaternary structural interactions in the type A PutA dimer interface. The model is tested by mutagenesis of the dimerization hairpin of a type A PutA and the CTDUF hairpin of RcPutA. Similar functional phenotypes are observed in the two sets of variants, supporting the hypothesis that the CTDUF mimics the type A PutA dimer interface. These results suggest annotation of the CTDUF as an ALDH superfamily domain that facilitates P5CDH activity and substrate channeling by stabilizing the aldehyde-binding site and sealing the substrate-channeling tunnel from the bulk medium. PMID:25137435

  9. Bruchpilot and Synaptotagmin collaborate to drive rapid glutamate release and active zone differentiation.

    PubMed

    Paul, Mila M; Pauli, Martin; Ehmann, Nadine; Hallermann, Stefan; Sauer, Markus; Kittel, Robert J; Heckmann, Manfred

    2015-01-01

    The active zone (AZ) protein Bruchpilot (Brp) is essential for rapid glutamate release at Drosophila melanogaster neuromuscular junctions (NMJs). Quantal time course and measurements of action potential-waveform suggest that presynaptic fusion mechanisms are altered in brp null mutants (brp(69) ). This could account for their increased evoked excitatory postsynaptic current (EPSC) delay and rise time (by about 1 ms). To test the mechanism of release protraction at brp(69) AZs, we performed knock-down of Synaptotagmin-1 (Syt) via RNAi (syt(KD) ) in wildtype (wt), brp(69) and rab3 null mutants (rab3(rup) ), where Brp is concentrated at a small number of AZs. At wt and rab3(rup) synapses, syt(KD) lowered EPSC amplitude while increasing rise time and delay, consistent with the role of Syt as a release sensor. In contrast, syt(KD) did not alter EPSC amplitude at brp(69) synapses, but shortened delay and rise time. In fact, following syt(KD) , these kinetic properties were strikingly similar in wt and brp(69) , which supports the notion that Syt protracts release at brp(69) synapses. To gain insight into this surprising role of Syt at brp(69) AZs, we analyzed the structural and functional differentiation of synaptic boutons at the NMJ. At 'tonic' type Ib motor neurons, distal boutons contain more AZs, more Brp proteins per AZ and show elevated and accelerated glutamate release compared to proximal boutons. The functional differentiation between proximal and distal boutons is Brp-dependent and reduced after syt(KD) . Notably, syt(KD) boutons are smaller, contain fewer Brp positive AZs and these are of similar number in proximal and distal boutons. In addition, super-resolution imaging via dSTORM revealed that syt(KD) increases the number and alters the spatial distribution of Brp molecules at AZs, while the gradient of Brp proteins per AZ is diminished. In summary, these data demonstrate that normal structural and functional differentiation of Drosophila AZs requires

  10. Bruchpilot and Synaptotagmin collaborate to drive rapid glutamate release and active zone differentiation

    PubMed Central

    Paul, Mila M.; Pauli, Martin; Ehmann, Nadine; Hallermann, Stefan; Sauer, Markus; Kittel, Robert J.; Heckmann, Manfred

    2015-01-01

    The active zone (AZ) protein Bruchpilot (Brp) is essential for rapid glutamate release at Drosophila melanogaster neuromuscular junctions (NMJs). Quantal time course and measurements of action potential-waveform suggest that presynaptic fusion mechanisms are altered in brp null mutants (brp69). This could account for their increased evoked excitatory postsynaptic current (EPSC) delay and rise time (by about 1 ms). To test the mechanism of release protraction at brp69 AZs, we performed knock-down of Synaptotagmin-1 (Syt) via RNAi (sytKD) in wildtype (wt), brp69 and rab3 null mutants (rab3rup), where Brp is concentrated at a small number of AZs. At wt and rab3rup synapses, sytKD lowered EPSC amplitude while increasing rise time and delay, consistent with the role of Syt as a release sensor. In contrast, sytKD did not alter EPSC amplitude at brp69 synapses, but shortened delay and rise time. In fact, following sytKD, these kinetic properties were strikingly similar in wt and brp69, which supports the notion that Syt protracts release at brp69synapses. To gain insight into this surprising role of Syt at brp69 AZs, we analyzed the structural and functional differentiation of synaptic boutons at the NMJ. At ‘tonic’ type Ib motor neurons, distal boutons contain more AZs, more Brp proteins per AZ and show elevated and accelerated glutamate release compared to proximal boutons. The functional differentiation between proximal and distal boutons is Brp-dependent and reduced after sytKD. Notably, sytKD boutons are smaller, contain fewer Brp positive AZs and these are of similar number in proximal and distal boutons. In addition, super-resolution imaging via dSTORM revealed that sytKD increases the number and alters the spatial distribution of Brp molecules at AZs, while the gradient of Brp proteins per AZ is diminished. In summary, these data demonstrate that normal structural and functional differentiation of Drosophila AZs requires concerted action of Brp and Syt. PMID

  11. Structure of Cryptosporidium IMP dehydrogenase bound to an inhibitor with in vivo antiparasitic activity

    DOE PAGES

    Kim, Youngchang; Makowska-Grzyska, Magdalena; Gorla, Suresh Kumar; Gollapalli, Deviprasad R.; Cuny, Gregory D.; Joachimiak, Andrzej; Hedstrom, Lizbeth

    2015-04-21

    Inosine 5´-monophosphate dehydrogenase (IMPDH) is a promising target for the treatment of Cryptosporidium infections. Here, the structure of C. parvum IMPDH (CpIMPDH) in complex with inosine 5´-monophosphate (IMP) and P131, an inhibitor with in vivo anticryptosporidial activity, is reported. P131 contains two aromatic groups, one of which interacts with the hypoxanthine ring of IMP, while the second interacts with the aromatic ring of a tyrosine in the adjacent subunit. In addition, the amine and NO2 moieties bind in hydrated cavities, forming water-mediated hydrogen bonds to the protein. The design of compounds to replace these water molecules is a new strategymore » for the further optimization of C. parvum inhibitors for both antiparasitic and antibacterial applications.« less

  12. Loss of succinate dehydrogenase activity results in dependency on pyruvate carboxylation for cellular anabolism.

    PubMed

    Lussey-Lepoutre, Charlotte; Hollinshead, Kate E R; Ludwig, Christian; Menara, Mélanie; Morin, Aurélie; Castro-Vega, Luis-Jaime; Parker, Seth J; Janin, Maxime; Martinelli, Cosimo; Ottolenghi, Chris; Metallo, Christian; Gimenez-Roqueplo, Anne-Paule; Favier, Judith; Tennant, Daniel A

    2015-11-02

    The tricarboxylic acid (TCA) cycle is a central metabolic pathway responsible for supplying reducing potential for oxidative phosphorylation and anabolic substrates for cell growth, repair and proliferation. As such it thought to be essential for cell proliferation and tissue homeostasis. However, since the initial report of an inactivating mutation in the TCA cycle enzyme complex, succinate dehydrogenase (SDH) in paraganglioma (PGL), it has become clear that some cells and tissues are not only able to survive with a truncated TCA cycle, but that they are also able of supporting proliferative phenotype observed in tumours. Here, we show that loss of SDH activity leads to changes in the metabolism of non-essential amino acids. In particular, we demonstrate that pyruvate carboxylase is essential to re-supply the depleted pool of aspartate in SDH-deficient cells. Our results demonstrate that the loss of SDH reduces the metabolic plasticity of cells, suggesting vulnerabilities that can be targeted therapeutically.

  13. Structure of Cryptosporidium IMP dehydrogenase bound to an inhibitor with in vivo antiparasitic activity.

    PubMed

    Kim, Youngchang; Makowska-Grzyska, Magdalena; Gorla, Suresh Kumar; Gollapalli, Deviprasad R; Cuny, Gregory D; Joachimiak, Andrzej; Hedstrom, Lizbeth

    2015-05-01

    Inosine 5'-monophosphate dehydrogenase (IMPDH) is a promising target for the treatment of Cryptosporidium infections. Here, the structure of C. parvum IMPDH (CpIMPDH) in complex with inosine 5'-monophosphate (IMP) and P131, an inhibitor with in vivo anticryptosporidial activity, is reported. P131 contains two aromatic groups, one of which interacts with the hypoxanthine ring of IMP, while the second interacts with the aromatic ring of a tyrosine in the adjacent subunit. In addition, the amine and NO2 moieties bind in hydrated cavities, forming water-mediated hydrogen bonds to the protein. The design of compounds to replace these water molecules is a new strategy for the further optimization of C. parvum inhibitors for both antiparasitic and antibacterial applications. PMID:25945705

  14. Chaperones rejuvenate folding and activity of 3-β hydroxysteroid dehydrogenase 2.

    PubMed

    Rajapaksha, Maheshinie; Prasad, Manoj; Thomas, James L; Whittal, Randy M; Bose, Himangshu S

    2013-05-17

    The steroidogenic enzyme 3-β hydroxysteroid dehydrogenase 2 (3βHSD2) mediates the conversion of pregnenolone to progesterone and dehydroepiandrosterone to androstenedione through both its dehydrogenase and isomerase activities, making it necessary for the protein to undergo a reversible conformational change. We hypothesized that chaperones assist 3βHSD2 in switching between the conformations to initiate, enhance, and maintain activity. In the presence of the chaperone lauryl maltoside (LM), 3βHSD2 immediately converted pregnenolone to progesterone, with a 6.4-fold increase in synthesis. Using far-UV circular dichroism (CD), we found that addition of LM increased 3βHSD2's α-helical content, which over time reverted to control levels, suggesting the formation of a stable but reversible conformation possibly due to hydrophobic interactions of the protein with LM micelles. We also found that LM increased fluorescence resonance energy transfer (FRET) about 11-fold between 3βHSD2 and fluorescing ANS molecules. This observation supports the idea that detergent(s) act as chaperones to assist 3βHSD2 in forming stable complexes, which in turn promotes proper folding. Mass spectrometric fingerprinting illustrated that LM incubation resulted in an ordered fragmentation of molecular mass from 39 to 13 kDa, as compared to limited or no proteolysis in the absence of LM. In addition, space-filling modeling demonstrated that 3βHSD2 association with detergents likely exposed the hydrophobic region, leading to its proteolysis. We conclude that detergents help 3βHSD2 to refold in order to rejuvenate, contributing to the ability of cells to rapidly produce steroids when needed.

  15. The carboxy-terminal tail of pyruvate dehydrogenase kinase 2 is required for the kinase activity.

    PubMed

    Klyuyeva, Alla; Tuganova, Alina; Popov, Kirill M

    2005-10-18

    Pyruvate dehydrogenase kinase 2 (PDK2) is a prototypical mitochondrial protein kinase that regulates the activity of the pyruvate dehydrogenase complex. Recent structural studies have established that PDK2 consists of a catalytic core built of the B and K domains and the relatively long amino and carboxyl tails of unknown function. Here, we show that the carboxy-terminal truncation variants of PDK2 display a greatly diminished capacity for phosphorylation of holo-PDC. This effect is due largely to the inability of the transacetylase component of PDC to promote the phosphorylation reaction catalyzed by the truncated PDK2 variants. Furthermore, the truncated forms of PDK2 bind poorly to the lipoyl-bearing domain(s) provided by the transacetylase component. Taken together, these data strongly suggest that the carboxyl tails of PDK isozymes contribute to the lipoyl-bearing domain-binding site of the kinase molecule. We also show that the carboxyl tails derived from isozymes PDK1, PDK3, and PDK4 are capable of supporting the kinase activity of the kinase core derived from PDK2 as well as binding of the respective PDK2 chimeras to the lipoyl-bearing domain. Furthermore, the chimera carrying the carboxyl tail of PDK3 displays a stronger response to the addition of the transacetylase component along with a better binding to the lipoyl-bearing domain, suggesting that, at least in part, the differences in the amino acid sequences of the carboxyl tails account for the differences between PDK isozymes. PMID:16216081

  16. Reduced activity of 11β-hydroxysteroid dehydrogenase in patients with cholestasis

    PubMed Central

    Quattropani, Cristiana; Vogt, Bruno; Odermatt, Alex; Dick, Bernhard; Frey, Brigitte M.; Frey, Felix J.

    2001-01-01

    Enhanced renal sodium retention and potassium loss in patients with cirrhosis is due to activation of mineralocorticoid receptors (MRs). Increased aldosterone concentrations, however, do not entirely explain the activation of MR in cirrhosis. Here, we hypothesize that cortisol activates MRs in patients with cholestasis. We present evidence that access of cortisol to MRs is a result of bile acid−mediated inhibition of 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2), an MR-protecting enzyme that converts cortisol to cortisone. Twelve patients with biliary obstruction and high plasma bile acid levels were studied before and after removal of the obstruction. The urinary ratio of (tetrahydrocortisol + 5α-tetrahydrocortisol)/tetrahydrocortisone, a measure of 11β-HSD2 activity, decreased from a median of 1.91 during biliary obstruction to 0.78 at 4 and 8 weeks after removal of the obstruction and normalization of plasma bile acid concentrations. In order to demonstrate that bile acids facilitate access of cortisol to the MR by inhibiting 11β-HSD2, an MR translocation assay was performed in HEK-293 cells transfected with human 11β-HSD2 and tagged MR. Increasing concentrations of chenodeoxycholic acid led to cortisol-induced nuclear translocation of MR. In conclusion, 11β-HSD2 activity is reduced in cholestasis, which results in MR activation by cortisol. PMID:11696574

  17. Muscular cholinesterase and lactate dehydrogenase activities in deep-sea fish from the NW Mediterranean.

    PubMed

    Koenig, Samuel; Solé, Montserrat

    2014-03-01

    Organisms inhabiting submarine canyons can be potentially exposed to higher inputs of anthropogenic chemicals than their counterparts from the adjacent areas. To find out to what extend this observation applies to a NW Mediterranean canyon (i.e. Blanes canyon) off the Catalan coast, four deep-sea fish species were collected from inside the canyon (BC) and the adjacent open slope (OS). The selected species were: Alepocephalus rostratus, Lepidion lepidion, Coelorinchus mediterraneus and Bathypterois mediterraneus. Prior to the choice of an adequate sentinel species, the natural variation of the selected parameters (biomarkers) in relation to factors such as size, sex, sampling depth and seasonality need to be characterised. In this study, the activities of cholinesterases (ChEs) and lactate dehydrogenase (LDH) enzymes were determined in the muscle of the four deep-sea fish. Of all ChEs, acetylcholinesterase (AChE) activity was dominant and selected for further monitoring. Overall, AChE activity exhibited a significant relationship with fish size whereas LDH activity was mostly dependent on the sex and gonadal development status, although in a species-dependent manner. The seasonal variability of LDH activity was more marked than for AChE activity, and inside-outside canyon (BC-OS) differences were not consistent in all contrasted fish species, and in fact they were more dependent on biological traits. Thus, they did not suggest a differential stress condition between sites inside and outside the canyon. PMID:24296242

  18. Muscular cholinesterase and lactate dehydrogenase activities in deep-sea fish from the NW Mediterranean.

    PubMed

    Koenig, Samuel; Solé, Montserrat

    2014-03-01

    Organisms inhabiting submarine canyons can be potentially exposed to higher inputs of anthropogenic chemicals than their counterparts from the adjacent areas. To find out to what extend this observation applies to a NW Mediterranean canyon (i.e. Blanes canyon) off the Catalan coast, four deep-sea fish species were collected from inside the canyon (BC) and the adjacent open slope (OS). The selected species were: Alepocephalus rostratus, Lepidion lepidion, Coelorinchus mediterraneus and Bathypterois mediterraneus. Prior to the choice of an adequate sentinel species, the natural variation of the selected parameters (biomarkers) in relation to factors such as size, sex, sampling depth and seasonality need to be characterised. In this study, the activities of cholinesterases (ChEs) and lactate dehydrogenase (LDH) enzymes were determined in the muscle of the four deep-sea fish. Of all ChEs, acetylcholinesterase (AChE) activity was dominant and selected for further monitoring. Overall, AChE activity exhibited a significant relationship with fish size whereas LDH activity was mostly dependent on the sex and gonadal development status, although in a species-dependent manner. The seasonal variability of LDH activity was more marked than for AChE activity, and inside-outside canyon (BC-OS) differences were not consistent in all contrasted fish species, and in fact they were more dependent on biological traits. Thus, they did not suggest a differential stress condition between sites inside and outside the canyon.

  19. Ghrelin Regulates Glucose and Glutamate Transporters in Hypothalamic Astrocytes

    PubMed Central

    Fuente-Martín, Esther; García-Cáceres, Cristina; Argente-Arizón, Pilar; Díaz, Francisca; Granado, Miriam; Freire-Regatillo, Alejandra; Castro-González, David; Ceballos, María L.; Frago, Laura M.; Dickson, Suzanne L.; Argente, Jesús; Chowen, Julie A.

    2016-01-01

    Hypothalamic astrocytes can respond to metabolic signals, such as leptin and insulin, to modulate adjacent neuronal circuits and systemic metabolism. Ghrelin regulates appetite, adiposity and glucose metabolism, but little is known regarding the response of astrocytes to this orexigenic hormone. We have used both in vivo and in vitro approaches to demonstrate that acylated ghrelin (acyl-ghrelin) rapidly stimulates glutamate transporter expression and glutamate uptake by astrocytes. Moreover, acyl-ghrelin rapidly reduces glucose transporter (GLUT) 2 levels and glucose uptake by these glial cells. Glutamine synthetase and lactate dehydrogenase decrease, while glycogen phosphorylase and lactate transporters increase in response to acyl-ghrelin, suggesting a change in glutamate and glucose metabolism, as well as glycogen storage by astrocytes. These effects are partially mediated through ghrelin receptor 1A (GHSR-1A) as astrocytes do not respond equally to desacyl-ghrelin, an isoform that does not activate GHSR-1A. Moreover, primary astrocyte cultures from GHSR-1A knock-out mice do not change glutamate transporter or GLUT2 levels in response to acyl-ghrelin. Our results indicate that acyl-ghrelin may mediate part of its metabolic actions through modulation of hypothalamic astrocytes and that this effect could involve astrocyte mediated changes in local glucose and glutamate metabolism that alter the signals/nutrients reaching neighboring neurons. PMID:27026049

  20. Ghrelin Regulates Glucose and Glutamate Transporters in Hypothalamic Astrocytes.

    PubMed

    Fuente-Martín, Esther; García-Cáceres, Cristina; Argente-Arizón, Pilar; Díaz, Francisca; Granado, Miriam; Freire-Regatillo, Alejandra; Castro-González, David; Ceballos, María L; Frago, Laura M; Dickson, Suzanne L; Argente, Jesús; Chowen, Julie A

    2016-03-30

    Hypothalamic astrocytes can respond to metabolic signals, such as leptin and insulin, to modulate adjacent neuronal circuits and systemic metabolism. Ghrelin regulates appetite, adiposity and glucose metabolism, but little is known regarding the response of astrocytes to this orexigenic hormone. We have used both in vivo and in vitro approaches to demonstrate that acylated ghrelin (acyl-ghrelin) rapidly stimulates glutamate transporter expression and glutamate uptake by astrocytes. Moreover, acyl-ghrelin rapidly reduces glucose transporter (GLUT) 2 levels and glucose uptake by these glial cells. Glutamine synthetase and lactate dehydrogenase decrease, while glycogen phosphorylase and lactate transporters increase in response to acyl-ghrelin, suggesting a change in glutamate and glucose metabolism, as well as glycogen storage by astrocytes. These effects are partially mediated through ghrelin receptor 1A (GHSR-1A) as astrocytes do not respond equally to desacyl-ghrelin, an isoform that does not activate GHSR-1A. Moreover, primary astrocyte cultures from GHSR-1A knock-out mice do not change glutamate transporter or GLUT2 levels in response to acyl-ghrelin. Our results indicate that acyl-ghrelin may mediate part of its metabolic actions through modulation of hypothalamic astrocytes and that this effect could involve astrocyte mediated changes in local glucose and glutamate metabolism that alter the signals/nutrients reaching neighboring neurons.

  1. The relationship between human skeletal muscle pyruvate dehydrogenase phosphatase activity and muscle aerobic capacity.

    PubMed

    Love, Lorenzo K; LeBlanc, Paul J; Inglis, J Greig; Bradley, Nicolette S; Choptiany, Jon; Heigenhauser, George J F; Peters, Sandra J

    2011-08-01

    Pyruvate dehydrogenase (PDH) is a mitochondrial enzyme responsible for regulating the conversion of pyruvate to acetyl-CoA for use in the tricarboxylic acid cycle. PDH is regulated through phosphorylation and inactivation by PDH kinase (PDK) and dephosphorylation and activation by PDH phosphatase (PDP). The effect of endurance training on PDK in humans has been investigated; however, to date no study has examined the effect of endurance training on PDP in humans. Therefore, the purpose of this study was to examine differences in PDP activity and PDP1 protein content in human skeletal muscle across a range of muscle aerobic capacities. This association is important as higher PDP activity and protein content will allow for increased activation of PDH, and carbohydrate oxidation. The main findings of this study were that 1) PDP activity (r(2) = 0.399, P = 0.001) and PDP1 protein expression (r(2) = 0.153, P = 0.039) were positively correlated with citrate synthase (CS) activity as a marker for muscle aerobic capacity; 2) E1α (r(2) = 0.310, P = 0.002) and PDK2 protein (r(2) = 0.229, P =0.012) are positively correlated with muscle CS activity; and 3) although it is the most abundant isoform, PDP1 protein content only explained ∼ 18% of the variance in PDP activity (r(2) = 0.184, P = 0.033). In addition, PDP1 in combination with E1α explained ∼ 38% of the variance in PDP activity (r(2) = 0.383, P = 0.005), suggesting that there may be alternative regulatory mechanisms of this enzyme other than protein content. These data suggest that with higher muscle aerobic capacity (CS activity) there is a greater capacity for carbohydrate oxidation (E1α), in concert with higher potential for PDH activation (PDP activity). PMID:21596918

  2. Activity of soil dehydrogenases, urease, and acid and alkaline phosphatases in soil polluted with petroleum.

    PubMed

    Wyszkowska, Jadwiga; Wyszkowski, Mirosław

    2010-01-01

    This study was undertaken to (1) determine the effects of petroleum pollution on changes in the biochemical properties of soil and (2) demonstrate whether the application of compost, bentonite, and calcium oxide is likely to restore biological balance. Petroleum soil pollution at a dose ranging from 2.5 to 10 cm(3)/kg disturbed the biochemical balance as evidenced by inhibition of the activities of soil dehydrogenases (SDH), urease (URE), and acid phosphatase (ACP). The greatest change was noted in the activity of SDH, whereas the least change occurred in URE. Petroleum significantly increased the activity of soil alkaline phosphatase (ALP) in soil used for spring rape, whereas in soil used for oat harvest there was decreased ALP activity. The application of compost, bentonite, and calcium oxide to soil proved effective in mitigating the adverse effects of petroleum on the activities of soil enzymes. Soil enrichment with compost, bentonite, and calcium oxide was found to stimulate the activities of URE and ALP and inhibit the activity of ACP. The influence of bentonite and calcium oxide was greater than that of compost. Calcium oxide and, to a lesser extent, compost were found to increase the activity of SDH, whereas bentonite exerted the opposite effect, especially in the case of the main crop, spring rape. The activities of SDH, URE, and ACP were higher in soil used for rape than that for oats. In contrast the activity of ALP was higher in soil used for oats. Data thus indicate that compost and especially bentonite and calcium oxide exerted a positive effect on activities of some enzymes in soil polluted with petroleum. Application of neutralizing additives to soil restored soil biological balance by counteracting the negative influence of petroleum on activities of URE and ALP. PMID:20706945

  3. Activation of metabotropic glutamate receptor 5 in the amygdala modulates pain-like behavior.

    PubMed

    Kolber, Benedict J; Montana, Michael C; Carrasquillo, Yarimar; Xu, Jian; Heinemann, Stephen F; Muglia, Louis J; Gereau, Robert W

    2010-06-16

    The central nucleus of the amygdala (CeA) has been identified as a site of nociceptive processing important for sensitization induced by peripheral injury. However, the cellular signaling components underlying this function remain unknown. Here, we identify metabotropic glutamate receptor 5 (mGluR5) as an integral component of nociceptive processing in the CeA. Pharmacological activation of mGluRs with (R,S)-3,5-dihydroxyphenylglycine (DHPG) in the CeA of mice is sufficient to induce peripheral hypersensitivity in the absence of injury. DHPG-induced peripheral hypersensitivity is reduced via pharmacological blockade of mGluR5 or genetic disruption of mGluR5. Furthermore, pharmacological blockade or conditional deletion of mGluR5 in the CeA abrogates inflammation-induced hypersensitivity, demonstrating the necessity of mGluR5 in CeA-mediated pain modulation. Moreover, we demonstrate that phosphorylation of extracellular-signal regulated kinase 1/2 (ERK1/2) is downstream of mGluR5 activation in the CeA and is necessary for the full expression of peripheral inflammation-induced behavioral sensitization. Finally, we present evidence of right hemispheric lateralization of mGluR5 modulation of amygdalar nociceptive processing. We demonstrate that unilateral pharmacological activation of mGluR5 in the CeA produces distinct behavioral responses depending on whether the right or left amygdala is injected. We also demonstrate significantly higher levels of mGluR5 expression in the right amygdala compared with the left under baseline conditions, suggesting a potential mechanism for right hemispheric lateralization of amygdala function in pain processing. Together, these results establish an integral role for mGluR5 and ERK1/2 in nociceptive processing in the CeA. PMID:20554871

  4. 2-Methylcitric acid impairs glutamate metabolism and induces permeability transition in brain mitochondria.

    PubMed

    Amaral, Alexandre Umpierrez; Cecatto, Cristiane; Castilho, Roger Frigério; Wajner, Moacir

    2016-04-01

    Accumulation of 2-methylcitric acid (2MCA) is observed in methylmalonic and propionic acidemias, which are clinically characterized by severe neurological symptoms. The exact pathogenetic mechanisms of brain abnormalities in these diseases are poorly established and very little has been reported on the role of 2MCA. In the present work we found that 2MCA markedly inhibited ADP-stimulated and uncoupled respiration in mitochondria supported by glutamate, with a less significant inhibition in pyruvate plus malate respiring mitochondria. However, no alterations occurred when α-ketoglutarate or succinate was used as respiratory substrates, suggesting a defect on glutamate oxidative metabolism. It was also observed that 2MCA decreased ATP formation in glutamate plus malate or pyruvate plus malate-supported mitochondria. Furthermore, 2MCA inhibited glutamate dehydrogenase activity at concentrations as low as 0.5 mM. Kinetic studies revealed that this inhibitory effect was competitive in relation to glutamate. In contrast, assays of osmotic swelling in non-respiring mitochondria suggested that 2MCA did not significantly impair mitochondrial glutamate transport. Finally, 2MCA provoked a significant decrease in mitochondrial membrane potential and induced swelling in Ca(2+)-loaded mitochondria supported by different substrates. These effects were totally prevented by cyclosporine A plus ADP or ruthenium red, indicating induction of mitochondrial permeability transition. Taken together, our data strongly indicate that 2MCA behaves as a potent inhibitor of glutamate oxidation by inhibiting glutamate dehydrogenase activity and as a permeability transition inducer, disturbing mitochondrial energy homeostasis. We presume that 2MCA-induced mitochondrial deleterious effects may contribute to the pathogenesis of brain damage in patients affected by methylmalonic and propionic acidemias. We propose that brain glutamate oxidation is disturbed by 2-methylcitric acid (2MCA), which

  5. 2-Methylcitric acid impairs glutamate metabolism and induces permeability transition in brain mitochondria.

    PubMed

    Amaral, Alexandre Umpierrez; Cecatto, Cristiane; Castilho, Roger Frigério; Wajner, Moacir

    2016-04-01

    Accumulation of 2-methylcitric acid (2MCA) is observed in methylmalonic and propionic acidemias, which are clinically characterized by severe neurological symptoms. The exact pathogenetic mechanisms of brain abnormalities in these diseases are poorly established and very little has been reported on the role of 2MCA. In the present work we found that 2MCA markedly inhibited ADP-stimulated and uncoupled respiration in mitochondria supported by glutamate, with a less significant inhibition in pyruvate plus malate respiring mitochondria. However, no alterations occurred when α-ketoglutarate or succinate was used as respiratory substrates, suggesting a defect on glutamate oxidative metabolism. It was also observed that 2MCA decreased ATP formation in glutamate plus malate or pyruvate plus malate-supported mitochondria. Furthermore, 2MCA inhibited glutamate dehydrogenase activity at concentrations as low as 0.5 mM. Kinetic studies revealed that this inhibitory effect was competitive in relation to glutamate. In contrast, assays of osmotic swelling in non-respiring mitochondria suggested that 2MCA did not significantly impair mitochondrial glutamate transport. Finally, 2MCA provoked a significant decrease in mitochondrial membrane potential and induced swelling in Ca(2+)-loaded mitochondria supported by different substrates. These effects were totally prevented by cyclosporine A plus ADP or ruthenium red, indicating induction of mitochondrial permeability transition. Taken together, our data strongly indicate that 2MCA behaves as a potent inhibitor of glutamate oxidation by inhibiting glutamate dehydrogenase activity and as a permeability transition inducer, disturbing mitochondrial energy homeostasis. We presume that 2MCA-induced mitochondrial deleterious effects may contribute to the pathogenesis of brain damage in patients affected by methylmalonic and propionic acidemias. We propose that brain glutamate oxidation is disturbed by 2-methylcitric acid (2MCA), which

  6. Glutamate signalling in roots.

    PubMed

    Forde, Brian G

    2014-03-01

    As a signalling molecule, glutamate is best known for its role as a fast excitatory neurotransmitter in the mammalian nervous system, a role that requires the activity of a family of ionotropic glutamate receptors (iGluRs). The unexpected discovery in 1998 that Arabidopsis thaliana L. possesses a family of iGluR-related (GLR) genes laid the foundations for an assessment of glutamate's potential role as a signalling molecule in plants that is still in progress. Recent advances in elucidating the function of Arabidopsis GLR receptors has revealed similarities with iGluRs in their channel properties, but marked differences in their ligand specificities. The ability of plant GLR receptors to act as amino-acid-gated Ca(2+) channels with a broad agonist profile, combined with their expression throughout the plant, makes them strong candidates for a multiplicity of amino acid signalling roles. Although root growth is inhibited in the presence of a number of amino acids, only glutamate elicits a specific sequence of changes in growth, root tip morphology, and root branching. The recent finding that the MEKK1 gene is a positive regulator of glutamate sensitivity at the root tip has provided genetic evidence for the existence in plants of a glutamate signalling pathway analogous to those found in animals. This short review will discuss the most recent advances in understanding glutamate signalling in roots, considering them in the context of previous work in plants and animals.

  7. Alcohol dehydrogenase activity in Lactococcus chungangensis: application in cream cheese to moderate alcohol uptake.

    PubMed

    Konkit, Maytiya; Choi, Woo Jin; Kim, Wonyong

    2015-09-01

    Many human gastrointestinal facultative anaerobic and aerobic bacteria possess alcohol dehydrogenase (ADH) activity and are therefore capable of oxidizing ethanol to acetaldehyde. However, the ADH activity of Lactococcus spp., except Lactococcus lactis ssp. lactis, has not been widely determined, though they play an important role as the starter for most cheesemaking technologies. Cheese is a functional food recognized as an aid to digestion. In the current study, the ADH activity of Lactococcus chungangensis CAU 28(T) and 11 reference strains from the genus Lactococcus was determined. Only 5 strains, 3 of dairy origin, L. lactis ssp. lactis KCTC 3769(T), L. lactis ssp. cremoris KCCM 40699(T), and Lactococcus raffinolactis DSM 20443(T), and 2 of nondairy origin, Lactococcus fujiensis NJ317(T) and Lactococcus chungangensis CAU 28(T) KCTC 13185(T), showed ADH activity and possessed the ADH gene. All these strains were capable of making cheese, but the highest level of ADH activity was found in L. chungangensis, with 45.9nmol/min per gram in tryptic soy broth and 65.8nmol/min per gram in cream cheese. The extent that consumption of cheese, following imbibing alcohol, reduced alcohol uptake was observed by following the level of alcohol in the serum of mice. The results show a potential novel benefit of cheese as a dairy functional food.

  8. Expression of 20 alpha-hydroxysteroid dehydrogenase activity in human lymphoid and non lymphoid cells.

    PubMed Central

    Carbone, A; Piantelli, M; Musiani, P; Larocca, L M; Revoltella, R P; Ranelletti, F O

    1986-01-01

    Expression of 20-alpha-hydroxysteroid dehydrogenase (20 alpha-SDH), a putative T cell marker in the murine system, has been examined in human haematopoietic cells. Higher levels of enzymatic activity were expressed by human peripheral blood mononuclear cells (PBMC) in comparison with thymocytes. When PBMC were fractionated into T and non T cell subsets, the greatest values of 20 alpha-SDH activity were associated with the non T cell population. Cells from bone marrow exhibited lower levels of 20 alpha-SDH than PBMC and thymocytes. Both myeloid and lymphoid leukaemic cells expressed significant 20 alpha-SDH activity. In addition our data in U-937 and CM-S human cell lines indicate that cells belonging to the myelomonocytic precursor compartment constitutively expressed 20 alpha-SDH activity. Furthermore, this activity was less expressed when these cells were induced to monocyte-macrophage differentiation. In conclusion, our data in the human system indicate that 20 alpha-SDH should not be viewed as a lymphoid lineage-restricted marker enzyme. PMID:3485485

  9. Recipient pretransplant inosine monophosphate dehydrogenase activity in nonmyeloablative hematopoietic cell transplantation.

    PubMed

    Bemer, Meagan J; Risler, Linda J; Phillips, Brian R; Wang, Joanne; Storer, Barry E; Sandmaier, Brenda M; Duan, Haichuan; Raccor, Brianne S; Boeckh, Michael J; McCune, Jeannine S

    2014-10-01

    Mycophenolic acid, the active metabolite of mycophenolate mofetil (MMF), inhibits inosine monophosphate dehydrogenase (IMPDH) activity. IMPDH is the rate-limiting enzyme involved in de novo synthesis of guanosine nucleotides and catalyzes the oxidation of inosine 5'-monophosphate to xanthosine 5'-monophosphate (XMP). We developed a highly sensitive liquid chromatography-mass spectrometry method to quantitate XMP concentrations in peripheral blood mononuclear cells (PMNCs) isolated from the recipient pretransplant and used this method to determine IMPDH activity in 86 nonmyeloablative allogeneic hematopoietic cell transplantation (HCT) patients. The incubation procedure and analytical method yielded acceptable within-sample and within-individual variability. Considerable between-individual variability was observed (12.2-fold). Low recipient pretransplant IMPDH activity was associated with increased day +28 donor T cell chimerism, more acute graft-versus-host disease (GVHD), lower neutrophil nadirs, and more cytomegalovirus reactivation but not with chronic GVHD, relapse, nonrelapse mortality, or overall mortality. We conclude that quantitation of the recipient's pretransplant IMPDH activity in PMNC lysate could provide a useful biomarker to evaluate a recipient's sensitivity to MMF. Further trials should be conducted to confirm our findings and to optimize postgrafting immunosuppression in nonmyeloablative HCT recipients.

  10. PHARMACOKINETIC AND PHARMACODYNAMIC ANALYSIS OF INOSINE MONOPHOSPHATE DEHYDROGENASE (IMPDH) ACTIVITY IN MMF-TREATED HCT RECIPIENTS

    PubMed Central

    Li, Hong; Mager, Donald E.; Sandmaier, Brenda M.; Storer, Barry E.; Boeckh, Michael J.; Bemer, Meagan J.; Phillips, Brian R.; Risler, Linda J.; McCune, Jeannine S.

    2014-01-01

    A novel approach to personalizing postgrafting immunosuppression in hematopoietic cell transplant (HCT) recipients is evaluating inosine monophosphate dehydrogenase (IMPDH) activity as a drug-specific biomarker of mycophenolic acid (MPA)-induced immunosuppression. This prospective study evaluated total MPA, unbound MPA, and total MPA glucuronide plasma concentrations and IMPDH activity in peripheral blood mononuclear cells (PMNC) at five time points after the morning dose of oral mycophenolate mofetil (MMF) on day +21 in 56 nonmyeloablative HCT recipients. Substantial interpatient variability in the pharmacokinetics and pharmacodynamics was observed and accurately characterized by the population pharmacokinetic/dynamic model. IMPDH activity decreased with increasing MPA plasma concentration, with maximum inhibition coinciding with maximum MPA concentration in most patients. The overall relationship between MPA concentration and IMPDH activity was described by a direct inhibitory Emax model with an IC50 = 3.23 mg/L total MPA and 57.3 ng/mL unbound MPA. The day +21 IMPDH area under the effect curve (AUEC) was associated with cytomegalovirus reactivation, non-relapse mortality, and overall mortality. In conclusion, a pharmacokinetic/dynamic model was developed that relates plasma MPA concentrations with PMNC IMPDH activity after an MMF dose in HCT recipients. Future studies should validate this model and confirm that day +21 IMPDH AUEC is a predictive biomarker. PMID:24727337

  11. Nicotine promotes Streptococcus mutans extracellular polysaccharide synthesis, cell aggregation and overall lactate dehydrogenase activity.

    PubMed

    Huang, R; Li, M; Gregory, R L

    2015-08-01

    Several epidemiology studies have reported a positive relationship between smoking and dental caries. Nicotine, an alkaloid component of tobacco, has been demonstrated to stimulate biofilm formation and metabolic activity of Streptococcus mutans, one of the most important pathogens of dental caries. The first aim of the present study was to explore the possible mechanisms leading to increased biofilm by nicotine treatment from three aspects, extracellular polysaccharides (EPS) synthesis, glucosyltransferase (Gtf) synthesis and glucan-binding protein (Gbp) synthesis at the mRNA and protein levels. The second aim was to investigate how nicotine affects S. mutans virulence, particular in lactate dehydrogenase (LDH) activity. Confocal laser scanning microscopy results demonstrated that both biofilm bacterial cell numbers and EPS were increased by nicotine. Gtf and GbpA protein expression of S. mutans planktonic cells were upregulated while GbpB protein expression of biofilm cells were downregulated by nicotine. The mRNA expression trends of those genes were mostly consistent with results on protein level but not statistically significant, and gtfD and gbpD of biofilm cells were inhibited. Nicotine was not directly involved in S. mutans LDH activity. However, since it increases the total number of bacterial cells in biofilm, the overall LDH activity of S. mutans biofilm is increased. In conclusion, nicotine stimulates S. mutans planktonic cell Gtf and Gbp expression. This leads to more planktonic cells attaching to the dental biofilm. Increased cell numbers within biofilm results in higher overall LDH activity. This contributes to caries development in smokers.

  12. Cytochrome b5 augments 3β-hydroxysteroid dehydrogenase/Δ5-Δ4 isomerase activity.

    PubMed

    Goosen, Pierre; Storbeck, Karl-Heinz; Swart, Amanda C; Conradie, Riaan; Swart, Pieter

    2011-11-01

    During adrenal steroidogenesis the competition between 3β-hydroxysteroid dehydrogenase/Δ(5)-Δ(4) isomerase (3βHSD) and cytochrome P450 17α-hydroxylase/17,20 lyase (CYP17A1) for Δ(5) steroid intermediates greatly influences steroidogenic output. Cytochrome-b(5) (Cyt-b(5)), a small electron transfer hemoprotein, known to augment the lyase activity of CYP17A1, has been shown to alter the steroidogenic outcome of this competition. In this study, the influence of Cyt-b(5) on 3βHSD activity was investigated. In COS-1 cells, Cyt-b(5) was shown to significantly increase the activity of both caprine and ovine 3βHSD towards pregnenolone, 17-OH pregnenolone and dehydroepiandrosterone in a substrate and species specific manner. Furthermore, kinetic studies revealed Cyt-b(5) to have no influence on the K(m) values while significantly increasing the V(max) values of ovine 3βHSD for all its respective substrates. In addition, the activity of ovine 3βHSD in microsomal preparations was significantly influenced by the addition of either purified Cyt-b(5) or anti-Cyt-b(5) IgG. The results presented in this study indicate that Cyt-b(5) augments 3βHSD activity and represents the first documentation of such augmentation in any species. PMID:21930205

  13. Influence of spaceflight on succinate dehydrogenase activity and soma size of rat ventral horn neurons

    NASA Technical Reports Server (NTRS)

    Ishihara, A.; Ohira, Y.; Roy, R. R.; Nagaoka, S.; Sekiguchi, C.; Hinds, W. E.; Edgerton, V. R.

    1996-01-01

    Succinate dehydrogenase (SDH) activities and soma cross-sectional areas (CSA) of neurons in the dorsolateral region of the ventral horn at the L5 segmental level of the spinal cord in the rat were determined after 14 days of spaceflight and after 9 days of recovery on earth. The results were compared to those in age-matched ground-based control rats. Spinal cords were quick-frozen, and the SDH activity and CSA of a sample of neurons with a visible nucleus were determined using a digitizer and a computer-assisted image analysis system. An inverse relationship between CSA and SDH activity of neurons was observed in all groups of rats. No change in mean CSA or mean SDH activity or in the size distribution of neurons was observed following spaceflight or recovery. However, there was a selective decrease in the SDH activity of neurons with soma CSA between 500 and 800 microns2 in the flight rats, and this effect persisted for at least 9 days following return to 1 g. It remains to be determined whether the selected population of motoneurons or the specific motor pools affected by spaceflight may be restricted to specific muscles.

  14. Enzymatic Activity Analysis and Catalytic Essential Residues Identification of Brucella abortus Malate Dehydrogenase

    PubMed Central

    Han, Xiangan; Tong, Yongliang; Tian, Mingxing; Zhang, Yuxi; Sun, Xiaoqing; Wang, Shaohui; Qiu, Xusheng; Ding, Chan; Yu, Shengqing

    2014-01-01

    Malate dehydrogenase (MDH) plays important metabolic roles in bacteria. In this study, the recombinant MDH protein (His-MDH) of Brucella abortus was purified and its ability to catalyze the conversion of oxaloacetate (OAA) to L-malate (hereon referred to as MDH activity) was analyzed. Michaelis Constant (Km) and Maximum Reaction Velocity (Vmax) of the reaction were determined to be 6.45 × 10−3 M and 0.87 mM L−1 min−1, respectively. In vitro studies showed that His-MDH exhibited maximal MDH activity in pH 6.0 reaction buffer at 40°C. The enzymatic activity was 100%, 60%, and 40% inhibited by Cu2+, Zn2+, and Pb2+, respectively. In addition, six amino acids in the MDH were mutated to investigate their roles in the enzymatic activity. The results showed that the substitutions of amino acids Arg 89, Asp 149, Arg 152, His 176, or Thr 231 almost abolished the activity of His-MDH. The present study will help to understand MDH's roles in B. abortus metabolism. PMID:24895685

  15. Recipient pretransplant inosine monophosphate dehydrogenase activity in nonmyeloablative hematopoietic cell transplantation.

    PubMed

    Bemer, Meagan J; Risler, Linda J; Phillips, Brian R; Wang, Joanne; Storer, Barry E; Sandmaier, Brenda M; Duan, Haichuan; Raccor, Brianne S; Boeckh, Michael J; McCune, Jeannine S

    2014-10-01

    Mycophenolic acid, the active metabolite of mycophenolate mofetil (MMF), inhibits inosine monophosphate dehydrogenase (IMPDH) activity. IMPDH is the rate-limiting enzyme involved in de novo synthesis of guanosine nucleotides and catalyzes the oxidation of inosine 5'-monophosphate to xanthosine 5'-monophosphate (XMP). We developed a highly sensitive liquid chromatography-mass spectrometry method to quantitate XMP concentrations in peripheral blood mononuclear cells (PMNCs) isolated from the recipient pretransplant and used this method to determine IMPDH activity in 86 nonmyeloablative allogeneic hematopoietic cell transplantation (HCT) patients. The incubation procedure and analytical method yielded acceptable within-sample and within-individual variability. Considerable between-individual variability was observed (12.2-fold). Low recipient pretransplant IMPDH activity was associated with increased day +28 donor T cell chimerism, more acute graft-versus-host disease (GVHD), lower neutrophil nadirs, and more cytomegalovirus reactivation but not with chronic GVHD, relapse, nonrelapse mortality, or overall mortality. We conclude that quantitation of the recipient's pretransplant IMPDH activity in PMNC lysate could provide a useful biomarker to evaluate a recipient's sensitivity to MMF. Further trials should be conducted to confirm our findings and to optimize postgrafting immunosuppression in nonmyeloablative HCT recipients. PMID:24923537

  16. Altered acetylation and succinylation profiles in Corynebacterium glutamicum in response to conditions inducing glutamate overproduction.

    PubMed

    Mizuno, Yuta; Nagano-Shoji, Megumi; Kubo, Shosei; Kawamura, Yumi; Yoshida, Ayako; Kawasaki, Hisashi; Nishiyama, Makoto; Yoshida, Minoru; Kosono, Saori

    2016-02-01

    The bacterium Corynebacterium glutamicum is utilized during industrial fermentation to produce amino acids such as L-glutamate. During L-glutamate fermentation, C. glutamicum changes the flux of central carbon metabolism to favor L-glutamate production, but the molecular mechanisms that explain these flux changes remain largely unknown. Here, we found that the profiles of two major lysine acyl modifications were significantly altered upon glutamate overproduction in C. glutamicum; acetylation decreased, whereas succinylation increased. A label-free semi-quantitative proteomic analysis identified 604 acetylated proteins with 1328 unique acetylation sites and 288 succinylated proteins with 651 unique succinylation sites. Acetylation and succinylation targeted enzymes in central carbon metabolic pathways that are directly related to glutamate production, including the 2-oxoglutarate dehydrogenase complex (ODHC), a key enzyme regulating glutamate overproduction. Structural mapping revealed that several critical lysine residues in the ODHC components were susceptible to acetylation and succinylation. Furthermore, induction of glutamate production was associated with changes in the extent of acetylation and succinylation of lysine, suggesting that these modifications may affect the activity of enzymes involved in glutamate production. Deletion of phosphotransacetylase decreased the extent of protein acetylation in nonproducing condition, suggesting that acetyl phosphate-dependent acetylation is active in C. glutamicum. However, no effect was observed on the profiles of acetylation and succinylation in glutamate-producing condition upon disruption of acetyl phosphate metabolism or deacetylase homologs. It was considered likely that the reduced acetylation in glutamate-producing condition may reflect metabolic states where the flux through acid-producing pathways is very low, and substrates for acetylation do not accumulate in the cell. Succinylation would occur more

  17. Periplasmic nitrate reductase and formate dehydrogenase: similar molecular architectures with very different enzymatic activities.

    PubMed

    Cerqueira, Nuno M F S A; Gonzalez, Pablo J; Fernandes, Pedro A; Moura, José J G; Ramos, Maria João

    2015-11-17

    It is remarkable how nature has been able to construct enzymes that, despite sharing many similarities, have simple but key differences that tune them for completely different functions in living cells. Periplasmic nitrate reductase (Nap) and formate dehydrogenase (Fdh) from the DMSOr family are representative examples of this. Both enzymes share almost identical three-dimensional protein foldings and active sites, in terms of coordination number, geometry and nature of the ligands. The substrates of both enzymes (nitrate and formate) are polyatomic anions that also share similar charge and stereochemistry. In terms of the catalytic mechanism, both enzymes have a common activation mechanism (the sulfur-shift mechanism) that ensures a constant coordination number around the metal ion during the catalytic cycle. In spite of these similarities, they catalyze very different reactions: Nap abstracts an oxygen atom from nitrate releasing nitrite, whereas FdH catalyzes a hydrogen atom transfer from formate and releases carbon dioxide. In this Account, a critical analysis of structure, function, and catalytic mechanism of the molybdenum enzymes periplasmic nitrate reductase (Nap) and formate dehydrogenase (Fdh) is presented. We conclude that the main structural driving force that dictates the type of reaction, catalyzed by each enzyme, is a key difference on one active site residue that is located in the top region of the active sites of both enzymes. In both enzymes, the active site is centered on the metal ion of the cofactor (Mo in Nap and Mo or W in Fdh) that is coordinated by four sulfur atoms from two pyranopterin guanosine dinucleotide (PGD) molecules and by a sulfido. However, while in Nap there is a Cys directly coordinated to the Mo ion, in FdH there is a SeCys instead. In Fdh there is also an important His that interacts very closely with the SeCys, whereas in Nap the same position is occupied by a Met. The role of Cys in Nap and SeCys in FdH is similar in both

  18. Effect of feeding and of DDT on the activity of hepatic glucose 6- phosphate dehydrogenase in two salmonids

    USGS Publications Warehouse

    Buhler, Donald R.; Benville, P.

    1969-01-01

    The specific activity of liver glucose 6-phosphate dehydrogenase in yearling rainbow trout remained unchanged when the fish were starved for periods as long as 8 weeks and when starved animals were fed diets of various compositions. Injection of insulin concurrently with refeeding also failed to alter the specific activity of the enzyme in trout. The absence of a dietary or insulin influence on the teleost enzyme system is to be contrasted with studies in mammals in which the activity of hepatic glucose 6-P dehydrogenase was markedly stimulated after refeeding starved animals or injection of insulin.Ingestion of the pesticide DDT by juvenile coho salmon or adult rainbow trout also had no effect on the specific activity of liver glucose 6-P dehydrogenase and DDT failed to inhibit the rainbow trout enzyme in vitro. These results also differ considerably from those found in higher animals.These results suggest that the glucose 6-P dehydrogenase enzyme in teleosts may be under a different type of regulatory control from that found in mammals.

  19. Effects of low molecular-weight organic acids and dehydrogenase activity in rhizosphere sediments of mangrove plants on phytoremediation of polycyclic aromatic hydrocarbons.

    PubMed

    Wang, Yuanyuan; Fang, Ling; Lin, Li; Luan, Tiangang; Tam, Nora F Y

    2014-03-01

    This work evaluated the roles of the low-molecular-weight organic acids (LMWOAs) from root exudates and the dehydrogenase activity in the rhizosphere sediments of three mangrove plant species on the removal of mixed PAHs. The results showed that the concentrations of LMWOAs and dehydrogenase activity changed species-specifically with the levels of PAH contamination. In all plant species, the concentration of citric acid was the highest, followed by succinic acid. For these acids, succinic acid was positively related to the removal of all the PAHs except Chr. Positive correlations were also found between the removal percentages of 4-and 5-ring PAHs and all LMWOAs, except citric acid. LMWOAs enhanced dehydrogenase activity, which positively related to PAH removal percentages. These findings suggested that LMWOAs and dehydrogenase activity promoted the removal of PAHs. Among three mangrove plants, Bruguiera gymnorrhiza, the plant with the highest root biomass, dehydrogenase activity and concentrations of LMWOAs, was most efficient in removing PAHs. PMID:24287262

  20. Effects of low molecular-weight organic acids and dehydrogenase activity in rhizosphere sediments of mangrove plants on phytoremediation of polycyclic aromatic hydrocarbons.

    PubMed

    Wang, Yuanyuan; Fang, Ling; Lin, Li; Luan, Tiangang; Tam, Nora F Y

    2014-03-01

    This work evaluated the roles of the low-molecular-weight organic acids (LMWOAs) from root exudates and the dehydrogenase activity in the rhizosphere sediments of three mangrove plant species on the removal of mixed PAHs. The results showed that the concentrations of LMWOAs and dehydrogenase activity changed species-specifically with the levels of PAH contamination. In all plant species, the concentration of citric acid was the highest, followed by succinic acid. For these acids, succinic acid was positively related to the removal of all the PAHs except Chr. Positive correlations were also found between the removal percentages of 4-and 5-ring PAHs and all LMWOAs, except citric acid. LMWOAs enhanced dehydrogenase activity, which positively related to PAH removal percentages. These findings suggested that LMWOAs and dehydrogenase activity promoted the removal of PAHs. Among three mangrove plants, Bruguiera gymnorrhiza, the plant with the highest root biomass, dehydrogenase activity and concentrations of LMWOAs, was most efficient in removing PAHs.

  1. Mitochondrial transporters involved in oleic acid utilization and glutamate metabolism in yeast.

    PubMed

    Trotter, Pamela J; Adamson, Amy L; Ghrist, Angela C; Rowe, Lindsay; Scott, Lori R; Sherman, Matthew P; Stites, Nicole C; Sun, Yue; Tawiah-Boateng, Mary Anne; Tibbetts, Anne S; Wadington, Megan C; West, Aaron C

    2005-10-01

    Utilization of fatty acids such as oleic acid as sole carbon source by the yeast Saccharomyces cerevisiae requires coordinated function of peroxisomes, where the fatty acids are degraded, and the mitochondria, where oxidation is completed. We identified two mitochondrial oxodicarboxylate transporters, Odc1p and Odc2p, as important in efficient utilization of oleic acid in yeast [Tibbetts et al., Arch. Biochem. Biophys. 406 (2002) 96-104]. Yet, the growth phenotype of odc1delta odc2delta strains indicated that additional transporter(s) were also involved. Here, we identify two putative transporter genes, YMC1 and YMC2, as able to suppress the odc1delta odc2delta growth phenotype. The mRNA levels for both are elevated in the presence of glycerol or oleic acid, as compared to glucose. Ymc1p and Ymc2p are localized to the mitochondria in oleic acid-grown cells. Deletion of all four transporters (quad mutant) prevents growth on oleic acid as sole carbon source, while growth on acetate is retained. It is known that the glutamate-sensitive retrograde signaling pathway is important for upregulation of peroxisomal function in response to oleic acid and the oxodicarboxylate alpha-ketoglutarate is transported out of the mitochondria for synthesis of glutamate. So, citric acid cycle function and glutamate synthesis were examined in transporter mutants. The quad mutant has significantly decreased citrate synthase activity and whole cell alpha-ketoglutarate levels, while isocitrate dehydrogenase activity is unaffected and glutamate dehydrogenase activity is increased 10-fold. Strains carrying only two or three transporter deletions exhibit intermediate affects. 13C NMR metabolic enrichment experiments confirm a defect in glutamate biosynthesis in the quad mutant and, in double and triple mutants, suggest increased cycling of the glutamate backbone in the mitochondria before export. Taken together these studies indicate that these four transporters have overlapping activity, and

  2. Pyruvate dehydrogenase activity and quantity decreases after coronary artery bypass grafting: a prospective observational study

    PubMed Central

    Andersen, Lars W.; Liu, Xiaowen; Peng, Teng J.; Giberson, Tyler A.; Khabbaz, Kamal R.; Donnino, Michael W.

    2014-01-01

    Introduction Pyruvate dehydrogenase (PDH) is a key gatekeeper enzyme in aerobic metabolism. The main purpose of this study was to determine if PDH activity is affected by major stress in the form of coronary artery bypass grafting (CABG) which has previously been used as a model of critical illness. Methods We conducted a prospective, observational study of patients undergoing CABG at an urban, tertiary care hospital. We included adult patients undergoing CABG with or without concomitant valve surgery. Measurements of PDH activity and quantity and thiamine were obtained prior to surgery, at the completion of surgery, and 6 hours post-surgery. Results Fourteen patients were enrolled (age: 67 ± 10 years, 21 % female). Study subjects had a mean 41.7 % (SD: 27.7) reduction in PDH activity after surgery and a mean 32.0% (SD: 31.4) reduction 6 hours after surgery (p < 0.001). Eight patients were thiamine deficient (≤ 7 nmol/L) after surgery compared to none prior to surgery (p = 0.002). Thiamine level was a significantly associated with PDH quantity at all time points (p = 0.01). Post-surgery lactate levels were inversely correlated with post-surgery thiamine levels (r = −0.58 and p = 0.04). Conclusion The stress of major surgery causes decreased PDH activity and quantity, and depletion of thiamine levels. PMID:25526377

  3. Two mitochondrial alcohol dehydrogenase activities of Kluyveromyces lactis are differently expressed during respiration and fermentation.

    PubMed

    Saliola, M; Falcone, C

    1995-12-20

    The lactose-utilizing yeast Kluyveromyces lactis is an essentially aerobic organism in which both respiration and fermentation can coexist depending on the sugar concentration. Despite a low fermentative capacity as compared to Saccharomyces cerevisiae, four structural genes encoding alcohol dehydrogenase (ADH) activities are present in this yeast. Two of these activities, namely K1ADH III and K1ADH IV, are located within mitochondria and their presence is dependent on the carbon sources in the medium. In this paper we demonstrate by transcription and activity analysis that KlADH3 is expressed in the presence of low glucose concentrations and in the presence of respiratory carbon sources other than ethanol. Indeed ethanol acts as a strong repressor of this gene. On the other hand, KlADH4 is induced by the presence of ethanol and not by other respiratory carbon sources. We also demonstrate that the presence of KLADH III and KLADH IV in K. lactis cells is dependent on glucose concentration, glucose uptake and the amount of ethanol produced. As a consequence, these activities can be used as markers for the onset of respiratory and fermentative metabolism in this yeast.

  4. Identification of Active Retinaldehyde Dehydrogenase Isoforms in the Postnatal Human Eye

    PubMed Central

    Harper, Angelica R.; Wiechmann, Allan F.; Moiseyev, Gennadiy; Ma, Jian-Xing; Summers, Jody A.

    2015-01-01

    Background/Objectives Retinaldehyde dehydrogenase 2 (RALDH2) has been implicated in regulating all-trans-retinoic acid (atRA) synthesis in response to visual signals in animal models of myopia. To explore the potential role of retinaldehyde dehydrogenase (RALDH) enzymes and atRA in human postnatal ocular growth, RALDH activity, along with the distribution of RALDH1, RALDH2, and RALDH3 in the postnatal eye was determined. Methodology Retina, retinal pigment epithelium (RPE), choroid, and sclera were isolated from donor human eyes. RALDH catalytic activity was measured in tissue homogenates using an in vitro atRA synthesis assay together with HPLC quantification of synthesized atRA. Homogenates were compared by western blotting for RALDH1, RALDH2, and RALDH3 protein. Immunohistochemistry was used to determine RALDH1 and RALDH2 localization in posterior fundal layers of the human eye. Principal Findings In the postnatal human eye, RALDH catalytic activity was detected in the choroid (6.84 ± 1.20 pmol/hr/ug), RPE (5.46 ± 1.18 pmol/hr/ug), and retina (4.21 ± 1.55 pmol/hr/ug), indicating the presence of active RALDH enzymes in these tissues. RALDH2 was most abundant in the choroid and RPE, in moderate abundance in the retina, and in relatively low abundance in sclera. RALDH1 was most abundant in the choroid, in moderate abundance in the sclera, and substantially reduced in the retina and RPE. RALDH3 was undetectable in human ocular fundal tissues. In the choroid, RALDH1 and RALDH2 localized to slender cells in the stroma, some of which were closely associated with blood vessels. Conclusions/Significance Results of this study demonstrated that: 1) Catalytically active RALDH is present in postnatal human retina, RPE, and choroid, 2) RALDH1 and RALDH2 isoforms are present in these ocular tissues, and 3) RALDH1 and RALDH2 are relatively abundant in the choroid and/or RPE. Taken together, these results suggest that RALDH1 and 2 may play a role in the regulation of

  5. Dengue Virus NS1 Protein Modulates Cellular Energy Metabolism by Increasing Glyceraldehyde-3-Phosphate Dehydrogenase Activity

    PubMed Central

    Allonso, Diego; Andrade, Iamara S.; Conde, Jonas N.; Coelho, Diego R.; Rocha, Daniele C. P.; da Silva, Manuela L.; Ventura, Gustavo T.

    2015-01-01

    ABSTRACT Dengue is one of the main public health concerns worldwide. Recent estimates indicate that over 390 million people are infected annually with the dengue virus (DENV), resulting in thousands of deaths. Among the DENV nonstructural proteins, the NS1 protein is the only one whose function during replication is still unknown. NS1 is a 46- to 55-kDa glycoprotein commonly found as both a membrane-associated homodimer and a soluble hexameric barrel-shaped lipoprotein. Despite its role in the pathogenic process, NS1 is essential for proper RNA accumulation and virus production. In the present study, we identified that glyceraldehyde-3-phosphate dehydrogenase (GAPDH) interacts with intracellular NS1. Molecular docking revealed that this interaction occurs through the hydrophobic protrusion of NS1 and the hydrophobic residues located at the opposite side of the catalytic site. Moreover, addition of purified recombinant NS1 enhanced the glycolytic activity of GAPDH in vitro. Interestingly, we observed that DENV infection promoted the relocalization of GAPDH to the perinuclear region, where NS1 is commonly found. Both DENV infection and expression of NS1 itself resulted in increased GAPDH activity. Our findings indicate that the NS1 protein acts to increase glycolytic flux and, consequently, energy production, which is consistent with the recent finding that DENV induces and requires glycolysis for proper replication. This is the first report to propose that NS1 is an important modulator of cellular energy metabolism. The data presented here provide new insights that may be useful for further drug design and the development of alternative antiviral therapies against DENV. IMPORTANCE Dengue represents a serious public health problem worldwide and is caused by infection with dengue virus (DENV). Estimates indicate that half of the global population is at risk of infection, with almost 400 million cases occurring per year. The NS1 glycoprotein is found in both the

  6. Lactate dehydrogenase activity in Bacteroides fragilis group strains with induced resistance to metronidazole.

    PubMed

    Presečki Stanko, Aleksandra; Sóki, Jozsef; Varda Brkić, Dijana; Plečko, Vanda

    2016-06-01

    The aims of this study were to induce in vitro metronidazole resistance in nim-negative Bacteroides fragilis group strains and to determine the lactate dehydrogenase (LDH) activity of the induced strains. A collection of B. fragilis group strains were identified by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF/MS). Minimum inhibitory concentrations (MICs) for metronidazole were determined by the agar dilution technique. The presence of nim genes was screened by PCR. A sample of 52 nim-negative metronidazole-susceptible strains were selected at random and were exposed to metronidazole in the resistance induction experiment. LDH activity was measured by spectrophotometry. Of the 52 selected strains, 12 (23.1%) acquired resistance to metronidazole. MICs ranged from 8mg/L to 96mg/L. Eight of the twelve induced strains displayed decreased LDH activity, whilst only one expressed a significant increase in LDH activity with LDH values of 49.1U/mg and 222.0U/mg, respectively. In conclusion, in vitro induction of metronidazole resistance could be selected in nim-negative B. fragilis group strains. A statistically significant decrease in LDH activity was in contrast to previous findings in which, underlying higher metronidazole MICs, an increase in LDH activity compensated for the decreased activity of pyruvate-ferredoxin oxidoreductase (PFOR). These findings could be explained if the induction caused only physiological and not genetic changes. We believe that genetic mutations in the B. fragilis strain that demonstrated an emergent increase in LDH activity were responsible for the increased activity. PMID:27436459

  7. ACTIVITIES OF AMMONIA ASSIMILATION ENZYMES AS INDICATORS OF THE RELATIVE SUPPLY OF NITROGEN SUBSTRATES FOR MARINE BACTERIOPLANKTON IN SUB-TROPICAL COASTAL WATER

    EPA Science Inventory

    The supply of nitrogen substrates available for bacterial production in seawater was determined using the activities of ammonia assimilation enzymes, glutamine synthetase (GS) and glutamate dehydrogenase (GDH). Expression of GS and GDH by bacteria in pure culture is generally ind...

  8. Physiological Regulation of Isocitrate Dehydrogenase and the Role of 2-Oxoglutarate in Prochlorococcus sp. Strain PCC 9511

    PubMed Central

    Diez, Jesús; Gómez-Baena, Guadalupe; Rangel-Zúñiga, Oriol Alberto; García-Fernández, José Manuel

    2014-01-01

    The enzyme isocitrate dehydrogenase (ICDH; EC 1.1.1.42) catalyzes the oxidative decarboxylation of isocitrate, to produce 2-oxoglutarate. The incompleteness of the tricarboxylic acids cycle in marine cyanobacteria confers a special importance to isocitrate dehydrogenase in the C/N balance, since 2-oxoglutarate can only be metabolized through the glutamine synthetase/glutamate synthase pathway. The physiological regulation of isocitrate dehydrogenase was studied in cultures of Prochlorococcus sp. strain PCC 9511, by measuring enzyme activity and concentration using the NADPH production assay and Western blotting, respectively. The enzyme activity showed little changes under nitrogen or phosphorus starvation, or upon addition of the inhibitors DCMU, DBMIB and MSX. Azaserine, an inhibitor of glutamate synthase, induced clear increases in the isocitrate dehydrogenase activity and icd gene expression after 24 h, and also in the 2-oxoglutarate concentration. Iron starvation had the most significant effect, inducing a complete loss of isocitrate dehydrogenase activity, possibly mediated by a process of oxidative inactivation, while its concentration was unaffected. Our results suggest that isocitrate dehydrogenase responds to changes in the intracellular concentration of 2-oxoglutarate and to the redox status of the cells in Prochlorococcus. PMID:25061751

  9. Glutamate transporters alterations in the reorganizing dentate gyrus are associated with progressive seizure activity in chronic epileptic rats.

    PubMed

    Gorter, Jan A; Van Vliet, Erwin A; Proper, Evelien A; De Graan, Pierre N E; Ghijsen, Wim E J M; Lopes Da Silva, Fernando H; Aronica, Eleonora

    2002-01-21

    The expression of glial and neuronal glutamate transporter proteins was investigated in the hippocampal region at different time points after electrically induced status epilepticus (SE) in the rat. This experimental rat model for mesial temporal lobe epilepsy is characterized by cell loss, gliosis, synaptic reorganization, and chronic seizures after a latent period. Despite extensive gliosis, immunocytochemistry revealed only an up-regulation of both glial transporters localized at the outer aspect of the inner molecular layer (iml) in chronic epileptic rats. The neuronal EAAC1 transporter was increased in many somata of individual CA1-3 neurons and granule cells that had survived after SE; this up-regulation was still present in the chronic epileptic phase. In contrast, a permanent decrease of EAAC1 immunoreactivity was observed in the iml of the dentate gyrus. This permanent decrease in EAAC1 expression, which was only observed in rats that experienced progressive spontaneous seizure activity, could lead to abnormal glutamate levels in the iml once new abnormal glutamatergic synaptic contacts are formed by means of sprouted mossy fibers. Considering the steady growth of reorganizing mossy fibers in the iml, the absence of a glutamate reuptake mechanism in this region could contribute to progression of spontaneous seizure activity, which occurs with a similar time course.

  10. Pyridoxine Supplementation Improves the Activity of Recombinant Glutamate Decarboxylase and the Enzymatic Production of Gama-Aminobutyric Acid

    PubMed Central

    Huang, Yan; Su, Lingqia; Wu, Jing

    2016-01-01

    Glutamate decarboxylase (GAD) catalyzes the irreversible decarboxylation of L-glutamate to the valuable food supplement γ-aminobutyric acid (GABA). In this study, GAD from Escherichia coli K12, a pyridoxal phosphate (PLP)-dependent enzyme, was overexpressed in E. coli. The GAD produced in media supplemented with 0.05 mM soluble vitamin B6 analog pyridoxine hydrochloride (GAD-V) activity was 154.8 U mL-1, 1.8-fold higher than that of GAD obtained without supplementation (GAD-C). Purified GAD-V exhibited increased activity (193.4 U mg-1, 1.5-fold higher than that of GAD-C), superior thermostability (2.8-fold greater than that of GAD-C), and higher kcat/Km (1.6-fold higher than that of GAD-C). Under optimal conditions in reactions mixtures lacking added PLP, crude GAD-V converted 500 g L-1 monosodium glutamate (MSG) to GABA with a yield of 100%, and 750 g L-1 MSG with a yield of 88.7%. These results establish the utility of pyridoxine supplementation and lay the foundation for large-scale enzymatic production of GABA. PMID:27438707

  11. Activation of presynaptic oxytocin receptors enhances glutamate release in the ventral hippocampus of prenatally restraint stressed rats.

    PubMed

    Mairesse, Jérôme; Gatta, Eleonora; Reynaert, Marie-Line; Marrocco, Jordan; Morley-Fletcher, Sara; Soichot, Marion; Deruyter, Lucie; Camp, Gilles Van; Bouwalerh, Hammou; Fagioli, Francesca; Pittaluga, Anna; Allorge, Delphine; Nicoletti, Ferdinando; Maccari, Stefania

    2015-12-01

    Oxytocin receptors are known to modulate synaptic transmission and network activity in the hippocampus, but their precise function has been only partially elucidated. Here, we have found that activation of presynaptic oxytocin receptor with the potent agonist, carbetocin, enhanced depolarization-evoked glutamate release in the ventral hippocampus with no effect on GABA release. This evidence paved the way for examining the effect of carbetocin treatment in "prenatally restraint stressed" (PRS) rats, i.e., the offspring of dams exposed to repeated episodes of restraint stress during pregnancy. Adult PRS rats exhibit an anxious/depressive-like phenotype associated with an abnormal glucocorticoid feedback regulation of the hypothalamus-pituitary-adrenal (HPA) axis, and, remarkably, with a reduced depolarization-evoked glutamate release in the ventral hippocampus. Chronic systemic treatment with carbetocin (1mg/kg, i.p., once a day for 2-3 weeks) in PRS rats corrected the defect in glutamate release, anxiety- and depressive-like behavior, and abnormalities in social behavior, in the HPA response to stress, and in the expression of stress-related genes in the hippocampus and amygdala. Of note, carbetocin treatment had no effect on these behavioral and neuroendocrine parameters in prenatally unstressed (control) rats, with the exception of a reduced expression of the oxytocin receptor gene in the amygdala. These findings disclose a novel function of oxytocin receptors in the hippocampus, and encourage the use of oxytocin receptor agonists in the treatment of stress-related psychiatric disorders in adult life.

  12. Pyridoxine Supplementation Improves the Activity of Recombinant Glutamate Decarboxylase and the Enzymatic Production of Gama-Aminobutyric Acid.

    PubMed

    Huang, Yan; Su, Lingqia; Wu, Jing

    2016-01-01

    Glutamate decarboxylase (GAD) catalyzes the irreversible decarboxylation of L-glutamate to the valuable food supplement γ-aminobutyric acid (GABA). In this study, GAD from Escherichia coli K12, a pyridoxal phosphate (PLP)-dependent enzyme, was overexpressed in E. coli. The GAD produced in media supplemented with 0.05 mM soluble vitamin B6 analog pyridoxine hydrochloride (GAD-V) activity was 154.8 U mL-1, 1.8-fold higher than that of GAD obtained without supplementation (GAD-C). Purified GAD-V exhibited increased activity (193.4 U mg-1, 1.5-fold higher than that of GAD-C), superior thermostability (2.8-fold greater than that of GAD-C), and higher kcat/Km (1.6-fold higher than that of GAD-C). Under optimal conditions in reactions mixtures lacking added PLP, crude GAD-V converted 500 g L-1 monosodium glutamate (MSG) to GABA with a yield of 100%, and 750 g L-1 MSG with a yield of 88.7%. These results establish the utility of pyridoxine supplementation and lay the foundation for large-scale enzymatic production of GABA. PMID:27438707

  13. II. Glutamine and glutamate.

    PubMed

    Tapiero, H; Mathé, G; Couvreur, P; Tew, K D

    2002-11-01

    plays a role in the formation and function of the cytoskeleton. Glutamine via glutamate is converted to alpha-ketoglutarate, an integral component of the citric acid cycle. It is a component of the antioxidant glutathione and of the polyglutamated folic acid. The cyclization of glutamate produces proline, an amino acid important for synthesis of collagen and connective tissue. Our aim here is to review on some amino acids with high functional priority such as glutamine and to define their effective activity in human health and pathologies.

  14. Activity and Conformation of Yeast Alcohol Dehydrogenase (YADH) Entrapped in Reverse Micelles.

    PubMed

    Das; Mozumdar; Maitra

    2000-10-15

    Yeast alcohol dehydrogenase (YADH) solubilized in reverse micelles of aerosol OT (i.e., AOT or sodium bis (2-ethyl hexyl) sulfosuccinate) in isooctane has been shown to be catalytically more active than that in aqueous buffer under optimum conditions of pH, temperature, and water content in reverse micelles. Studies of the secondary structure conformational changes of the enzyme in reverse micelles have been made from circular dichroism spectroscopy. It has been seen that the conformation of YADH in reverse micelles is extremely sensitive to pH, temperature, and water content. A comparison has been made between the catalytic activity of the enzyme and the alpha-helix content in the conformation and it has been observed that the enzyme is most active at the maximum alpha-helix content. While the beta-sheet content in the conformation of the entrapped enzyme was found to be dependent on the enzyme-micelle interface interaction, the alpha-helix and random coil conformations are governed by the degree of entrapment and the extent of rigidity provided by the micelle core to the enzyme structure. Copyright 2000 Academic Press.

  15. Aldosterone impairs vascular reactivity by decreasing glucose-6-phosphate dehydrogenase activity

    PubMed Central

    Leopold, Jane A.; Dam, Aamir; Maron, Bradley A.; Scribner, Anne W.; Liao, Ronglih; Handy, Diane E.; Stanton, Robert C.; Pitt, Bertram; Loscalzo, Joseph

    2013-01-01

    Hyperaldosteronism is associated with impaired vascular reactivity; however, the mechanism by which aldosterone promotes endothelial dysfunction remains unknown. Glucose-6-phosphate dehydrogenase (G6pd), the principal source of Nadph, modulates vascular function by limiting oxidant stress to preserve bioavailable nitric oxide (NO•). In these studies, we show that aldosterone (10−9-10−7 mol/l) decreases endothelial G6pd expression and activity in vitro resulting in increased oxidant stress and decreased cGMP levels similar to what is observed in G6pd-deficient cells. Aldosterone decreases G6pd expression by protein kinase A activation to increase expression of Crem, which interferes with Creb binding to the G6pd promoter. In vivo, infusion of aldosterone decreases vascular G6pd expression and impairs vascular reactivity. These effects are abrogated by spironolactone or vascular gene transfer of G6pd. These studies demonstrate that aldosterone induces a G6pd-deficient phenotype to impair endothelial function; aldosterone antagonism or gene transfer of G6pd improves vascular reactivity by restoring G6pd activity. PMID:17273168

  16. E4F1-mediated control of pyruvate dehydrogenase activity is essential for skin homeostasis.

    PubMed

    Goguet-Rubio, Perrine; Seyran, Berfin; Gayte, Laurie; Bernex, Florence; Sutter, Anne; Delpech, Hélène; Linares, Laetitia Karine; Riscal, Romain; Repond, Cendrine; Rodier, Geneviève; Kirsh, Olivier; Touhami, Jawida; Noel, Jean; Vincent, Charles; Pirot, Nelly; Pavlovic, Guillaume; Herault, Yann; Sitbon, Marc; Pellerin, Luc; Sardet, Claude; Lacroix, Matthieu; Le Cam, Laurent

    2016-09-27

    The multifunctional protein E4 transcription factor 1 (E4F1) is an essential regulator of epidermal stem cell (ESC) maintenance. Here, we found that E4F1 transcriptionally regulates a metabolic program involved in pyruvate metabolism that is required to maintain skin homeostasis. E4F1 deficiency in basal keratinocytes resulted in deregulated expression of dihydrolipoamide acetyltransferase (Dlat), a gene encoding the E2 subunit of the mitochondrial pyruvate dehydrogenase (PDH) complex. Accordingly, E4f1 knock-out (KO) keratinocytes exhibited impaired PDH activity and a redirection of the glycolytic flux toward lactate production. The metabolic reprogramming of E4f1 KO keratinocytes associated with remodeling of their microenvironment and alterations of the basement membrane, led to ESC mislocalization and exhaustion of the ESC pool. ShRNA-mediated depletion of Dlat in primary keratinocytes recapitulated defects observed upon E4f1 inactivation, including increased lactate secretion, enhanced activity of extracellular matrix remodeling enzymes, and impaired clonogenic potential. Altogether, our data reveal a central role for Dlat in the metabolic program regulated by E4F1 in basal keratinocytes and illustrate the importance of PDH activity in skin homeostasis. PMID:27621431

  17. Serratia marcescens Quinoprotein Glucose Dehydrogenase Activity Mediates Medium Acidification and Inhibition of Prodigiosin Production by Glucose

    PubMed Central

    Fender, James E.; Bender, Cody M.; Stella, Nicholas A.; Lahr, Roni M.; Kalivoda, Eric J.

    2012-01-01

    Serratia marcescens is a model organism for the study of secondary metabolites. The biologically active pigment prodigiosin (2-methyl-3-pentyl-6-methoxyprodiginine), like many other secondary metabolites, is inhibited by growth in glucose-rich medium. Whereas previous studies indicated that this inhibitory effect was pH dependent and did not require cyclic AMP (cAMP), there is no information on the genes involved in mediating this phenomenon. Here we used transposon mutagenesis to identify genes involved in the inhibition of prodigiosin by glucose. Multiple genetic loci involved in quinoprotein glucose dehydrogenase (GDH) activity were found to be required for glucose inhibition of prodigiosin production, including pyrroloquinoline quinone and ubiquinone biosynthetic genes. Upon assessing whether the enzymatic products of GDH activity were involved in the inhibitory effect, we observed that d-glucono-1,5-lactone and d-gluconic acid, but not d-gluconate, were able to inhibit prodigiosin production. These data support a model in which the oxidation of d-glucose by quinoprotein GDH initiates a reduction in pH that inhibits prodigiosin production through transcriptional control of the prodigiosin biosynthetic operon, providing new insight into the genetic pathways that control prodigiosin production. Strains generated in this report may be useful in large-scale production of secondary metabolites. PMID:22752173

  18. Abscisic acid effects on activity and expression of barley (Hordeum vulgare) plastidial glucose-6-phosphate dehydrogenase

    PubMed Central

    Cardi, Manuela; Chibani, Kamel; Cafasso, Donata; Rouhier, Nicolas; Jacquot, Jean-Pierre; Esposito, Sergio

    2011-01-01

    Total glucose-6-phosphate dehydrogenase (G6PDH) activity, protein abundance, and transcript levels of G6PDH isoforms were measured in response to exogenous abscisic acid (ABA) supply to barley (Hordeum vulgare cv Nure) hydroponic culture. Total G6PDH activity increased by 50% in roots treated for 12 h with exogenous 0.1 mM ABA. In roots, a considerable increase (35%) in plastidial P2-G6PDH transcript levels was observed during the first 3 h of ABA treatment. Similar protein variations were observed in immunoblotting analyses. In leaves, a 2-fold increase in total G6PDH activity was observed after ABA treatment, probably related to an increase in the mRNA level (increased by 50%) and amount of protein (increased by 85%) of P2-G6PDH. Together these results suggest that the plastidial P2-isoform plays an important role in ABA-treated barley plants. PMID:21464159

  19. Serratia marcescens quinoprotein glucose dehydrogenase activity mediates medium acidification and inhibition of prodigiosin production by glucose.

    PubMed

    Fender, James E; Bender, Cody M; Stella, Nicholas A; Lahr, Roni M; Kalivoda, Eric J; Shanks, Robert M Q

    2012-09-01

    Serratia marcescens is a model organism for the study of secondary metabolites. The biologically active pigment prodigiosin (2-methyl-3-pentyl-6-methoxyprodiginine), like many other secondary metabolites, is inhibited by growth in glucose-rich medium. Whereas previous studies indicated that this inhibitory effect was pH dependent and did not require cyclic AMP (cAMP), there is no information on the genes involved in mediating this phenomenon. Here we used transposon mutagenesis to identify genes involved in the inhibition of prodigiosin by glucose. Multiple genetic loci involved in quinoprotein glucose dehydrogenase (GDH) activity were found to be required for glucose inhibition of prodigiosin production, including pyrroloquinoline quinone and ubiquinone biosynthetic genes. Upon assessing whether the enzymatic products of GDH activity were involved in the inhibitory effect, we observed that d-glucono-1,5-lactone and d-gluconic acid, but not d-gluconate, were able to inhibit prodigiosin production. These data support a model in which the oxidation of d-glucose by quinoprotein GDH initiates a reduction in pH that inhibits prodigiosin production through transcriptional control of the prodigiosin biosynthetic operon, providing new insight into the genetic pathways that control prodigiosin production. Strains generated in this report may be useful in large-scale production of secondary metabolites.

  20. Glutamic acid as anticancer agent: An overview.

    PubMed

    Dutta, Satyajit; Ray, Supratim; Nagarajan, K

    2013-10-01

    The objective of the article is to highlight various roles of glutamic acid like endogenic anticancer agent, conjugates to anticancer agents, and derivatives of glutamic acid as possible anticancer agents. Besides these emphases are given especially for two endogenous derivatives of glutamic acid such as glutamine and glutamate. Glutamine is a derivative of glutamic acid and is formed in the body from glutamic acid and ammonia in an energy requiring reaction catalyzed by glutamine synthase. It also possesses anticancer activity. So the transportation and metabolism of glutamine are also discussed for better understanding the role of glutamic acid. Glutamates are the carboxylate anions and salts of glutamic acid. Here the roles of various enzymes required for the metabolism of glutamates are also discussed.

  1. Relationships within the aldehyde dehydrogenase extended family.

    PubMed

    Perozich, J; Nicholas, H; Wang, B C; Lindahl, R; Hempel, J

    1999-01-01

    One hundred-forty-five full-length aldehyde dehydrogenase-related sequences were aligned to determine relationships within the aldehyde dehydrogenase (ALDH) extended family. The alignment reveals only four invariant residues: two glycines, a phenylalanine involved in NAD binding, and a glutamic acid that coordinates the nicotinamide ribose in certain E-NAD binary complex crystal structures, but which may also serve as a general base for the catalytic reaction. The cysteine that provides the catalytic thiol and its closest neighbor in space, an asparagine residue, are conserved in all ALDHs with demonstrated dehydrogenase activity. Sixteen residues are conserved in at least 95% of the sequences; 12 of these cluster into seven sequence motifs conserved in almost all ALDHs. These motifs cluster around the active site of the enzyme. Phylogenetic analysis of these ALDHs indicates at least 13 ALDH families, most of which have previously been identified but not grouped separately by alignment. ALDHs cluster into two main trunks of the phylogenetic tree. The largest, the "Class 3" trunk, contains mostly substrate-specific ALDH families, as well as the class 3 ALDH family itself. The other trunk, the "Class 1/2" trunk, contains mostly variable substrate ALDH families, including the class 1 and 2 ALDH families. Divergence of the substrate-specific ALDHs occurred earlier than the division between ALDHs with broad substrate specificities. A site on the World Wide Web has also been devoted to this alignment project.

  2. Glycine activated ion channel subunits encoded by ctenophore glutamate receptor genes.

    PubMed

    Alberstein, Robert; Grey, Richard; Zimmet, Austin; Simmons, David K; Mayer, Mark L

    2015-11-01

    Recent genome projects for ctenophores have revealed the presence of numerous ionotropic glutamate receptors (iGluRs) in Mnemiopsis leidyi and Pleurobrachia bachei, among our earliest metazoan ancestors. Sequence alignments and phylogenetic analysis show that these form a distinct clade from the well-characterized AMPA, kainate, and NMDA iGluR subtypes found in vertebrates. Although annotated as glutamate and kainate receptors, crystal structures of the ML032222a and PbiGluR3 ligand-binding domains (LBDs) reveal endogenous glycine in the binding pocket, whereas ligand-binding assays show that glycine binds with nanomolar affinity; biochemical assays and structural analysis establish that glutamate is occluded from the binding cavity. Further analysis reveals ctenophore-specific features, such as an interdomain Arg-Glu salt bridge, present only in subunits that bind glycine, but also a conserved disulfide in loop 1 of the LBD that is found in all vertebrate NMDA but not AMPA or kainate receptors. We hypothesize that ctenophore iGluRs are related to an early ancestor of NMDA receptors, suggesting a common evolutionary path for ctenophores and bilaterian species, and suggest that future work should consider both glycine and glutamate as candidate neurotransmitters in ctenophore species. PMID:26460032

  3. Glycine activated ion channel subunits encoded by ctenophore glutamate receptor genes

    PubMed Central

    Alberstein, Robert; Grey, Richard; Zimmet, Austin; Simmons, David K.; Mayer, Mark L.

    2015-01-01

    Recent genome projects for ctenophores have revealed the presence of numerous ionotropic glutamate receptors (iGluRs) in Mnemiopsis leidyi and Pleurobrachia bachei, among our earliest metazoan ancestors. Sequence alignments and phylogenetic analysis show that these form a distinct clade from the well-characterized AMPA, kainate, and NMDA iGluR subtypes found in vertebrates. Although annotated as glutamate and kainate receptors, crystal structures of the ML032222a and PbiGluR3 ligand-binding domains (LBDs) reveal endogenous glycine in the binding pocket, whereas ligand-binding assays show that glycine binds with nanomolar affinity; biochemical assays and structural analysis establish that glutamate is occluded from the binding cavity. Further analysis reveals ctenophore-specific features, such as an interdomain Arg-Glu salt bridge, present only in subunits that bind glycine, but also a conserved disulfide in loop 1 of the LBD that is found in all vertebrate NMDA but not AMPA or kainate receptors. We hypothesize that ctenophore iGluRs are related to an early ancestor of NMDA receptors, suggesting a common evolutionary path for ctenophores and bilaterian species, and suggest that future work should consider both glycine and glutamate as candidate neurotransmitters in ctenophore species. PMID:26460032

  4. Bovine neuronal vesicular glutamate transporter activity is inhibited by ergovaline and other ergopeptines

    Technology Transfer Automated Retrieval System (TEKTRAN)

    L-Glutamate (Glu) is the major excitatory neurotransmitter responsible for neurotransmission in the vertebrate central nervous system, including the gastrointestinal tract (GIT) of cattle. Vesicular Glu transporters VGLUT1 and VGLUT2 concentrate (50 mM) Glu (Km = 1 to 4 mM) into synaptic vesicles (S...

  5. Glycine activated ion channel subunits encoded by ctenophore glutamate receptor genes.

    PubMed

    Alberstein, Robert; Grey, Richard; Zimmet, Austin; Simmons, David K; Mayer, Mark L

    2015-11-01

    Recent genome projects for ctenophores have revealed the presence of numerous ionotropic glutamate receptors (iGluRs) in Mnemiopsis leidyi and Pleurobrachia bachei, among our earliest metazoan ancestors. Sequence alignments and phylogenetic analysis show that these form a distinct clade from the well-characterized AMPA, kainate, and NMDA iGluR subtypes found in vertebrates. Although annotated as glutamate and kainate receptors, crystal structures of the ML032222a and PbiGluR3 ligand-binding domains (LBDs) reveal endogenous glycine in the binding pocket, whereas ligand-binding assays show that glycine binds with nanomolar affinity; biochemical assays and structural analysis establish that glutamate is occluded from the binding cavity. Further analysis reveals ctenophore-specific features, such as an interdomain Arg-Glu salt bridge, present only in subunits that bind glycine, but also a conserved disulfide in loop 1 of the LBD that is found in all vertebrate NMDA but not AMPA or kainate receptors. We hypothesize that ctenophore iGluRs are related to an early ancestor of NMDA receptors, suggesting a common evolutionary path for ctenophores and bilaterian species, and suggest that future work should consider both glycine and glutamate as candidate neurotransmitters in ctenophore species.

  6. Communication between Thiamin Cofactors in the Escherichia coli Pyruvate Dehydrogenase Complex E1 Component Active Centers

    PubMed Central

    Nemeria, Natalia S.; Arjunan, Palaniappa; Chandrasekhar, Krishnamoorthy; Mossad, Madouna; Tittmann, Kai; Furey, William; Jordan, Frank

    2010-01-01

    Kinetic, spectroscopic, and structural analysis tested the hypothesis that a chain of residues connecting the 4′-aminopyrimidine N1′ atoms of thiamin diphosphates (ThDPs) in the two active centers of the Escherichia coli pyruvate dehydrogenase complex E1 component provides a signal transduction pathway. Substitution of the three acidic residues (Glu571, Glu235, and Glu237) and Arg606 resulted in impaired binding of the second ThDP, once the first active center was filled, suggesting a pathway for communication between the two ThDPs. 1) Steady-state kinetic and fluorescence quenching studies revealed that upon E571A, E235A, E237A, and R606A substitutions, ThDP binding in the second active center was affected. 2) Analysis of the kinetics of thiazolium C2 hydrogen/deuterium exchange of enzyme-bound ThDP suggests half-of-the-sites reactivity for the E1 component, with fast (activated site) and slow exchanging sites (dormant site). The E235A and E571A variants gave no evidence for the slow exchanging site, indicating that only one of two active sites is filled with ThDP. 3) Titration of the E235A and E237A variants with methyl acetylphosphonate monitored by circular dichroism suggested that only half of the active sites were filled with a covalent predecarboxylation intermediate analog. 4) Crystal structures of E235A and E571A in complex with ThDP revealed the structural basis for the spectroscopic and kinetic observations and showed that either substitution affects cofactor binding, despite the fact that Glu235 makes no direct contact with the cofactor. The role of the conserved Glu571 residue in both catalysis and cofactor orientation is revealed by the combined results for the first time. PMID:20106967

  7. Enzymatic synthesis of γ-glutamylmethylamide from glutamic acid γ-methyl ester and methylamine catalyzed by Escherichia coli having γ-glutamyltranspeptidase activity.

    PubMed

    Xu, Lisheng; Gao, Guizhen; Wengen, Cao; Xu, Jigui; Zhao, Liang; Shi, Hongwei; Zhang, Xingtao

    2014-06-01

    A new method for the synthesis of γ-glutamylmethylamide is presented. Glutamic acid γ-methyl ester was used as substrate for γ-glutamylmethylamide synthesis catalyzed by Escherichia coli with γ-glutamyltranspeptidase activity. Reaction conditions were optimized by using 300 mM glutamic acid γ-methyl ester and 3,000 mM methylamine at pH 10 and 40 °C. Bioconversion rate of γ-glutamylmethylamide reached 87 % after 10 h. γ-Glutamyltranspeptidase was reversibly inhibited only when glutamic acid γ-methyl ester was above 300 mM.

  8. Optimization of enzyme assisted extraction of Fructus Mori polysaccharides and its activities on antioxidant and alcohol dehydrogenase.

    PubMed

    Deng, Qingfang; Zhou, Xin; Chen, Huaguo

    2014-10-13

    In the present study, enzyme assisted extraction of Fructus Mori polysaccharides (FMPS) from F. mori using four kinds of enzymes and three compound enzymes were examined. Research found that glucose oxidase offered a better performance in enhancement of the extraction yields of FMPS, antioxidant and activate alcohol dehydrogenase activities. The glucose oxidase assisted extraction process was further optimized by using response surface method (RSM) to obtain maximum yield of crude FMPS. The results showed that optimized extraction conditions were ratio of enzyme amount 0.40%, enzyme treated time 38 min, treated temperature 58 °C and liquid-solid radio 11.0. Under these conditions, the mean experimental value of extraction yield (16.16 ± 0.14%) corresponded well with the predicted values and increased 160% than none enzyme treated ones. Pharmacological verification tests showed that F. mori crude polysaccharides had good antioxidant and activate alcohol dehydrogenase activities in vitro. PMID:25037415

  9. Differential inhibition of PDKs by phenylbutyrate and enhancement of pyruvate dehydrogenase complex activity by combination with dichloroacetate.

    PubMed

    Ferriero, Rosa; Iannuzzi, Clara; Manco, Giuseppe; Brunetti-Pierri, Nicola

    2015-09-01

    Pyruvate dehydrogenase complex (PDHC) is a key enzyme in metabolism linking glycolysis to tricarboxylic acid cycle and its activity is tightly regulated by phosphorylation catalyzed by four pyruvate dehydrogenase kinase (PDK) isoforms. PDKs are pharmacological targets for several human diseases including cancer, diabetes, obesity, heart failure, and inherited PDHC deficiency. We investigated the inhibitory activity of phenylbutyrate toward PDKs and found that PDK isoforms 1-to-3 are inhibited whereas PDK4 is unaffected. Moreover, docking studies revealed putative binding sites of phenylbutyrate on PDK2 and 3 that are located on different sites compared to dichloroacetate (DCA), a previously known PDK inhibitor. Based on these findings, we showed both in cells and in mice that phenylbutyrate combined to DCA results in greater increase of PDHC activity compared to each drug alone. These results suggest that therapeutic efficacy can be enhanced by combination of drugs increasing PDHC enzyme activity. PMID:25601413

  10. Optimization of enzyme assisted extraction of Fructus Mori polysaccharides and its activities on antioxidant and alcohol dehydrogenase.

    PubMed

    Deng, Qingfang; Zhou, Xin; Chen, Huaguo

    2014-10-13

    In the present study, enzyme assisted extraction of Fructus Mori polysaccharides (FMPS) from F. mori using four kinds of enzymes and three compound enzymes were examined. Research found that glucose oxidase offered a better performance in enhancement of the extraction yields of FMPS, antioxidant and activate alcohol dehydrogenase activities. The glucose oxidase assisted extraction process was further optimized by using response surface method (RSM) to obtain maximum yield of crude FMPS. The results showed that optimized extraction conditions were ratio of enzyme amount 0.40%, enzyme treated time 38 min, treated temperature 58 °C and liquid-solid radio 11.0. Under these conditions, the mean experimental value of extraction yield (16.16 ± 0.14%) corresponded well with the predicted values and increased 160% than none enzyme treated ones. Pharmacological verification tests showed that F. mori crude polysaccharides had good antioxidant and activate alcohol dehydrogenase activities in vitro.

  11. Mitochondrial Dihydrolipoyl Dehydrogenase Activity Shapes Photosynthesis and Photorespiration of Arabidopsis thaliana.

    PubMed

    Timm, Stefan; Wittmiß, Maria; Gamlien, Sabine; Ewald, Ralph; Florian, Alexandra; Frank, Marcus; Wirtz, Markus; Hell, Rüdiger; Fernie, Alisdair R; Bauwe, Hermann

    2015-07-01

    Mitochondrial dihydrolipoyl dehydrogenase (mtLPD; L-protein) is an integral component of several multienzyme systems involved in the tricarboxylic acid (TCA) cycle, photorespiration, and the degradation of branched-chain α-ketoacids. The majority of the mtLPD present in photosynthesizing tissue is used for glycine decarboxylase (GDC), necessary for the high-flux photorespiratory glycine-into-serine conversion. We previously suggested that GDC activity could be a signal in a regulatory network that adjusts carbon flux through the Calvin-Benson cycle in response to photorespiration. Here, we show that elevated GDC L-protein activity significantly alters several diagnostic parameters of cellular metabolism and leaf gas exchange in Arabidopsis thaliana. Overexpressor lines displayed markedly decreased steady state contents of TCA cycle and photorespiratory intermediates as well as elevated NAD(P)(+)-to-NAD(P)H ratios. Additionally, increased rates of CO2 assimilation, photorespiration, and plant growth were observed. Intriguingly, however, day respiration rates remained unaffected. By contrast, respiration was enhanced in the first half of the dark phase but depressed in the second. We also observed enhanced sucrose biosynthesis in the light in combination with a lower diel magnitude of starch accumulation and breakdown. These data thus substantiate our prior hypothesis that facilitating flux through the photorespiratory pathway stimulates photosynthetic CO2 assimilation in the Calvin-Benson cycle. They furthermore suggest that this regulation is, at least in part, dependent on increased light-capture/use efficiency. PMID:26116608

  12. Hypoxic repression of pyruvate dehydrogenase activity is necessary for metabolic reprogramming and growth of model tumours.

    PubMed

    Golias, Tereza; Papandreou, Ioanna; Sun, Ramon; Kumar, Bhavna; Brown, Nicole V; Swanson, Benjamin J; Pai, Reetesh; Jaitin, Diego; Le, Quynh-Thu; Teknos, Theodoros N; Denko, Nicholas C

    2016-01-01

    Tumour cells fulfil the bioenergetic and biosynthetic needs of proliferation using the available environmental metabolites. Metabolic adaptation to hypoxia causes decreased mitochondrial function and increased lactate production. This work examines the biological importance of the hypoxia-inducible inhibitory phosphorylations on the pyruvate dehydrogenase E1α subunit. Pancreatic cancer cell lines were genetically manipulated to alter the net phosphorylation of PDH E1α through reduced kinase expression or enhanced phosphatase expression. The modified cells were tested for hypoxic changes in phosphorylated E1α, mitochondrial metabolism and growth as xenografted tumours. Even though there are four PDHK genes, PDHK1 is essential for inhibitory PDH phosphorylation of E1α at serine 232, is partially responsible for modification of serines 293 and 300, and these phosphorylations are necessary for model tumour growth. In order to determine the clinical relevance, a cohort of head and neck cancer patient biopsies was examined for phosphorylated E1α and expression of PDHK1. Patients with detectable 232 phosphorylation or expression of PDHK1 tend to have worse clinical outcome. These data show that PDHK1 activity is unique and non-redundant in the family of PHDK enzymes and a PDHK1 specific inhibitor would therefore have anti-cancer activity with reduced chance of side effects from inhibition of other PDHKs. PMID:27498883

  13. Hypoxic repression of pyruvate dehydrogenase activity is necessary for metabolic reprogramming and growth of model tumours

    PubMed Central

    Golias, Tereza; Papandreou, Ioanna; Sun, Ramon; Kumar, Bhavna; Brown, Nicole V.; Swanson, Benjamin J.; Pai, Reetesh; Jaitin, Diego; Le, Quynh-Thu; Teknos, Theodoros N.; Denko, Nicholas C.

    2016-01-01

    Tumour cells fulfil the bioenergetic and biosynthetic needs of proliferation using the available environmental metabolites. Metabolic adaptation to hypoxia causes decreased mitochondrial function and increased lactate production. This work examines the biological importance of the hypoxia-inducible inhibitory phosphorylations on the pyruvate dehydrogenase E1α subunit. Pancreatic cancer cell lines were genetically manipulated to alter the net phosphorylation of PDH E1α through reduced kinase expression or enhanced phosphatase expression. The modified cells were tested for hypoxic changes in phosphorylated E1α, mitochondrial metabolism and growth as xenografted tumours. Even though there are four PDHK genes, PDHK1 is essential for inhibitory PDH phosphorylation of E1α at serine 232, is partially responsible for modification of serines 293 and 300, and these phosphorylations are necessary for model tumour growth. In order to determine the clinical relevance, a cohort of head and neck cancer patient biopsies was examined for phosphorylated E1α and expression of PDHK1. Patients with detectable 232 phosphorylation or expression of PDHK1 tend to have worse clinical outcome. These data show that PDHK1 activity is unique and non-redundant in the family of PHDK enzymes and a PDHK1 specific inhibitor would therefore have anti-cancer activity with reduced chance of side effects from inhibition of other PDHKs. PMID:27498883

  14. Aldehyde Dehydrogenase Activity Identifies a Population of Human Skeletal Muscle Cells With High Myogenic Capacities

    PubMed Central

    Vauchez, Karine; Marolleau, Jean-Pierre; Schmid, Michel; Khattar, Patricia; Chapel, Alain; Catelain, Cyril; Lecourt, Séverine; Larghéro, Jérôme; Fiszman, Marc; Vilquin, Jean-Thomas

    2009-01-01

    Aldehyde dehydrogenase 1A1 (ALDH) activity is one hallmark of human bone marrow (BM), umbilical cord blood (UCB), and peripheral blood (PB) primitive progenitors presenting high reconstitution capacities in vivo. In this study, we have identified ALDH+ cells within human skeletal muscles, and have analyzed their phenotypical and functional characteristics. Immunohistofluorescence analysis of human muscle tissue sections revealed rare endomysial cells. Flow cytometry analysis using the fluorescent substrate of ALDH, Aldefluor, identified brightly stained (ALDHbr) cells with low side scatter (SSClo), in enzymatically dissociated muscle biopsies, thereafter abbreviated as SMALD+ (for skeletal muscle ALDH+) cells. Phenotypical analysis discriminated two sub-populations according to CD34 expression: SMALD+/CD34− and SMALD+/CD34+ cells. These sub-populations did not initially express endothelial (CD31), hematopoietic (CD45), and myogenic (CD56) markers. Upon sorting, however, whereas SMALD+/CD34+ cells developed in vitro as a heterogeneous population of CD56− cells able to differentiate in adipoblasts, the SMALD+/CD34− fraction developed in vitro as a highly enriched population of CD56+ myoblasts able to form myotubes. Moreover, only the SMALD+/CD34− population maintained a strong myogenic potential in vivo upon intramuscular transplantation. Our results suggest that ALDH activity is a novel marker for a population of new human skeletal muscle progenitors presenting a potential for cell biology and cell therapy. PMID:19738599

  15. Characterization of 10-Hydroxygeraniol Dehydrogenase from Catharanthus roseus Reveals Cascaded Enzymatic Activity in Iridoid Biosynthesis

    PubMed Central

    Krithika, Ramakrishnan; Srivastava, Prabhakar Lal; Rani, Bajaj; Kolet, Swati P.; Chopade, Manojkumar; Soniya, Mantri; Thulasiram, Hirekodathakallu V.

    2015-01-01

    Catharanthus roseus [L.] is a major source of the monoterpene indole alkaloids (MIAs), which are of significant interest due to their therapeutic value. These molecules are formed through an intermediate, cis-trans-nepetalactol, a cyclized product of 10-oxogeranial. One of the key enzymes involved in the biosynthesis of MIAs is an NAD(P)+ dependent oxidoreductase system, 10-hydroxygeraniol dehydrogenase (Cr10HGO), which catalyses the formation of 10-oxogeranial from 10-hydroxygeraniol via 10-oxogeraniol or 10-hydroxygeranial. This work describes the cloning and functional characterization of Cr10HGO from C. roseus and its role in the iridoid biosynthesis. Substrate specificity studies indicated that, Cr10HGO has good activity on substrates such as 10-hydroxygeraniol, 10-oxogeraniol or 10-hydroxygeranial over monohydroxy linear terpene derivatives. Further it was observed that incubation of 10-hydroxygeraniol with Cr10HGO and iridoid synthase (CrIDS) in the presence of NADP+ yielded a major metabolite, which was characterized as (1R, 4aS, 7S, 7aR)-nepetalactol by comparing its retention time, mass fragmentation pattern, and co-injection studies with that of the synthesized compound. These results indicate that there is concerted activity of Cr10HGO with iridoid synthase in the formation of (1R, 4aS, 7S, 7aR)-nepetalactol, an important intermediate in iridoid biosynthesis. PMID:25651761

  16. Mitochondrial Dihydrolipoyl Dehydrogenase Activity Shapes Photosynthesis and Photorespiration of Arabidopsis thaliana.

    PubMed

    Timm, Stefan; Wittmiß, Maria; Gamlien, Sabine; Ewald, Ralph; Florian, Alexandra; Frank, Marcus; Wirtz, Markus; Hell, Rüdiger; Fernie, Alisdair R; Bauwe, Hermann

    2015-07-01

    Mitochondrial dihydrolipoyl dehydrogenase (mtLPD; L-protein) is an integral component of several multienzyme systems involved in the tricarboxylic acid (TCA) cycle, photorespiration, and the degradation of branched-chain α-ketoacids. The majority of the mtLPD present in photosynthesizing tissue is used for glycine decarboxylase (GDC), necessary for the high-flux photorespiratory glycine-into-serine conversion. We previously suggested that GDC activity could be a signal in a regulatory network that adjusts carbon flux through the Calvin-Benson cycle in response to photorespiration. Here, we show that elevated GDC L-protein activity significantly alters several diagnostic parameters of cellular metabolism and leaf gas exchange in Arabidopsis thaliana. Overexpressor lines displayed markedly decreased steady state contents of TCA cycle and photorespiratory intermediates as well as elevated NAD(P)(+)-to-NAD(P)H ratios. Additionally, increased rates of CO2 assimilation, photorespiration, and plant growth were observed. Intriguingly, however, day respiration rates remained unaffected. By contrast, respiration was enhanced in the first half of the dark phase but depressed in the second. We also observed enhanced sucrose biosynthesis in the light in combination with a lower diel magnitude of starch accumulation and breakdown. These data thus substantiate our prior hypothesis that facilitating flux through the photorespiratory pathway stimulates photosynthetic CO2 assimilation in the Calvin-Benson cycle. They furthermore suggest that this regulation is, at least in part, dependent on increased light-capture/use efficiency.

  17. Mitochondrial Dihydrolipoyl Dehydrogenase Activity Shapes Photosynthesis and Photorespiration of Arabidopsis thaliana

    PubMed Central

    Timm, Stefan; Wittmiß, Maria; Gamlien, Sabine; Ewald, Ralph; Florian, Alexandra; Frank, Marcus; Wirtz, Markus; Hell, Rüdiger; Fernie, Alisdair R.; Bauwe, Hermann

    2015-01-01

    Mitochondrial dihydrolipoyl dehydrogenase (mtLPD; L-protein) is an integral component of several multienzyme systems involved in the tricarboxylic acid (TCA) cycle, photorespiration, and the degradation of branched-chain α-ketoacids. The majority of the mtLPD present in photosynthesizing tissue is used for glycine decarboxylase (GDC), necessary for the high-flux photorespiratory glycine-into-serine conversion. We previously suggested that GDC activity could be a signal in a regulatory network that adjusts carbon flux through the Calvin-Benson cycle in response to photorespiration. Here, we show that elevated GDC L-protein activity significantly alters several diagnostic parameters of cellular metabolism and leaf gas exchange in Arabidopsis thaliana. Overexpressor lines displayed markedly decreased steady state contents of TCA cycle and photorespiratory intermediates as well as elevated NAD(P)+-to-NAD(P)H ratios. Additionally, increased rates of CO2 assimilation, photorespiration, and plant growth were observed. Intriguingly, however, day respiration rates remained unaffected. By contrast, respiration was enhanced in the first half of the dark phase but depressed in the second. We also observed enhanced sucrose biosynthesis in the light in combination with a lower diel magnitude of starch accumulation and breakdown. These data thus substantiate our prior hypothesis that facilitating flux through the photorespiratory pathway stimulates photosynthetic CO2 assimilation in the Calvin-Benson cycle. They furthermore suggest that this regulation is, at least in part, dependent on increased light-capture/use efficiency. PMID:26116608

  18. Evaluation on the inhibition of pyrrol-2-yl ethanone derivatives to lactate dehydrogenase and anticancer activities

    NASA Astrophysics Data System (ADS)

    Lu, Na-Na; Weng, Zhao-Yue; Chen, Qiu-Yun; Boison, Daniel; Xiao, Xin-Xin; Gao, Jing

    2016-08-01

    Lactate dehydrogenase A (LDH-A) is a potentially important metabolic target for the inhibition of the highly activated glycolysis pathway in cancer cells. In order to develop bifunctional compounds as inhibitor of LDH-A and anticancer agents, two pyrrol-2-yl methanone (or ethanone) derivatives (PM1 and PM2) were synthesized and evaluated as inhibitors of LDH-A based on the enzyme assay and cell assay by spectroscopy analysis. Fluorescence and CD spectra results demonstrated that both the change of second structure of LDH-A and the affinity interaction for compounds to LDH-A gave great effect on the activity of LDH-A. In particular, low concentration of compounds (1 μμ-25 μμ) could change the level of pyruvate in cancer cells. Moreover, the in vitro assay results demonstrated that pyrrol-2-yl ethanone derivatives can inhibit the proliferation of cancer cells. Therefore, pyrrol-2-yl ethanone derivatives (PM2) can be both LDH-A inhibitor and anticancer agents.

  19. Biophysical and mutagenic analysis of Thermoanaerobacter ethanolicus secondary-alcohol dehydrogenase activity and specificity.

    PubMed Central

    Burdette, D S; Secundo, F; Phillips, R S; Dong, J; Scott, R A; Zeikus, J G

    1997-01-01

    The Thermoanaerobacter ethanolicus 39E adhB gene encoding the secondary-alcohol dehydrogenase (secondary ADH) was overexpressed in Escherichia coli at more than 10% of total protein. The recombinant enzyme was purified in high yield (67%) by heat-treatment at 85 degrees C and (NH4)2SO4 precipitation. Site-directed mutants (C37S, H59N, D150N, D150Eand D150C were analysed to test the peptide sequence comparison-based predictions of amino acids responsible for putative catalytic Zn binding. X-ray absorption spectroscopy confirmed the presence of a protein-bound Zn atom with ZnS1(imid)1(N,O)3 co-ordination sphere. Inductively coupled plasma atomic emission spectrometry measured 0.48 Zn atoms per wild-type secondary ADH subunit. The C37S, H59N and D150N mutant enzymes bound only 0.11, 0.13 and 0.33 Zn per subunit respectively,suggesting that these residues are involved in Zn liganding. The D150E and D150C mutants retained 0.47 and 1.2 Zn atoms per subunit, indicating that an anionic side-chain moiety at this position preserves the bound Zn. All five mutant enzymes had activity, suggesting that the T. ethanolicus secondary ADH requires a properly co-ordinated catalytic Zn atom. The His-59 and Asp-150 mutations also altered secondary ADH affinity for propan-2-ol over a 140-fold range, whereas the overall change in affinity for ethanol spanned a range of only 7-fold, supporting the importance of the metal in secondary ADH substrate binding. The lack of significant changes in cofactor affinity as a result of these catalytic Zn ligand mutations suggested that secondary ADH substrate-and cofactor-binding sites are structurally distinct. Altering Gly198 to Asp reduced the enzyme specific activity 2.7-fold, increased the Km(app) for NADP+ 225-fold, and decreased the Km(app) for NAD+ 3-fold, supporting the prediction that the enzyme binds nicotinamide cofactor in a Rossmann fold. Our data indicate therefore that, unlike the liver primary ADH

  20. The pathways of glutamate and glutamine oxidation by tumor cell mitochondria. Role of mitochondrial NAD(P)+-dependent malic enzyme.

    PubMed

    Moreadith, R W; Lehninger, A L

    1984-05-25

    Little evidence has been available on the oxidative pathways of glutamine and glutamate, the major respiratory substrates of cancer cells. Glutamate formed from glutamine by phosphate-dependent glutaminase undergoes quantitative transamination by aerobic tumor mitochondria to yield aspartate. However, when malate is also added there is a pronounced decrease in aspartate production and a large formation of citrate and alanine, in both state 3 and 4 conditions. In contrast, addition of malate to normal rat heart, liver, or kidney mitochondria oxidizing glutamate causes a marked increase in aspartate production. Further analysis showed that extramitochondrial malate is oxidized almost quantitatively to pyruvate + CO2 by NAD(P)+-linked malic enzyme, present in the mitochondria of all tumors tested, but absent in heart, liver, and kidney mitochondria. On the other hand intramitochondrial malate generated from glutamate is oxidized quantitatively to oxalacetate by mitochondrial malate dehydrogenase of tumors. Acetyl-CoA derived from extramitochondrial malate via pyruvate and oxalacetate derived from glutamate via intramitochondrial malate are quantitatively converted into citrate, which is extruded. No evidence was found that malic enzyme of tumor mitochondria converts glutamate-derived malate into pyruvate as postulated in other reports. Possible mechanisms for the integration of mitochondrial malic enzyme and malate dehydrogenase activities in tumors are discussed.

  1. Burst firing in gonadotrophin-releasing hormone neurones does not require ionotrophic GABA or glutamate receptor activation.

    PubMed

    Lee, K; Liu, X; Herbison, A E

    2012-12-01

    Burst firing is a feature of many neuroendocrine cell types, including the hypothalamic gonadotrophin-releasing hormone (GnRH) neurones that control fertility. The role of intrinsic and extrinsic influences in generating GnRH neurone burst firing is presently unclear. In the present study, we investigated the role of fast amino acid transmission in burst firing by examining the effects of receptor antagonists on bursting displayed by green fluorescent protein GnRH neurones in sagittal brain slices prepared from adult male mice. Blockade of AMPA and NMDA glutamate receptors with a cocktail of CNQX and AP5 was found to have no effects on burst firing in GnRH neurones. The frequency of bursts, dynamics of individual bursts, or percentage of firing clustered in bursts was not altered. Similarly, GABA(A) receptor antagonists bicuculline and picrotoxin had no effects upon burst firing in GnRH neurones. To examine the importance of both glutamate and GABA ionotrophic signalling, a cocktail including picrotoxin, CNQX and AP5 was used but, again, this was found to have no effects on GnRH neurone burst firing. To further question the impact of endogenous amino acid release on burst firing, electrical activation of anteroventral periventricular nuclei GABA/glutamate inputs to GnRH neurones was undertaken and found to have no impact on burst firing. Taken together, these observations indicate that bursting in GnRH neurones is not dependent upon acute ionotrophic GABA and glutamate signalling and suggest that extrinsic inputs to GnRH neurones acting through AMPA, NMDA and GABA(A) receptors are unlikely to be required for burst initiation in these cells.

  2. Medial Septal NMDA Glutamate Receptors are Involved in Modulation of Blood Natural Killer Cell Activity in Rats.

    PubMed

    Podlacha, Magdalena; Glac, Wojciech; Listowska, Magdalena; Grembecka, Beata; Majkutewicz, Irena; Myślińska, Dorota; Plucińska, Karolina; Jerzemowska, Grażyna; Grzybowska, Maria; Wrona, Danuta

    2016-03-01

    The purpose of the present study was to determine the specific role of the medial septal (MS) NMDA glutamate receptors on peripheral blood natural killer cell cytotoxicity (NKCC) and their (large granular lymphocyte, LGL) number, as well as the plasma concentration of tumor necrosis factor α (TNF-α) and corticosterone in male Wistar rats exposed to elevated plus maze (EPM) stress or non-stress conditions. The NMDA groups were injected with NMDA glutamate receptor agonist (N-methyl-D-aspartate; 0.25 μg/rat), the D-AP7 group was injected with DL-2-amino-7-phosphoheptanoate (0.1 μg/rat), an antagonist of NMDA glutamate receptors, and the control Sal group with saline (0.5 μl/rat) via previously implanted cannulae into the MS. There was an increase in the NKCC, NK/LGL number and plasma TNF-α concentration after the NMDA injections, being much stronger within the rats under non-stress conditions rather than the rats exposed to EPM stress. These parameters were decreased in the D-AP7 rats, suggesting receptor/ion channel specificity. Moreover, a lower plasma corticosterone concentration within the NMDA rather than the Sal and D-AP7 groups was found. The obtained results suggest that activation of the NMDA glutamate receptors in the MS, accompanied by changes in the corticosterone and cytokine responses, may be involved in modulation of the blood natural anti-tumor response, under EPM stress and non-stress conditions. PMID:26454750

  3. Functional response of the isolated, perfused normoxic heart to pyruvate dehydrogenase activation by dichloroacetate and pyruvate

    PubMed Central

    Jaimes, Rafael; Kuzmiak-Glancy, Sarah; Brooks, Daina M.; Swift, Luther M.; Posnack, Nikki G.; Kay, Matthew W.

    2015-01-01

    Dichloroacetate (DCA) and pyruvate activate pyruvate dehydrogenase (PDH), a key enzyme that modulates glucose oxidation and mitochondrial NADH production. Both compounds improve recovery after ischemia in isolated hearts. However, the action of DCA and pyruvate in normoxic myocardium is incompletely understood. We measured the effect of DCA and pyruvate on contraction, mitochondrial redox state, and intracellular calcium cycling in isolated rat hearts during normoxic perfusion. Normalized epicardial NADH fluorescence (nNADH) and left ventricular developed pressure (LVDP) were measured before and after administering DCA (5 mM) or pyruvate (5 mM). Optical mapping of Rhod-2AM was used to measure cytosolic calcium kinetics. DCA maximally activated PDH, increasing the ratio of active to total PDH from 0.48±0.03 to 1.03 ±0.03. Pyruvate sub-maximally activated PDH to a ratio of 0.75±0.02. DCA and pyruvate increased LVDP. When glucose was the only exogenous fuel, pyruvate increased nNADH by 21.4±2.9 % while DCA reduced nNADH by 21.4±6.1 % and elevated the incidence of premature ventricular contractions (PVCs). When lactate, pyruvate, and glucose were provided together as exogenous fuels, nNADH increased with DCA, indicating that PDH activation with glucose as the only exogenous fuel depletes PDH substrate. Calcium transient time-to-peak was shortened by DCA and pyruvate and SR calcium re-uptake was 30 % longer. DCA and pyruvate increased SR calcium load in myocyte monolayers. Overall, during normoxia when glucose is the only exogenous fuel, DCA elevates SR calcium, increases LVDP and contractility, and diminishes mitochondrial NADH. Administering DCA with plasma levels of lactate and pyruvate mitigates the drop in mitochondrial NADH and prevents PVCs. PMID:26142699

  4. Subcellular Characterization of Porcine Oocytes with Different Glucose-6-phosphate Dehydrogenase Activities

    PubMed Central

    Fu, Bo; Ren, Liang; Liu, Di; Ma, Jian-Zhang; An, Tie-Zhu; Yang, Xiu-Qin; Ma, Hong; Zhang, Dong-Jie; Guo, Zhen-Hua; Guo, Yun-Yun; Zhu, Meng; Bai, Jing

    2015-01-01

    The in vitro maturation (IVM) efficiency of porcine embryos is still low because of poor oocyte quality. Although brilliant cresyl blue positive (BCB+) oocytes with low glucose-6-phosphate dehydrogenase (G6PDH) activity have shown superior quality than BCB negative (−) oocytes with high G6PDH activity, the use of a BCB staining test before IVM is still controversial. This study aimed to shed more light on the subcellular characteristics of porcine oocytes after selection using BCB staining. We assessed germinal vesicle chromatin configuration, cortical granule (CG) migration, mitochondrial distribution, the levels of acetylated lysine 9 of histone H3 (AcH3K9) and nuclear apoptosis features to investigate the correlation between G6PDH activity and these developmentally related features. A pattern of chromatin surrounding the nucleoli was seen in 53.0% of BCB+ oocytes and 77.6% of BCB+ oocytes showed peripherally distributed CGs. After IVM, 48.7% of BCB+ oocytes had a diffused mitochondrial distribution pattern. However, there were no significant differences in the levels of AcH3K9 in the nuclei of blastocysts derived from BCB+ and BCB− oocytes; at the same time, we observed a similar incidence of apoptosis in the BCB+ and control groups. Although this study indicated that G6PDH activity in porcine oocytes was correlated with several subcellular characteristics such as germinal vesicle chromatin configuration, CG migration and mitochondrial distribution, other features such as AcH3K9 level and nuclear apoptotic features were not associated with G6PDH activity and did not validate the BCB staining test. In using this test for selecting porcine oocytes, subcellular characteristics such as the AcH3K9 level and apoptotic nuclear features should also be considered. Adding histone deacetylase inhibitors or apoptosis inhibitors into the culture medium used might improve the efficiency of IVM of BCB+ oocytes. PMID:26580437

  5. Activation of synaptic group II metabotropic glutamate receptors induces long-term depression at GABAergic synapses in CNS neurons.

    PubMed

    Tang, Zheng-Quan; Liu, Yu-Wei; Shi, Wei; Dinh, Emilie Hoang; Hamlet, William R; Curry, Rebecca J; Lu, Yong

    2013-10-01

    Metabotropic glutamate receptor (mGluR)-dependent homosynaptic long-term depression (LTD) has been studied extensively at glutamatergic synapses in the CNS. However, much less is known about heterosynaptic long-term plasticity induced by mGluRs at inhibitory synapses. Here we report that pharmacological or synaptic activation of group II mGluRs (mGluR II) induces LTD at GABAergic synapses without affecting the excitatory glutamatergic transmission in neurons of the chicken cochlear nucleus. Coefficient of variation and failure rate analysis suggested that the LTD was expressed presynaptically. The LTD requires presynaptic spike activity, but does not require the activation of NMDA receptors. The classic cAMP-dependent protein kinase A signaling is involved in the transduction pathway. Remarkably, blocking mGluR II increased spontaneous GABA release, indicating the presence of tonic activation of mGluR II by ambient glutamate. Furthermore, synaptically released glutamate induced by electrical stimulations that concurrently activated both the glutamatergic and GABAergic pathways resulted in significant and constant suppression of GABA release at various stimulus frequencies (3.3, 100, and 300 Hz). Strikingly, low-frequency stimulation (1 Hz, 15 min) of the glutamatergic synapses induced heterosynaptic LTD of GABAergic transmission, and the LTD was blocked by mGluR II antagonist, indicating that synaptic activation of mGluR II induced the LTD. This novel form of long-term plasticity in the avian auditory brainstem may play a role in the development as well as in temporal processing in the sound localization circuit.

  6. The regulation of adipose tissue pyruvate dehydrogenase activity of dietary fiber.

    PubMed

    Ogunwole, J O; Knight, E M; Adkins, J S; Thomaskutty, K G; Pointer, R H

    1987-05-01

    In vitro studies have established that insulin enhances the oxidation of pyruvate to acetyl CoA by the stimulation of mitochondrial pyruvate dehydrogenase (PDH) activity through plasma membrane binding response (Jarett and Seals 1979; Kiechle, Jarett, Dennis and Kotagal 1980). In the present study adipose tissue PDH activity was utilized as a marker for insulin responsiveness. The metabolic response of this enzyme to exogenous insulin was employed to test the hypothesis that dietary fiber enhances tissue responsiveness to insulin using adipose tissue from male weanling Sprague Dawley rats. Eight groups of rats (n = 5 per group) were fed ad libitum various diets containing different levels of cellulose and protein as already reported elsewhere (Ogunwole, Knight, Adkins, Thomaskutty and Pointer 1985). Percent insulin stimulation of PDH from basal activity (PDS) was utilized as an index of insulin responsiveness. Compared to all fiber treated groups, both basal (PDB) and insulin stimulated (PDI) activities were significantly lower (P less than 0.05) in the fiber free groups at both low (10%) and high (20%) protein levels. At all fiber levels tested (0, 5, 15 and 30%) protein intake resulted in a significant increase in both PDB and PDI. Gradual increase in cellulose intake resulted in a biphasic increase in PDS in both protein groups at the 5% and 30% fiber levels. PDS was higher (P less than 0.05) in the 10% protein groups than the 20% protein group at all fiber levels tested. A significant interaction effect of protein and fiber was observed on PDB (P less than 0.001) and PDI (P less than 0.04) when caloric intake was held constant as a covariate.(ABSTRACT TRUNCATED AT 250 WORDS)

  7. Postnatal Chick Choroids Exhibit Increased Retinaldehyde Dehydrogenase Activity During Recovery From Form Deprivation Induced Myopia

    PubMed Central

    Harper, Angelica R.; Wang, Xiang; Moiseyev, Gennadiy; Ma, Jian-Xing; Summers, Jody A.

    2016-01-01

    Purpose Increases in retinaldehyde dehydrogenase 2 (RALDH2) transcript in the chick choroid suggest that RALDH2 may be responsible for increases observed in all-trans-retinoic acid (atRA) synthesis during recovery from myopic defocus. The purpose of the present study was to examine RALDH2 protein expression, RALDH activity, and distribution of RALDH2 cells in control and recovering chick ocular tissues. Methods Myopia was induced in White Leghorn chicks for 10 days, followed by up to 15 days of unrestricted vision (recovery). Expression of RALDH isoforms in chick ocular tissues was evaluated by Western blot. Catalytic activity of RALDH was measured in choroidal cytosol fractions using an in vitro atRA synthesis assay together with HPLC quantification of synthesized atRA. Distribution of RALDH2 cells throughout the choroid was evaluated by immunohistochemistry. Results RALDH2 was expressed predominately in the chick choroid (P < 0.001) and increased after 24 hours and 4 days of recovery (76%, 74%, and 165%, respectively; P < 0.05). Activity of RALDH was detected solely in the choroid and was elevated at 3 and 7 days of recovery compared to controls (70% and 48%, respectively; P < 0.05). The number of RALDH2 immunopositive cells in recovering choroids was increased at 24 hours and 4 to 15 days of recovery (P < 0.05) and were concentrated toward the RPE side compared to controls. Conclusions The results of this study suggest that RALDH2 is the major RALDH isoform in the chick choroid and is responsible for the increased RALDH activity seen during recovery. PMID:27654415

  8. Lack of Skeletal Muscle IL-6 Affects Pyruvate Dehydrogenase Activity at Rest and during Prolonged Exercise

    PubMed Central

    Gudiksen, Anders; Schwartz, Camilla Lindgren; Bertholdt, Lærke; Joensen, Ella; Knudsen, Jakob G.; Pilegaard, Henriette

    2016-01-01

    Pyruvate dehydrogenase (PDH) plays a key role in the regulation of skeletal muscle substrate utilization. IL-6 is produced in skeletal muscle during exercise in a duration dependent manner and has been reported to increase whole body fatty acid oxidation, muscle glucose uptake and decrease PDHa activity in skeletal muscle of fed mice. The aim of the present study was to examine whether muscle IL-6 contributes to exercise-induced PDH regulation in skeletal muscle. Skeletal muscle-specific IL-6 knockout (IL-6 MKO) mice and floxed littermate controls (control) completed a single bout of treadmill exercise for 10, 60 or 120 min, with rested mice of each genotype serving as basal controls. The respiratory exchange ratio (RER) was overall higher (P<0.05) in IL-6 MKO than control mice during the 120 min of treadmill exercise, while RER decreased during exercise independent of genotype. AMPK and ACC phosphorylation also increased with exercise independent of genotype. PDHa activity was in control mice higher (P<0.05) at 10 and 60 min of exercise than at rest but remained unchanged in IL-6 MKO mice. In addition, PDHa activity was higher (P<0.05) in IL-6 MKO than control mice at rest and 60 min of exercise. Neither PDH phosphorylation nor acetylation could explain the genotype differences in PDHa activity. Together, this provides evidence that skeletal muscle IL-6 contributes to the regulation of PDH at rest and during prolonged exercise and suggests that muscle IL-6 normally dampens carbohydrate utilization during prolonged exercise via effects on PDH. PMID:27327080

  9. The activity of liver alcohol dehydrogenase with nicotinamide–adenine dinucleotide phosphate as coenzyme

    PubMed Central

    Dalziel, K.; Dickinson, F. M.

    1965-01-01

    1. The separation of nucleotide impurities from commercial NADP preparations by chromatography is described. All the preparations studied contained 0·1–0·2% of NAD. 2. The activity of pure crystalline liver alcohol dehydrogenase with NADP as coenzyme has been confirmed. Initial-rate data are reported for the reaction at pH 6·0 and 7·0 with ethanol and acetaldehyde as substrates. With NADP and NADPH2 of high purity, the maximal specific rates were similar to those obtained with NAD and NADH2, but the Michaelis constants for the former coenzymes were much greater than those for the latter. 3. The oxidation of ethanol by NADP is greatly inhibited by NADH2, and this accounts for low values of certain initial-rate parameters obtained with commercial NADP preparations containing NAD. The kinetics of the inhibition are consistent with competitive inhibition in a compulsory-order mechanism. 4. Initial-rate data with NAD and NADPH2 do not conform to the requirements of the mechanism proposed by Theorell & Chance (1951), in contrast with results previously obtained with NAD and NADH2. The possibility that the deviations are due to competing nucleotide impurity in the oxidized coenzyme cannot be excluded. The data show that the enzyme reacts more slowly with, and has a smaller affinity for, NADP and NADPH2 than NAD and NADH2. 5. Phosphate behaves as a competitive inhibitor towards NADP. PMID:14340079

  10. Estrogen-related receptor alpha modulates lactate dehydrogenase activity in thyroid tumors.

    PubMed

    Mirebeau-Prunier, Delphine; Le Pennec, Soazig; Jacques, Caroline; Fontaine, Jean-Fred; Gueguen, Naig; Boutet-Bouzamondo, Nathalie; Donnart, Audrey; Malthièry, Yves; Savagner, Frédérique

    2013-01-01

    Metabolic modifications of tumor cells are hallmarks of cancer. They exhibit an altered metabolism that allows them to sustain higher proliferation rates in hostile environment outside the cell. In thyroid tumors, the expression of the estrogen-related receptor α (ERRα), a major factor of metabolic adaptation, is closely related to the oxidative metabolism and the proliferative status of the cells. To elucidate the role played by ERRα in the glycolytic adaptation of tumor cells, we focused on the regulation of lactate dehydrogenases A and B (LDHA, LDHB) and the LDHA/LDHB ratio. Our study included tissue samples from 10 classical and 10 oncocytic variants of follicular thyroid tumors and 10 normal thyroid tissues, as well as samples from three human thyroid tumor cell lines: FTC-133, XTC.UC1 and RO82W-1. We identified multiple cis-acting promoter elements for ERRα, in both the LDHA and LDHB genes. The interaction between ERRα and LDH promoters was confirmed by chromatin immunoprecipitation assays and in vitro analysis for LDHB. Using knock-in and knock-out cellular models, we found an inverse correlation between ERRα expression and LDH activity. This suggests that thyroid tumor cells may reprogram their metabolic pathways through the up-regulation of ERRα by a process distinct from that proposed by the recently revisited Warburg hypothesis.

  11. Estimating the number of viable animal cells in multi-well cultures based on their lactate dehydrogenase activities.

    PubMed

    Haslam, G; Wyatt, D; Kitos, P A

    2000-01-01

    A method is described for estimating the numbers ofanimal cells in multi-well culture by simultaneouslymeasuring the lactate dehydrogenase activity of thetotal culture and the medium. The difference betweenthe two reflects the dehydrogenase content of thecells and correlates with cell number. This LDH/INTmethod was tested using several lines of normal andtransformed suspension and adherent cells. Thelactate dehydrogenase activities of duplicate cultureswere determined colourimetrically using reactioncocktails containing lactate, NAD(+), diaphorase,and p-iodonitrotetrazolium violet, with and withoutTriton X-100. The difference in absorbance at 490 nm(DeltaA(490) = A(490, test) - A(490, control)) was used to calculate the lactatedehydrogenase activity of the total culture (+ Triton)and the medium (- Triton). The cellular lactatedehydrogenase activity (difference between totaland medium dehydrogenaseactivities) was proportional to viable cell number. The effects on cell growth of four metabolicinhibitors, sodium azide, actinomycin D,cycloheximide, and taxol, were determined using theLDH/INT assay and direct cell counting. The inhibitorconcentrations that caused decreases in the LDHactivity and cell number by 50% were similar. TheLDH/INT assay is quick and sensitive, works equallywell for adherent and suspension cells, and providesinformation about LDH activities of both the mediumand cells. It is particularly useful for screeningpotential cell-growth inhibitors. PMID:19002967

  12. Regulation of the activity of lactate dehydrogenases from four lactic acid bacteria.

    PubMed

    Feldman-Salit, Anna; Hering, Silvio; Messiha, Hanan L; Veith, Nadine; Cojocaru, Vlad; Sieg, Antje; Westerhoff, Hans V; Kreikemeyer, Bernd; Wade, Rebecca C; Fiedler, Tomas

    2013-07-19

    Despite high similarity in sequence and catalytic properties, the l-lactate dehydrogenases (LDHs) in lactic acid bacteria (LAB) display differences in their regulation that may arise from their adaptation to different habitats. We combined experimental and computational approaches to investigate the effects of fructose 1,6-bisphosphate (FBP), phosphate (Pi), and ionic strength (NaCl concentration) on six LDHs from four LABs studied at pH 6 and pH 7. We found that 1) the extent of activation by FBP (Kact) differs. Lactobacillus plantarum LDH is not regulated by FBP, but the other LDHs are activated with increasing sensitivity in the following order: Enterococcus faecalis LDH2 ≤ Lactococcus lactis LDH2 < E. faecalis LDH1 < L. lactis LDH1 ≤ Streptococcus pyogenes LDH. This trend reflects the electrostatic properties in the allosteric binding site of the LDH enzymes. 2) For L. plantarum, S. pyogenes, and E. faecalis, the effects of Pi are distinguishable from the effect of changing ionic strength by adding NaCl. 3) Addition of Pi inhibits E. faecalis LDH2, whereas in the absence of FBP, Pi is an activator of S. pyogenes LDH, E. faecalis LDH1, and L. lactis LDH1 and LDH2 at pH 6. These effects can be interpreted by considering the computed binding affinities of Pi to the catalytic and allosteric binding sites of the enzymes modeled in protonation states corresponding to pH 6 and pH 7. Overall, the results show a subtle interplay among the effects of Pi, FBP, and pH that results in different regulatory effects on the LDHs of different LABs.

  13. Lowering effect of firefly squid powder on triacylglycerol content and glucose-6-phosphate dehydrogenase activity in rat liver.

    PubMed

    Takeuchi, Hiroyuki; Morita, Ritsuko; Shirai, Yoko; Nakagawa, Yoshihisa; Terashima, Teruya; Ushikubo, Shun; Matsuo, Tatsuhiro

    2014-01-01

    Effects of dietary firefly squid on serum and liver lipid levels were investigated. Male Wistar rats were fed a diet containing 5% freeze-dried firefly squid or Japanese flying squid for 2 weeks. There was no significant difference in the liver triacylglycerol level between the control and Japanese flying squid groups, but the rats fed the firefly squid diet had a significantly lower liver triacylglycerol content than those fed the control diet. No significant difference was observed in serum triacylglycerol levels between the control and firefly squid groups. The rats fed the firefly squid had a significantly lower activity of liver glucose-6-phosphate dehydrogenase compared to the rats fed the control diet. There was no significant difference in liver fatty acid synthetase activity among the three groups. Hepatic gene expression and lipogenic enzyme activity were investigated; a DNA microarray showed that the significantly enriched gene ontology category of down-regulated genes in the firefly squid group was "lipid metabolic process". The firefly squid group had lower mRNA level of glucose-6-phosphate dehydrogenase compared to the controls. These results suggest that an intake of firefly squid decreases hepatic triacylglycerol in rats, and the reduction of mRNA level and enzyme activity of glucose-6-phosphate dehydrogenase might be related to the mechanisms.

  14. Display of Bombyx mori Alcohol Dehydrogenases on the Bacillus subtilis Spore Surface to Enhance Enzymatic Activity under Adverse Conditions

    PubMed Central

    Wang, Nan; Chang, Cheng; Yao, Qin; Li, Guohui; Qin, Lvgao; Chen, Liang; Chen, Keping

    2011-01-01

    Alcohol dehydrogenases (ADHs) are oxidoreductases catalyzing the reversible oxidation of alcohols to corresponding aldehydes or ketones accompanied by nicotinamide adenine dinucleotide (NAD) or nicotinamide adenine dinucleotide phosphate (NADP) as coenzyme. ADHs attract major scientific and industrial interest for the evolutionary perspectives, afforded by their wide occurrence in nature, and for their use in industrial synthesis. However, the low activity of ADHs under extremes of pH and temperature often limits their application. To obtain ADH with high activity, in this study, we used Bombyx mori alcohol dehydrogenases (BmADH) as foreign gene and constructed a recombinant integrative plasmid pJS700-BmADH. This pJS700-BmADH was transformed into Bacillus subtilis by double cross-over and produced an amylase inactivated mutant. The fusion protein containing BmADH was expressed on the spore surface and recognized by BmADH-specific antibody. We also assayed the alcohol dehydrogenase activity of the fusion protein together with the native BmADH at different pH and temperature levels, which indicated the recombinant enzyme exhibits activity over wider ranges of temperature and pH than its native form, perhaps due to the resistance properties of B. subtilis spores against adverse conditions. PMID:21738670

  15. Methylmalonate impairs mitochondrial respiration supported by NADH-linked substrates: involvement of mitochondrial glutamate metabolism.

    PubMed

    Melo, Daniela R; Mirandola, Sandra R; Assunção, Nilson A; Castilho, Roger F

    2012-06-01

    The neurodegeneration that occurs in methylmalonic acidemia is proposed to be associated with impairment of mitochondrial oxidative metabolism resulting from methylmalonate (MMA) accumulation. The present study evaluated the effects of MMA on oxygen consumption by isolated rat brain mitochondria in the presence of NADH-linked substrates (α-ketoglutarate, citrate, isocitrate, glutamate, malate, and pyruvate). Respiration supported either by glutamate or glutamate plus malate was significantly inhibited by MMA (1-10 mM), whereas no inhibition was observed when a cocktail of NADH-linked substrates was used. Measurements of glutamate transport revealed that the inhibitory effect of MMA on respiration maintained by this substrate is not due to inhibition of its mitochondrial uptake. In light of this result, the effect of MMA on the activity of relevant enzymes involved in mitochondrial glutamate metabolism was investigated. MMA had minor inhibitory effects on glutamate dehydrogenase and aspartate aminotransferase, whereas α-ketoglutarate dehydrogenase was significantly inhibited by this metabolite (K(i) = 3.65 mM). Moreover, measurements of α-ketoglutarate transport and mitochondrial MMA accumulation indicated that MMA/α-ketoglutarate exchange depletes mitochondria from this substrate, which may further contribute to the inhibition of glutamate-sustained respiration. To study the effect of chronic in vivo MMA treatment on mitochondrial function, young rats were intraperitoneally injected with MMA. No significant difference was observed in respiration between isolated brain mitochondria from control and MMA-treated rats, indicating that in vivo MMA treatment did not lead to permanent mitochondrial respiratory defects. Taken together, these findings indicate that the inhibitory effect of MMA on mitochondrial oxidative metabolism can be ascribed to concurrent inhibition of specific enzymes and lower availability of respiratory substrates. PMID:22488725

  16. Effect of metals and other inorganic ions on soil microbial activity: soil dehydrogenase assay as a simple toxicity test

    SciTech Connect

    Rogers, J.E.; Li, S.W.

    1985-06-01

    The purpose of this report is to illustrate the utility of the soil dehydrogenase assay as an effective primary test for assessing the potential toxicity of chemicals to soil microbial activity. In this manuscript the authors describe their use of the soil dehydrogenase assay in determining the effects of a number of potential toxic inorganic ions on soil microbial activity. The ions include Cu/sup 2 +/, Mg/sup 2 +/, Ni/sup 2 +/, Zn/sup 2 +/, NH/sub 4//sup +/, Cd/sup 2 +/, Cr/sup 32/, F/sup -/, AsO/sub 4//sup 3 -/, BO/sub 3//sup 3 -/, and SO/sub 4//sup 2 -/.

  17. Allelochemical L-DOPA induces quinoprotein adducts and inhibits NADH dehydrogenase activity and root growth of cucumber.

    PubMed

    Mushtaq, Muhammad Naeem; Sunohara, Yukari; Matsumoto, Hiroshi

    2013-09-01

    Allelochemical L-DOPA (l-3,4-dihydroxyphenylalanine) inhibits growth of several plant species. However, its mode of action is not well clarified in plants. The present studies were conducted to explore the action mechanism of L-DOPA in cucumber roots. The results revealed that L-DOPA suppressed the root growth of cucumber and induced quinoprotein and melanin formation in the roots. Moreover, L-DOPA not only decreased mitochondrial viability and NADH dehydrogenase (complex I) activity but also increased quinoprotein formation in vitro in isolated mitochondria from cucumber roots. Strong correlations were observed between quinoprotein formation and root growth inhibition, quinoprotein formation and NADH dehydrogenase activity, after L-DOPA treatment. The results suggest that quinoprotein formation and mitochondrial impairment might be involved in growth-inhibition mechanism of L-DOPA in cucumber roots. PMID:23831820

  18. The cannabinoid CB1 receptor biphasically modulates motor activity and regulates dopamine and glutamate release region dependently.

    PubMed

    Polissidis, Alexia; Galanopoulos, Andreas; Naxakis, George; Papahatjis, Demetris; Papadopoulou-Daifoti, Zeta; Antoniou, Katerina

    2013-03-01

    Cannabinoid administration modulates both dopaminergic and glutamatergic neurotransmission. The present study examines the effects of high and low dose WIN55,212-2, a CB1 receptor agonist, on extracellular dopamine and glutamate release in vivo via brain microdialysis in the nucleus accumbens (NAc), striatum and prefrontal cortex (PFC) in parallel to its effects on locomotor activity. WIN55,212-2 increased extracellular dopamine in the NAc (1 mg/kg i.p.), striatum (0.1 and 1 mg/kg i.p.) and PFC (1 mg/kg i.p.). Glutamate release was also elevated by WIN55,212-2 in the PFC (1 mg/kg i.p.) whereas in the NAc (0.1 and 1 mg/kg i.p.) and striatum, it was reduced (1 mg/kg i.p.). WIN55,212-2 administration produced hyperlocomotion at the lower dose (0.1 mg/kg i.p.) and hypolocomotion at the higher dose (1 mg/kg i.p.). Co-administration with the CB1 antagonist, SR-141716A (0.03 mg/kg i.p.), prevented the above effects. According to the present results, WIN55,212-2 affected locomotor activity biphasically while exerting converging effects on dopamine activity but diverging effects on glutamate release between cortical and subcortical regions, especially at the higher dose. These findings emphasize the involvement of the CB1 receptor in the simultaneous modulation of dopaminergic and glutamatergic neurotransmission in brain regions involved in reward and locomotion and suggest possible underlying mechanisms of acute cannabinoid exposure and its psychoactive and behavioural manifestations.

  19. Lysine and Arginine Reduce the Effects of Cerebral Ischemic Insults and Inhibit Glutamate-Induced Neuronal Activity in Rats

    PubMed Central

    Kondoh, Takashi; Kameishi, Makiko; Mallick, Hruda Nanda; Ono, Taketoshi; Torii, Kunio

    2010-01-01

    Intravenous administration of arginine was shown to be protective against cerebral ischemic insults via nitric oxide production and possibly via additional mechanisms. The present study aimed at evaluating the neuroprotective effects of oral administration of lysine (a basic amino acid), arginine, and their combination on ischemic insults (cerebral edema and infarction) and hemispheric brain swelling induced by transient middle cerebral artery occlusion/reperfusion in rats. Magnetic resonance imaging and 2,3,5-triphenyltetrazolium chloride staining were performed 2 days after ischemia induction. In control animals, the major edematous areas were observed in the cerebral cortex and striatum. The volumes associated with cortical edema were significantly reduced by lysine (2.0 g/kg), arginine (0.6 g/kg), or their combined administration (0.6 g/kg each). Protective effects of these amino acids on infarction were comparable to the inhibitory effects on edema formation. Interestingly, these amino acids, even at low dose (0.6 g/kg), were effective to reduce hemispheric brain swelling. Additionally, the effects of in vivo microiontophoretic (juxtaneuronal) applications of these amino acids on glutamate-evoked neuronal activity in the ventromedial hypothalamus were investigated in awake rats. Glutamate-induced neuronal activity was robustly inhibited by microiontophoretic applications of lysine or arginine onto neuronal membranes. Taken together, our results demonstrate the neuroprotective effects of oral ingestion of lysine and arginine against ischemic insults (cerebral edema and infarction), especially in the cerebral cortex, and suggest that suppression of glutamate-induced neuronal activity might be the primary mechanism associated with these neuroprotective effects. PMID:20589237

  20. Structure-Activity Relationship Studies and Biological Characterization of Human NAD+-dependent 15-Hydroxyprostaglandin Dehydrogenase Inhibitors

    PubMed Central

    Duveau, Damien Y.; Yasgar, Adam; Wang, Yuhong; Hu, Xin; Kouznetsova, Jennifer; Brimacombe, Kyle R.; Jadhav, Ajit; Simeonov, Anton; Thomas, Craig J.; Maloney, David J.

    2014-01-01

    The structure-activity relationship (SAR) study of two chemotypes identified as inhibitors of the human NAD+-dependent 15-hydroxyprostaglandin dehydrogenase (HPGD, 15-PGDH) was conducted. Top compounds from both series displayed potent inhibition (IC50 <50 nM), demonstrate excellent selectivity towards HPGD and potently induce PGE2 production in A549 lung cancer and LNCaP prostate cancer cells. PMID:24360556

  1. Structure-activity relationship studies and biological characterization of human NAD(+)-dependent 15-hydroxyprostaglandin dehydrogenase inhibitors.

    PubMed

    Duveau, Damien Y; Yasgar, Adam; Wang, Yuhong; Hu, Xin; Kouznetsova, Jennifer; Brimacombe, Kyle R; Jadhav, Ajit; Simeonov, Anton; Thomas, Craig J; Maloney, David J

    2014-01-15

    The structure-activity relationship (SAR) study of two chemotypes identified as inhibitors of the human NAD(+)-dependent 15-hydroxyprostaglandin dehydrogenase (HPGD, 15-PGDH) was conducted. Top compounds from both series displayed potent inhibition (IC50 <50 nM), demonstrate excellent selectivity towards HPGD and potently induce PGE2 production in A549 lung cancer and LNCaP prostate cancer cells. PMID:24360556

  2. Luteal 3beta-hydroxysteroid dehydrogenase and 20alpha-hydroxysteroid dehydrogenase activities in the rat corpus luteum of pseudopregnancy: Effect of the deciduoma reaction

    PubMed Central

    Clementi, Marisa A; Deis, Ricardo P; Telleria, Carlos M

    2004-01-01

    Background In the rat, the maintenance of gestation is dependent on progesterone production from the corpora lutea (CL), which are under the control of pituitary, decidual and placental hormones. The luteal metabolism of progesterone during gestation has been amply studied. However, the regulation of progesterone synthesis and degradation during pseudopregnancy (PSP), in which the CL are mainly under the control of pituitary prolactin (PRL), is not well known. The objectives of this investigation were: i) to study the luteal metabolism of progesterone during PSP by measuring the activities of the enzymes 3beta-hydroxysteroid dehydrogenase (3betaHSD), involved in progesterone biosynthesis, and that of 20alpha-hydroxysteroid dehydrogenase (20alphaHSD), involved in progesterone catabolism; and ii) to determine the role of decidualization on progesterone metabolism in PSP. Methods PSP was induced mechanically at 10:00 h on the estrus of 4-day cycling Wistar rats, and the stimulus for decidualization was provided by scratching the uterus on day 4 of PSP. 3betaHSD and 20alphaHSD activities were measured in the CL isolated from ovaries of PSP rats using a spectrophotometric method. Serum concentrations of progesterone, PRL, androstenedione, and estradiol were measured by radioimmunoassay (RIA). Results The PSP stage induced mechanically in cycling rats lasted 11.3 ± 0.09 days (n = 14). Serum progesterone concentration was high until day 10 of PSP, and declined thereafter. Serum PRL concentration was high on the first days of PSP but decreased significantly from days 6 to 9, having minimal values on days 10 and 11. Luteal 3betaHSD activities were elevated until day 6 of PSP, after which they progressively declined, reaching minimal values at the end of PSP. Luteal 20alphaHSD activities were very low until day 9, but abruptly increased at the end of PSP. When the deciduoma was induced by scratching the uterus of pseudopregnant animals on day 4 (PSP+D), PSP was extended to

  3. Inhibition of Cancer-Associated Mutant Isocitrate Dehydrogenases: Synthesis, Structure–Activity Relationship, and Selective Antitumor Activity

    PubMed Central

    2015-01-01

    Mutations of isocitrate dehydrogenase 1 (IDH1) are frequently found in certain cancers such as glioma. Different from the wild-type (WT) IDH1, the mutant enzymes catalyze the reduction of α-ketoglutaric acid to d-2-hydroxyglutaric acid (D2HG), leading to cancer initiation. Several 1-hydroxypyridin-2-one compounds were identified to be inhibitors of IDH1(R132H). A total of 61 derivatives were synthesized, and their structure–activity relationships were investigated. Potent IDH1(R132H) inhibitors were identified with Ki values as low as 140 nM, while they possess weak or no activity against WT IDH1. Activities of selected compounds against IDH1(R132C) were found to be correlated with their inhibitory activities against IDH1(R132H), as well as cellular production of D2HG, with R2 of 0.83 and 0.73, respectively. Several inhibitors were found to be permeable through the blood–brain barrier in a cell-based model assay and exhibit potent and selective activity (EC50 = 0.26–1.8 μM) against glioma cells with the IDH1 R132H mutation. PMID:25271760

  4. L-Malate dehydrogenase activity in the reductive arm of the incomplete citric acid cycle of Nitrosomonas europaea.

    PubMed

    Deutch, Charles E

    2013-11-01

    The autotrophic nitrifying bacterium Nitrosomonas europaea does not synthesize 2-oxoglutarate (α-ketoglutarate) dehydrogenase under aerobic conditions and so has an incomplete citric acid cycle. L-malate (S-malate) dehydrogenase (MDH) from N. europaea was predicted to show similarity to the NADP(+)-dependent enzymes from chloroplasts and was separated from the NAD(+)-dependent proteins from most other bacteria or mitochondria. MDH activity in a soluble fraction from N. europaea ATCC 19718 was measured spectrophotometrically and exhibited simple Michaelis-Menten kinetics. In the reductive direction, activity with NADH increased from pH 6.0 to 8.5 but activity with NADPH was consistently lower and decreased with pH. At pH 7.0, the K m for oxaloacetate was 20 μM; the K m for NADH was 22 μM but that for NADPH was at least 10 times higher. In the oxidative direction, activity with NAD(+) increased with pH but there was very little activity with NADP(+). At pH 7.0, the K m for L-malate was 5 mM and the K m for NAD(+) was 24 μM. The reductive activity was quite insensitive to inhibition by L-malate but the oxidative activity was very sensitive to oxaloacetate. MDH activity was not strongly activated or inhibited by glycolytic or citric acid cycle metabolites, adenine nucleotides, NaCl concentrations, or most metal ions, but increased with temperature up to about 55 °C. The reductive activity was consistently 10-20 times higher than the oxidative activity. These results indicate that the L-malate dehydrogenase in N. europaea is similar to other NAD(+)-dependent MDHs (EC 1.1.1.37) but physiologically adapted for its role in a reductive biosynthetic sequence.

  5. Isolation of an enzyme complex with carbon monoxide dehydrogenase activity containing corrinoid and nickel from acetate-grown Methanosarcina thermophila.

    PubMed Central

    Terlesky, K C; Nelson, M J; Ferry, J G

    1986-01-01

    Fast protein liquid chromatography of cell extract from methanol- or acetate-grown Methanosarcina thermophila resolved two peaks of CO dehydrogenase activity. The activity of one of the CO dehydrogenases was sixfold greater in acetate-grown compared with methanol-grown cells. This CO dehydrogenase was purified to apparent homogeneity (70 mumol of methyl viologen reduced per min per mg of protein) and made up greater than 10% of the cellular protein of acetate-grown cells. The native enzyme (Mr 250,000) formed aggregates with an Mr of approximately 1,000,000. The enzyme contained five subunits (Mrs 89,000, 71,000, 60,000, 58,000, and 19,000), suggesting a multifunctional enzyme complex. Nickel, iron, cobalt, zinc, inorganic sulfide, and a corrinoid were present in the complex. The UV-visible spectrum suggested the presence of iron-sulfur centers. The electron paramagnetic resonance spectrum contained g values of 2.073, 2.049, and 2.028; these features were broadened in enzyme that was purified from cells grown in the presence of medium enriched with 61Ni, indicating the involvement of this metal in the spectrum. The pattern of potassium cyanide inhibition indicated that cyanide binds at or near the CO binding site. The properties of the enzyme imply an involvement in the dissimilation of acetate to methane, possibly by cleavage of acetate or activated acetate. Images PMID:3023296

  6. Highly selective anti-Prelog synthesis of optically active aryl alcohols by recombinant Escherichia coli expressing stereospecific alcohol dehydrogenase.

    PubMed

    Li, Ming; Nie, Yao; Mu, Xiao Qing; Zhang, Rongzhen; Xu, Yan

    2016-07-01

    Biocatalytic asymmetric synthesis has been widely used for preparation of optically active chiral alcohols as the important intermediates and precursors of active pharmaceutical ingredients. However, the available whole-cell system involving anti-Prelog specific alcohol dehydrogenase is yet limited. A recombinant Escherichia coli system expressing anti-Prelog stereospecific alcohol dehydrogenase from Candida parapsilosis was established as a whole-cell system for catalyzing asymmetric reduction of aryl ketones to anti-Prelog configured alcohols. Using 2-hydroxyacetophenone as the substrate, reaction factors including pH, cell status, and substrate concentration had obvious impacts on the outcome of whole-cell biocatalysis, and xylose was found to be an available auxiliary substrate for intracellular cofactor regeneration, by which (S)-1-phenyl-1,2-ethanediol was achieved with an optical purity of 97%e.e. and yield of 89% under the substrate concentration of 5 g/L. Additionally, the feasibility of the recombinant cells toward different aryl ketones was investigated, and most of the corresponding chiral alcohol products were obtained with an optical purity over 95%e.e. Therefore, the whole-cell system involving recombinant stereospecific alcohol dehydrogenase was constructed as an efficient biocatalyst for highly enantioselective anti-Prelog synthesis of optically active aryl alcohols and would be promising in the pharmaceutical industry.

  7. Cryopreservation of glucose-6-phosphate dehydrogenase activity inside red blood cells: developing a specimen repository in support of development and evaluation of glucose-6-phosphate dehydrogenase deficiency tests

    PubMed Central

    2013-01-01

    Background Glucose-6-phosphate dehydrogenase (G6PD) deficiency is a common human enzyme deficiency. It is characterized by abnormally low levels of G6PD activity. Individuals with G6PD deficiency are at risk of undergoing acute haemolysis when exposed to 8‒aminoquinoline-based drugs, such as primaquine. For this reason it is imperative to identify individuals with G6PD deficiency prior to administering these anti-malarial drugs. There is a need for the development and evaluation of point-of-care G6PD deficiency screening tests suitable for areas of the developing world where malarial treatments are frequently administered. The development and evaluation of new G6PD tests will be greatly assisted with the availability of specimen repositories. Methods Cryopreservation of erythrocytes was evaluated as a means to preserve G6PD activity. Blood specimens from 31 patients including ten specimens with normal G6PD activity, three with intermediate activity, and 18 with deficient activity were cryopreserved for up to six months. Results Good correlation in G6PD activity between fresh and cryopreserved specimens (R2 = 0.95). The cryopreserved specimens show an overall small drop in mean G6PD activity of 0.23 U/g Hb (P=0.23). Cytochemical staining showed that intracellular G6PD activity distribution within the red blood cell populations is preserved during cryopreservation. Furthermore, the mosaic composition of red blood cells in heterozygous women is also preserved for six months or more. The fluorescent spot and the BinaxNOW qualitative tests for G6PD deficiency also showed high concordance in G6PD status determination between cryopreserved specimens and fresh specimens. Conclusions A methodology for establishing a specimen panel for evaluation of G6PD tests is described. The approach is similar to that used in several malaria research facilities for the cryopreservation of parasites in clinical specimens and axenic cultures. Specimens stored in this manner will aid

  8. Escherichia coli Pyruvate Dehydrogenase Complex Is an Important Component of CXCL10-Mediated Antimicrobial Activity

    PubMed Central

    Schutte, Kirsten M.; Fisher, Debra J.; Burdick, Marie D.; Mehrad, Borna; Mathers, Amy J.; Mann, Barbara J.; Nakamoto, Robert K.

    2015-01-01

    Chemokines are best recognized for their role within the innate immune system as chemotactic cytokines, signaling and recruiting host immune cells to sites of infection. Certain chemokines, such as CXCL10, have been found to play an additional role in innate immunity, mediating CXCR3-independent killing of a diverse array of pathogenic microorganisms. While this is still not clearly understood, elucidating the mechanisms underlying chemokine-mediated antimicrobial activity may facilitate the development of novel therapeutic strategies effective against antibiotic-resistant Gram-negative pathogens. Here, we show that CXCL10 exerts antibacterial effects on clinical and laboratory strains of Escherichia coli and report that disruption of pyruvate dehydrogenase complex (PDHc), which converts pyruvate to acetyl coenzyme A, enables E. coli to resist these antimicrobial effects. Through generation and screening of a transposon mutant library, we identified two mutants with increased resistance to CXCL10, both with unique disruptions of the gene encoding the E1 subunit of PDHc, aceE. Resistance to CXCL10 also occurred following deletion of either aceF or lpdA, genes that encode the remaining two subunits of PDHc. Although PDHc resides within the bacterial cytosol, electron microscopy revealed localization of immunogold-labeled CXCL10 to the bacterial cell surface in both the E. coli parent and aceE deletion mutant strains. Taken together, our findings suggest that while CXCL10 interacts with an as-yet-unidentified component on the cell surface, PDHc is an important mediator of killing by CXCL10. To our knowledge, this is the first description of PDHc as a key bacterial component involved in the antibacterial effect of a chemokine. PMID:26553462

  9. Escherichia coli Pyruvate Dehydrogenase Complex Is an Important Component of CXCL10-Mediated Antimicrobial Activity.

    PubMed

    Schutte, Kirsten M; Fisher, Debra J; Burdick, Marie D; Mehrad, Borna; Mathers, Amy J; Mann, Barbara J; Nakamoto, Robert K; Hughes, Molly A

    2016-01-01

    Chemokines are best recognized for their role within the innate immune system as chemotactic cytokines, signaling and recruiting host immune cells to sites of infection. Certain chemokines, such as CXCL10, have been found to play an additional role in innate immunity, mediating CXCR3-independent killing of a diverse array of pathogenic microorganisms. While this is still not clearly understood, elucidating the mechanisms underlying chemokine-mediated antimicrobial activity may facilitate the development of novel therapeutic strategies effective against antibiotic-resistant Gram-negative pathogens. Here, we show that CXCL10 exerts antibacterial effects on clinical and laboratory strains of Escherichia coli and report that disruption of pyruvate dehydrogenase complex (PDHc), which converts pyruvate to acetyl coenzyme A, enables E. coli to resist these antimicrobial effects. Through generation and screening of a transposon mutant library, we identified two mutants with increased resistance to CXCL10, both with unique disruptions of the gene encoding the E1 subunit of PDHc, aceE. Resistance to CXCL10 also occurred following deletion of either aceF or lpdA, genes that encode the remaining two subunits of PDHc. Although PDHc resides within the bacterial cytosol, electron microscopy revealed localization of immunogold-labeled CXCL10 to the bacterial cell surface in both the E. coli parent and aceE deletion mutant strains. Taken together, our findings suggest that while CXCL10 interacts with an as-yet-unidentified component on the cell surface, PDHc is an important mediator of killing by CXCL10. To our knowledge, this is the first description of PDHc as a key bacterial component involved in the antibacterial effect of a chemokine.

  10. Identification by mutagenesis of a conserved glutamate (Glu487) residue important for catalytic activity in rat liver carnitine palmitoyltransferase II.

    PubMed

    Zheng, Guolu; Dai, Jia; Woldegiorgis, Gebre

    2002-11-01

    Mammalian mitochondrial membranes express two active but distinct carnitine palmitoyltransferases: carnitine palmitoyltransferase I (CPTI), which is malonyl coA-sensitive and detergent-labile; and carnitine palmitoyltransferase II (CPTII), which is malonyl coA-insensitive and detergent-stable. To determine the role of the highly conserved C-terminal acidic residues glutamate 487 (Glu(487)) and glutamate 500 (Glu(500)) on catalytic activity in rat liver CPTII, we separately mutated these residues to alanine, aspartate, or lysine, and the effect of the mutations on CPTII activity was determined in the Escherichia coli-expressed mutants. Substitution of Glu(487) with alanine, aspartate, or lysine resulted in almost complete loss in CPTII activity. Because a conservative substitution mutation of this residue, Glu(487) with aspartate (E487D), resulted in a 97% loss in activity, we predicted that Glu(487) would be at the active-site pocket of CPTII. The substantial loss in CPTII activity observed with the E487K mutant, along with the previously reported loss in activity observed in a child with a CPTII deficiency disease, establishes that Glu(487) is crucial for maintaining the configuration of the liver isoform of the CPTII active site. Substitution of the conserved Glu(500) in CPTII with alanine or aspartate reduced the V(max) for both substrates, suggesting that Glu(500) may be important in stabilization of the enzyme-substrate complex. A conservative substitution of Glu(500) to aspartate resulted in a significant decrease in the V(max) for the substrates. Thus, Glu(500) may play a role in substrate binding and catalysis. Our site-directed mutagenesis studies demonstrate that Glu(487) in the liver isoform of CPTII is essential for catalysis. PMID:12200419

  11. 21 CFR 862.1445 - Lactate dehydrogenase isoenzymes test system.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... dehydrogenase isoenzymes test system is a device intended to measure the activity of lactate dehydrogenase isoenzymes (a group of enzymes with similar biological activity) in serum. Measurements of...

  12. 21 CFR 862.1445 - Lactate dehydrogenase isoenzymes test system.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... dehydrogenase isoenzymes test system is a device intended to measure the activity of lactate dehydrogenase isoenzymes (a group of enzymes with similar biological activity) in serum. Measurements of...

  13. 21 CFR 862.1445 - Lactate dehydrogenase isoenzymes test system.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... dehydrogenase isoenzymes test system is a device intended to measure the activity of lactate dehydrogenase isoenzymes (a group of enzymes with similar biological activity) in serum. Measurements of...

  14. 21 CFR 862.1445 - Lactate dehydrogenase isoenzymes test system.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... dehydrogenase isoenzymes test system is a device intended to measure the activity of lactate dehydrogenase isoenzymes (a group of enzymes with similar biological activity) in serum. Measurements of...

  15. 21 CFR 862.1445 - Lactate dehydrogenase isoenzymes test system.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... dehydrogenase isoenzymes test system is a device intended to measure the activity of lactate dehydrogenase isoenzymes (a group of enzymes with similar biological activity) in serum. Measurements of...

  16. Antipsychotic profiles of TASP0443294, a novel and orally active positive allosteric modulator of metabotropic glutamate 2 receptor.

    PubMed

    Hikichi, Hirohiko; Hiyoshi, Tetsuaki; Marumo, Toshiyuki; Tomishima, Yasumitsu; Kaku, Ayaka; Iida, Izumi; Urabe, Hiroki; Tamita, Tomoko; Yasuhara, Akito; Karasawa, Jun-ichi; Chaki, Shigeyuki

    2015-03-01

    Glutamatergic dysfunction has been implicated in psychiatric disorders such as schizophrenia. The stimulation of metabotropic glutamate (mGlu) 2 receptor has been shown to be effective in a number of animal models of schizophrenia. In this study, we investigated the antipsychotic profiles of (2S)-5-methyl-2-{[4-(1,1,1-trifluoro-2-methylpropan-2-yl)phenoxy]methyl}-2,3-dihydroimidazo[2,1-b][1,3]oxazole-6-carboxamide (TASP0443294), a newly synthesized positive allosteric modulator of the mGlu2 receptor. TASP0443294 potentiated the response of human mGlu2 and rat mGlu2 receptors to glutamate with EC50 values of 277 and 149 nM, respectively, without affecting the glutamate response of human mGlu3 receptor. TASP0443294 was distributed in the brain and cerebrospinal fluid after peroral administration in rats. The peroral administration of TASP0443294 inhibited methamphetamine-induced hyperlocomotion in rats, which was attenuated by an mGlu2/3 receptor antagonist, and improved social memory impairment induced by 5R,10S-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine hydrogen maleate (MK-801) in rats. Furthermore, TASP0443294 reduced the ketamine-induced basal gamma hyperactivity in the prefrontal cortex and suppressed rapid eye movement (REM) sleep in rats. These findings indicate that TASP0443294 is an mGlu2 receptor positive allosteric modulator with antipsychotic activity, and that the suppression of aberrant gamma oscillations and REM sleep could be considered as neurophysiological biomarkers for TASP0443294. PMID:25837934

  17. Activator Protein-1 Regulation of Murine Aldehyde Dehydrogenase 1a1

    PubMed Central

    Makia, N. L.; Amunom, I.; Falkner, K. C.; Conklin, D. J.; Surapureddi, S.; Goldstein, J. A.

    2012-01-01

    Previously we demonstrated that aldehyde dehydrogenase (ALDH) 1a1 is the major ALDH expressed in mouse liver and is an effective catalyst in metabolism of lipid aldehydes. Quantitative real-time polymerase chain reaction analysis revealed a ≈2.5- to 3-fold induction of the hepatic ALDH1A1 mRNA in mice administered either acrolein (5 mg/kg acrolein p.o.) or butylated hydroxylanisole (BHA) (0.45% in the diet) and of cytosolic NAD+-dependent ALDH activity. We observed ≈2-fold increases in ALDH1A1 mRNA levels in both Nrf2(+/+) and Nrf2(−/−) mice treated with BHA compared with controls, suggesting that BHA-induced expression is independent of nuclear factor E2-related factor 2 (Nrf2). The levels of activator protein-1 (AP-1) mRNA and protein, as well as the amount of phosphorylated c-Jun were significantly increased in mouse liver or Hepa1c1c7 cells treated with either BHA or acrolein. With use of luciferase reporters containing the 5′-flanking sequence of Aldh1a1 (−1963/+27), overexpression of c-Jun resulted in an ≈4-fold induction in luciferase activity, suggesting that c-Jun transactivates the Aldh1a1 promoter as a homodimer and not as a c-Jun/c-Fos heterodimer. Promoter deletion and mutagenesis analyses demonstrated that the AP-1 site at position −758 and possibly −1069 relative to the transcription start site was responsible for c-Jun-mediated transactivation. Electrophoretic mobility shift assay analysis with antibodies against c-Jun and c-Fos showed that c-Jun binds to the proximal AP-1 site at position −758 but not at −1069. Recruitment of c-Jun to this proximal AP-1 site by BHA was confirmed by chromatin immunoprecipitation analysis, indicating that recruitment of c-Jun to the mouse Aldh1a1 gene promoter results in increased transcription. This mode of regulation of an ALDH has not been described before. PMID:22740640

  18. Aldehyde dehydrogenase activity selects for the holoclone phenotype in prostate cancer cells

    SciTech Connect

    Doherty, R.E.; Haywood-Small, S.L.; Sisley, K.; Cross, N.A.

    2011-11-04

    Highlights: Black-Right-Pointing-Pointer Isolated ALDH{sup Hi} PC3 cells preferentially form primitive holoclone-type colonies. Black-Right-Pointing-Pointer Primitive holoclone colonies are predominantly ALDH{sup Lo} but contain rare ALDH{sup Hi} cells. Black-Right-Pointing-Pointer Holoclone-forming cells are not restricted to the ALDH{sup Hi} population. Black-Right-Pointing-Pointer ALDH phenotypic plasticity occurs in PC3 cells (ALDH{sup Lo} to ALDH{sup Hi} and vice versa). Black-Right-Pointing-Pointer ALDH{sup Hi} cells are observed but very rare in PC3 spheroids grown in stem cell medium. -- Abstract: Aldehyde dehydrogenase 1 (ALDH) activity is considered to be a marker of cancer stem cells (CSCs) in many tumour models, since these cells are more proliferative and tumourigenic than ALDH{sup Lo} cells in experimental models. However it is unclear whether all CSC-like cells are within the ALDH{sup Hi} population, or whether all ALDH{sup Hi} cells are highly proliferative and tumourigenic. The ability to establish a stem cell hierarchy in vitro, whereby sub-populations of cells have differing proliferative and differentiation capacities, is an alternate indication of the presence of stem cell-like populations within cell lines. In this study, we have examined the interaction between ALDH status and the ability to establish a stem cell hierarchy in PC3 prostate cancer cells. We demonstrate that PC3 cells contain a stem cell hierarchy, and isolation of ALDH{sup Hi} cells enriches for the most primitive holoclone population, however holoclone formation is not restricted to ALDH{sup Hi} cells. In addition, we show that ALDH activity undergoes phenotypic plasticity, since the ALDH{sup Lo} population can develop ALDH{sup Hi} populations comparable to parental cells within 2 weeks in culture. Furthermore, we show that the majority of ALDH{sup Hi} cells are found within the least primitive paraclone population, which is circumvented by culturing PC3 cells as spheroids in

  19. Vitamin E-Induced Changes in Glutamate and GABA Metabolizing Enzymes of Chick Embryo Cerebrum

    PubMed Central

    Dessai, Shanti N.; Pinto, Annaliza

    2013-01-01

    Vitamin E exists in eight different forms, four tocopherols and four tocotrienols. It forms an important component of our antioxidant system. The structure of Vitamin E makes it unique and indispensable in protecting cell membranes. α-tocopherol, one of the forms of Vitamin E, is also known to regulate signal transduction pathways by mechanisms that are independent of its antioxidant properties. Vitamin E compounds reduce the production of inflammatory compounds such as prostaglandins. Swollen, dystrophic axons are considered as the hallmark of Vitamin E deficiency in the brains of rats, monkeys, and humans. The present work aimed to study the Vitamin E- (α-tochopherol acetate-) induced alterations of enzymes involved in metabolism of Glutamate and GABA during developmental neurogenesis of cerebrum. Therefore, cytosolic and crude mitochondrial enzyme activities of glutamine synthetase, aspartate transaminase, alanine transaminase, GABA transaminase, succinic Semialdehyde dehydrogenase, glutamic dehydrogenase, and α-Ketoglutarate dehydrogenase were analysed. Vitamin E induced significant changes in these enzymes thus altering the normal levels of glutamate and GABA during developmental neurogenesis. Such changes are surely to disturb the expression and/or intensity of neurotransmitter signaling during critical periods of brain development. PMID:23984094

  20. A new role for α-ketoglutarate dehydrogenase complex: regulating metabolism through post-translational modification of other enzymes.

    PubMed

    McKenna, Mary C; Rae, Caroline D

    2015-07-01

    This Editorial highlights a study by Gibson et al. published in this issue of JNeurochem, in which the authors reveal a novel role for the α-ketoglutarate dehydrogenase complex (KGDHC) in post-translational modification of proteins. KGDHC may catalyze post-translational modification of itself as well as several other proteins by succinylation of lysine residues. The authors' report of an enzyme responsible for succinylation of key mitochondrial enzymes represents a major step toward our understanding of the complex functional metabolome. TCA, tricarboxylic acid; KG, α-ketoglutarate; KGDHC, α-ketoglutarate dehydrogenase complex; FUM, fumarase; MDH, malate dehydrogenase; ME, malic enzyme; GDH, glutamate dehydrogenase; AAT, aspartate aminotransferase; GS, glutamine synthetase; PAG, phosphate-activated glutaminase; SIRT3, silent information regulator 3; SIRT5, silent information regulator 5. PMID:26052752

  1. Neuroprotection Promoted by Guanosine Depends on Glutamine Synthetase and Glutamate Transporters Activity in Hippocampal Slices Subjected to Oxygen/Glucose Deprivation.

    PubMed

    Dal-Cim, Tharine; Martins, Wagner C; Thomaz, Daniel T; Coelho, Victor; Poluceno, Gabriela Godoy; Lanznaster, Débora; Vandresen-Filho, Samuel; Tasca, Carla I

    2016-05-01

    Guanosine (GUO) has been shown to act as a neuroprotective agent against glutamatergic excitotoxicity by increasing glutamate uptake and decreasing its release. In this study, a putative effect of GUO action on glutamate transporters activity modulation was assessed in hippocampal slices subjected to oxygen and glucose deprivation (OGD), an in vitro model of brain ischemia. Slices subjected to OGD showed increased excitatory amino acids release (measured by D-[(3)H]aspartate release) that was prevented in the presence of GUO (100 µM). The glutamate transporter blockers, DL-TBOA (10 µM), DHK (100 µM, selective inhibitor of GLT-1), and sulfasalazine (SAS, 250 µM, Xc(-) system inhibitor) decreased OGD-induced D-aspartate release. Interestingly, DHK or DL-TBOA blocked the decrease in glutamate release induced by GUO, whereas SAS did not modify the GUO effect. GUO protected hippocampal slices from cellular damage by modulation of glutamate transporters, however selective blockade of GLT-1 or Xc- system only did not affect this protective action of GUO. OGD decreased hippocampal glutamine synthetase (GS) activity and GUO recovered GS activity to control levels without altering the kinetic parameters of GS activity, thus suggesting GUO does not directly interact with GS. Additionally, the pharmacological inhibition of GS activity with methionine sulfoximine abolished the effect of GUO in reducing D-aspartate release and cellular damage evoked by OGD. Altogether, results in hippocampal slices subjected to OGD show that GUO counteracts the release of excitatory amino acids, stimulates the activity of GS, and decreases the cellular damage by modulation of glutamate transporters activity.

  2. Xanthurenic Acid Activates mGlu2/3 Metabotropic Glutamate Receptors and is a Potential Trait Marker for Schizophrenia.

    PubMed

    Fazio, Francesco; Lionetto, Luana; Curto, Martina; Iacovelli, Luisa; Cavallari, Michele; Zappulla, Cristina; Ulivieri, Martina; Napoletano, Flavia; Capi, Matilde; Corigliano, Valentina; Scaccianoce, Sergio; Caruso, Alessandra; Miele, Jessica; De Fusco, Antonio; Di Menna, Luisa; Comparelli, Anna; De Carolis, Antonella; Gradini, Roberto; Nisticò, Robert; De Blasi, Antonio; Girardi, Paolo; Bruno, Valeria; Battaglia, Giuseppe; Nicoletti, Ferdinando; Simmaco, Maurizio

    2015-12-08

    The kynurenine pathway of tryptophan metabolism has been implicated in the pathophysiology of psychiatric disorders, including schizophrenia. We report here that the kynurenine metabolite, xanturenic acid (XA), interacts with, and activates mGlu2 and mGlu3 metabotropic glutamate receptors in heterologous expression systems. However, the molecular nature of this interaction is unknown, and our data cannot exclude that XA acts primarily on other targets, such as the vesicular glutamate transporter, in the CNS. Systemic administration of XA in mice produced antipsychotic-like effects in the MK-801-induced model of hyperactivity. This effect required the presence of mGlu2 receptors and was abrogated by the preferential mGlu2/3 receptor antagonist, LY341495. Because the mGlu2 receptor is a potential drug target in the treatment of schizophrenia, we decided to measure serum levels of XA and other kynurenine metabolites in patients affected by schizophrenia. Serum XA levels were largely reduced in a large cohort of patients affected by schizophrenia, and, in patients with first-episode schizophrenia, levels remained low after 12 months of antipsychotic medication. As opposed to other kynurenine metabolites, XA levels were also significantly reduced in first-degree relatives of patients affected by schizophrenia. We suggest that lowered serum XA levels might represent a novel trait marker for schizophrenia.

  3. Xanthurenic Acid Activates mGlu2/3 Metabotropic Glutamate Receptors and is a Potential Trait Marker for Schizophrenia.

    PubMed

    Fazio, Francesco; Lionetto, Luana; Curto, Martina; Iacovelli, Luisa; Cavallari, Michele; Zappulla, Cristina; Ulivieri, Martina; Napoletano, Flavia; Capi, Matilde; Corigliano, Valentina; Scaccianoce, Sergio; Caruso, Alessandra; Miele, Jessica; De Fusco, Antonio; Di Menna, Luisa; Comparelli, Anna; De Carolis, Antonella; Gradini, Roberto; Nisticò, Robert; De Blasi, Antonio; Girardi, Paolo; Bruno, Valeria; Battaglia, Giuseppe; Nicoletti, Ferdinando; Simmaco, Maurizio

    2015-01-01

    The kynurenine pathway of tryptophan metabolism has been implicated in the pathophysiology of psychiatric disorders, including schizophrenia. We report here that the kynurenine metabolite, xanturenic acid (XA), interacts with, and activates mGlu2 and mGlu3 metabotropic glutamate receptors in heterologous expression systems. However, the molecular nature of this interaction is unknown, and our data cannot exclude that XA acts primarily on other targets, such as the vesicular glutamate transporter, in the CNS. Systemic administration of XA in mice produced antipsychotic-like effects in the MK-801-induced model of hyperactivity. This effect required the presence of mGlu2 receptors and was abrogated by the preferential mGlu2/3 receptor antagonist, LY341495. Because the mGlu2 receptor is a potential drug target in the treatment of schizophrenia, we decided to measure serum levels of XA and other kynurenine metabolites in patients affected by schizophrenia. Serum XA levels were largely reduced in a large cohort of patients affected by schizophrenia, and, in patients with first-episode schizophrenia, levels remained low after 12 months of antipsychotic medication. As opposed to other kynurenine metabolites, XA levels were also significantly reduced in first-degree relatives of patients affected by schizophrenia. We suggest that lowered serum XA levels might represent a novel trait marker for schizophrenia. PMID:26643205

  4. Xanthurenic Acid Activates mGlu2/3 Metabotropic Glutamate Receptors and is a Potential Trait Marker for Schizophrenia

    PubMed Central

    Fazio, Francesco; Lionetto, Luana; Curto, Martina; Iacovelli, Luisa; Cavallari, Michele; Zappulla, Cristina; Ulivieri, Martina; Napoletano, Flavia; Capi, Matilde; Corigliano, Valentina; Scaccianoce, Sergio; Caruso, Alessandra; Miele, Jessica; De Fusco, Antonio; Di Menna, Luisa; Comparelli, Anna; De Carolis, Antonella; Gradini, Roberto; Nisticò, Robert; De Blasi, Antonio; Girardi, Paolo; Bruno, Valeria; Battaglia, Giuseppe; Nicoletti, Ferdinando; Simmaco, Maurizio

    2015-01-01

    The kynurenine pathway of tryptophan metabolism has been implicated in the pathophysiology of psychiatric disorders, including schizophrenia. We report here that the kynurenine metabolite, xanturenic acid (XA), interacts with, and activates mGlu2 and mGlu3 metabotropic glutamate receptors in heterologous expression systems. However, the molecular nature of this interaction is unknown, and our data cannot exclude that XA acts primarily on other targets, such as the vesicular glutamate transporter, in the CNS. Systemic administration of XA in mice produced antipsychotic-like effects in the MK-801-induced model of hyperactivity. This effect required the presence of mGlu2 receptors and was abrogated by the preferential mGlu2/3 receptor antagonist, LY341495. Because the mGlu2 receptor is a potential drug target in the treatment of schizophrenia, we decided to measure serum levels of XA and other kynurenine metabolites in patients affected by schizophrenia. Serum XA levels were largely reduced in a large cohort of patients affected by schizophrenia, and, in patients with first-episode schizophrenia, levels remained low after 12 months of antipsychotic medication. As opposed to other kynurenine metabolites, XA levels were also significantly reduced in first-degree relatives of patients affected by schizophrenia. We suggest that lowered serum XA levels might represent a novel trait marker for schizophrenia. PMID:26643205

  5. Curcumin attenuates glutamate neurotoxicity in the hippocampus by suppression of ER stress-associated TXNIP/NLRP3 inflammasome activation in a manner dependent on AMPK

    SciTech Connect

    Li, Ying; Li, Jia; Li, Shanshan; Li, Yi; Wang, Xiangxiang; Liu, Baolin; Fu, Qiang; Ma, Shiping

    2015-07-01

    Curcumin is a natural polyphenolic compound in Curcuma longa with beneficial effects on neuronal protection. This study aims to investigate the action of curcumin in the hippocampus subjected to glutamate neurotoxicity. Glutamate stimulation induced reactive oxygen species (ROS), endoplasmic reticulum stress (ER stress) and TXNIP/NLRP3 inflammasome activation, leading to damage in the hippocampus. Curcumin treatment in the hippocampus or SH-SY5Y cells inhibited IRE1α and PERK phosphorylation with suppression of intracellular ROS production. Curcumin increased AMPK activity and knockdown of AMPKα with specific siRNA abrogated its inhibitory effects on IRE1α and PERK phosphorylation, indicating that AMPK activity was essential for the suppression of ER stress. As a result, curcumin reduced TXNIP expression and inhibited NLRP3 inflammasome activation by downregulation of NLRP3 and cleaved caspase-1 induction, and thus reduced IL-1β secretion. Specific fluorescent probe and flow cytometry analysis showed that curcumin prevented mitochondrial malfunction and protected cell survival from glutamate neurotoxicity. Moreover, oral administration of curcumin reduced brain infarct volume and attenuated neuronal damage in rats subjected to middle cerebral artery occlusion. Immunohistochemistry showed that curcumin inhibited p-IRE1α, p-PERK and NLRP3 expression in hippocampus CA1 region. Together, these results showed that curcumin attenuated glutamate neurotoxicity by inhibiting ER stress-associated TXNIP/NLRP3 inflammasome activation via the regulation of AMPK, and thereby protected the hippocampus from ischemic insult. - Highlights: • Curcumin attenuates glutamate neurotoxicity in the hippocampus. • Curcumin suppresses ER stress in glutamate-induced hippocampus slices. • Curcumin inhibits TXNIP/NLRP3 inflammasome activation. • Regulation of AMPK by curcumin contributes to suppressing ER stress.

  6. Behaviour of mesotrione in maize and soil system and its influence on soil dehydrogenase activity.

    PubMed

    Kaczynski, Piotr; Lozowicka, Bozena; Hrynko, Izabela; Wolejko, Elzbieta

    2016-11-15

    The aim of this study was to investigate the dissipation of mesotrione and effect on dehydrogenase activity (DHA) in maize and soil system. The paper for the first time describes behaviour of this herbicide applied at various doses (separately or in mixture with other herbicide) in acidic and alkaline environment. The experiments were conducted using the method randomized blocks in four repetition cycles. Chemical application in seven variants at recommended doses of herbicide were performed. The sample preparation was performed by a modified QuEChERS method and the concentrations of mesotrione in maize and soil were determined by the liquid chromatography with tandem mass spectrometry (LC-MS/MS). The limit of detection was 0.0005mgkg(-1) and quantification 0.001mgkg(-1). The dissipation of mesotrione were described according to first-order (FO) kinetics equation with R(2) were between 0.8794 and 0.9934. The initial deposit of herbicide in soil and maize was higher in an acidic environment (0.06-0.18mgkg(-1)). A positive correlation between an alkaline pH and the rate of dissipation in soil was observed. The results showed that the time after which 50% (DT50) of substance has been degraded was different for both plant and soil. DT50 for soil was within the range 3.2-6.0days and 2.9-4.4days, for the maize 3.9-4.8days and 3.4-4.5days in an alkaline and an acidic environment, respectively. Concentration of mesotrione at applicable MRL level of 0.05mgkg(-1) in maize was achieved at 0.5-5.9days and at proposed MRL of 0.01mgkg(-1) at 8.8-15.8days. The results indicate that the application of mesotrione affected on DHA in the soil. One day after application this herbicide, concentration of DHA in soil was lower than in control plots, but after 21days was observed trend of increasing DHA. PMID:27492351

  7. Liposomal encapsulation of yeast alcohol dehydrogenase with cofactor for stabilization of the enzyme structure and activity.

    PubMed

    Yoshimoto, Makoto; Sato, Mami; Yoshimoto, Noriko; Nakao, Katsumi

    2008-01-01

    Yeast alcohol dehydrogenase (YADH) with its cofactor nicotinamide adenine dinucleotide (NAD+) could be stably encapsulated in liposomes composed of POPC (1-palmitoyl-2-oleoyl-sn-glycero-3- phosphocholine). The YADH- and NAD+-containing liposomes (YADH-NADL) were 100 nm in mean diameter. The liposomal YADH and NAD+ concentrations were 2.3 mg/mL and 3.9 mM, respectively. A synergistic effect of the liposomal encapsulation and the presence of NAD+ was examined on the thermal stability of YADH at 45 and 50 degrees C. The enzyme stability of the YADH-NADL was compared to the stabilities of the liposomal YADH (YADHL) containing 3.3 mg/mL YADH without NAD+ as well as the free YADH with and without NAD+. Free YADH was increasingly deactivated during its incubation at 45 degrees C for 2 h with decrease of the enzyme concentration from 3.3 to 0.01 mg/mL because of the dissociation of tetrameric YADH into its subunits. At that temperature, the coexistence of free NAD+ at 3.9 mM improved the stability of free YADH at 2.3 mg/mL through forming their thermostable complex, although the stabilization effect of NAD+ was lowered at 50 degrees C. The turbidity measurements for the above free YADH solution with and without NAD+ revealed that the change in the enzyme tertiary structure was much more pronounced at 50 degrees C than at 45 degrees C even in the presence of NAD+. This suggests that YADH was readily deactivated in free solution due to a decrease in the inherent affinity of YADH with NAD+. On the other hand, both liposomal enzyme systems, YADH-NADL and YADHL, showed stabilities at both 45 and 50 degrees C much higher than those of the above free enzyme systems, YADH/NAD+ and YADH. These results imply that the liposome membranes stabilized the enzyme tertiary and thus quaternary structures. Furthermore, the enzyme activity of the YADH-NADL showed a stability higher than that of the YADHL with a more remarkable effect of NAD+ at 50 degrees C than at 45 degrees C. This was

  8. Exploring the active conformation of cyclohexane carboxylate positive allosteric modulators of the type 4 metabotropic glutamate receptor.

    PubMed

    Rovira, Xavier; Harrak, Youssef; Trapero, Ana; González-Bulnes, Patricia; Malhaire, Fanny; Pin, Jean-Philippe; Goudet, Cyril; Giraldo, Jesús; Llebaria, Amadeu

    2014-12-01

    The active conformation of a family of metabotropic glutamate receptor subtype 4 (mGlu4 ) positive allosteric modulators (PAMs) with the cyclohexane 1,2-dicarboxylic scaffold present in cis-2-(3,5-dichlorophenylcarbamoyl)cyclohexanecarboxylic acid (VU0155041) was investigated by testing structurally similar six-membered ring compounds that have a locked conformation. The norbornane and cyclohexane molecules designed as mGlu4 conformational probes and the enantiomers of the trans diastereomer were computationally characterized and tested in mGlu4 pharmacological assays. The results support a VU0155041 active conformation, with the chair cyclohexane having the aromatic amide substituent in an axial position and the carboxylate in an equatorial position. Moreover, the receptor displays enantiomeric discrimination of the chiral PAMs. The constructed pharmacophore characterized a highly constrained mGlu4 allosteric binding site, thus providing a step forward in structure-based drug design for mGlu4 PAMs.

  9. Enhanced degradation of benzene by percarbonate activated with Fe(II)-glutamate complex.

    PubMed

    Fu, Xiaori; Gu, Xiaogang; Lu, Shuguang; Miao, Zhouwei; Xu, Minhui; Zhang, Xiang; Danish, Muhammad; Cui, Hang; Farooq, Usman; Qiu, Zhaofu; Sui, Qian

    2016-04-01

    Effective degradation of benzene was achieved in sodium percarbonate (SPC)/Fe(II)-Glu system. The presence of glutamate (Glu) could enhance the regeneration of Fe(III) to Fe(II), which ensures the benzene degradation efficiency at wider pH range and eliminate the influence of HCO3 (-) in low concentration. Meanwhile, the significant scavenging effects of high HCO3 (-) concentration could also be overcome by increasing the Glu/SPC/Fe(II)/benzene molar ratio. Free radical probe compound tests, free radical scavenger tests, and electron paramagnetic resonance (EPR) analysis were conducted to explore the reaction mechanism for benzene degradation, in which hydroxyl radical (HO•) and superoxide anion radical (O2 (•-)) were confirmed as the predominant species responsible for benzene degradation. In addition, the results obtained in actual groundwater test strongly indicated that SPC/Fe(II)-Glu system is applicable for the remediation of benzene-contaminated groundwater in practice. PMID:26662563

  10. Enhanced degradation of benzene by percarbonate activated with Fe(II)-glutamate complex.

    PubMed

    Fu, Xiaori; Gu, Xiaogang; Lu, Shuguang; Miao, Zhouwei; Xu, Minhui; Zhang, Xiang; Danish, Muhammad; Cui, Hang; Farooq, Usman; Qiu, Zhaofu; Sui, Qian

    2016-04-01

    Effective degradation of benzene was achieved in sodium percarbonate (SPC)/Fe(II)-Glu system. The presence of glutamate (Glu) could enhance the regeneration of Fe(III) to Fe(II), which ensures the benzene degradation efficiency at wider pH range and eliminate the influence of HCO3 (-) in low concentration. Meanwhile, the significant scavenging effects of high HCO3 (-) concentration could also be overcome by increasing the Glu/SPC/Fe(II)/benzene molar ratio. Free radical probe compound tests, free radical scavenger tests, and electron paramagnetic resonance (EPR) analysis were conducted to explore the reaction mechanism for benzene degradation, in which hydroxyl radical (HO•) and superoxide anion radical (O2 (•-)) were confirmed as the predominant species responsible for benzene degradation. In addition, the results obtained in actual groundwater test strongly indicated that SPC/Fe(II)-Glu system is applicable for the remediation of benzene-contaminated groundwater in practice.

  11. Long-Term Activation of Group I Metabotropic Glutamate Receptors Increases Functional TRPV1-Expressing Neurons in Mouse Dorsal Root Ganglia

    PubMed Central

    Masuoka, Takayoshi; Kudo, Makiko; Yoshida, Junko; Ishibashi, Takaharu; Muramatsu, Ikunobu; Kato, Nobuo; Imaizumi, Noriko; Nishio, Matomo

    2016-01-01

    Damaged tissues release glutamate and other chemical mediators for several hours. These chemical mediators contribute to modulation of pruritus and pain. Herein, we investigated the effects of long-term activation of excitatory glutamate receptors on functional expression of transient receptor potential vaniloid type 1 (TRPV1) in dorsal root ganglion (DRG) neurons and then on thermal pain behavior. In order to detect the TRPV1-mediated responses in cultured DRG neurons, we monitored intracellular calcium responses to capsaicin, a TRPV1 agonist, with Fura-2. Long-term (4 h) treatment with glutamate receptor agonists (glutamate, quisqualate or DHPG) increased the proportion of neurons responding to capsaicin through activation of metabotropic glutamate receptor mGluR1, and only partially through the activation of mGluR5; engagement of these receptors was evident in neurons responding to allylisothiocyanate (AITC), a transient receptor potential ankyrin type 1 (TRPA1) agonist. Increase in the proportion was suppressed by phospholipase C (PLC), protein kinase C, mitogen/extracellular signal-regulated kinase, p38 mitogen-activated protein kinase or transcription inhibitors. Whole-cell recording was performed to record TRPV1-mediated membrane current; TRPV1 current density significantly increased in the AITC-sensitive neurons after the quisqualate treatment. To elucidate the physiological significance of this phenomenon, a hot plate test was performed. Intraplantar injection of quisqualate or DHPG induced heat hyperalgesia that lasted for 4 h post injection. This chronic hyperalgesia was attenuated by treatment with either mGluR1 or mGluR5 antagonists. These results suggest that long-term activation of mGluR1/5 by peripherally released glutamate may increase the number of neurons expressing functional TRPV1 in DRG, which may be strongly associated with chronic hyperalgesia. PMID:27064319

  12. The cystine/glutamate antiporter regulates indoleamine 2,3-dioxygenase protein levels and enzymatic activity in human dendritic cells.

    PubMed

    Mattox, Mildred L; D'Angelo, June A; Grimes, Zachary M; Fiebiger, Edda; Dickinson, Bonny L

    2012-11-30

    Indoleamine 2,3-dioxygenase (IDO) is the rate-limiting enzyme in the tryptophan-catabolizing pathway and a key regulator of peripheral immune tolerance. As the suppressive effects of IDO are predominantly mediated by dendritic cells (DCs) and IDO-competent DCs promote long-term immunologic tolerance, a detailed understanding of how IDO expression and activity is regulated in these cells is central to the rational design of therapies to induce robust immune tolerance. We previously reported that the cystine/glutamate antiporter modulates the functional expression of IDO in human monocyte-derived DCs. Specifically, we showed that blocking antiporter uptake of cystine significantly increased both IDO mRNA and IDO enzymatic activity and that this correlated with impaired DC presentation of exogenous antigen to T cells via MHC class II and the cross-presentation pathway. The antiporter regulates intracellular and extracellular redox by transporting cystine into the cell in exchange for glutamate. Intracellular cystine is reduced to cysteine to support biosynthesis of the major cellular antioxidant glutathione and cysteine is exported from the cell where it functions as an extracellular antioxidant. Here we show that antiporter control of IDO expression in DCs is reversible, independent of interferon-γ, regulated by redox, and requires active protein synthesis. These findings highlight a role for antiporter regulation of cellular redox as a critical control point for modulating IDO expression and activity in DCs. Thus, systemic disease and aging, processes that perturb redox homeostasis, may adversely affect immunity by promoting the generation of IDO-competent DCs.

  13. The cystine/glutamate antiporter regulates indoleamine 2,3-dioxygenase protein levels and enzymatic activity in human dendritic cells

    PubMed Central

    Mattox, Mildred L; D’Angelo, June A; Grimes, Zachary M; Fiebiger, Edda; Dickinson, Bonny L

    2012-01-01

    Indoleamine 2,3-dioxygenase (IDO) is the rate-limiting enzyme in the tryptophan-catabolizing pathway and a key regulator of peripheral immune tolerance. As the suppressive effects of IDO are predominantly mediated by dendritic cells (DCs) and IDO-competent DCs promote long-term immunologic tolerance, a detailed understanding of how IDO expression and activity is regulated in these cells is central to the rational design of therapies to induce robust immune tolerance. We previously reported that the cystine/glutamate antiporter modulates the functional expression of IDO in human monocyte-derived DCs. Specifically, we showed that blocking antiporter uptake of cystine significantly increased both IDO mRNA and IDO enzymatic activity and that this correlated with impaired DC presentation of exogenous antigen to T cells via MHC class II and the cross-presentation pathway. The antiporter regulates intracellular and extracellular redox by transporting cystine into the cell in exchange for glutamate. Intracellular cystine is reduced to cysteine to support biosynthesis of the major cellular antioxidant glutathione and cysteine is exported from the cell where it functions as an extracellular antioxidant. Here we show that antiporter control of IDO expression in DCs is reversible, independent of interferon-γ, regulated by redox, and requires active protein synthesis. These findings highlight a role for antiporter regulation of cellular redox as a critical control point for modulating IDO expression and activity in DCs. Thus, systemic disease and aging, processes that perturb redox homeostasis, may adversely affect immunity by promoting the generation of IDO-competent DCs. PMID:23243629

  14. Temperature and enzyme activity in poikilotherms. Isocitrate dehydrogenases in rainbow-trout liver

    PubMed Central

    Moon, Thomas W.; Hochachka, P. W.

    1971-01-01

    1. The kinetics of the thermally induced enzyme variants of the supernatant NADP–isocitrate dehydrogenase from rainbow-trout liver are investigated. 2. Fish acclimatized to 2°C (cold-adapted enzyme) and 17°C (warm-adapted enzyme) show different relative distributions of the three NADP–isocitrate dehydrogenase isoenzymes; this has been demonstrated with electrophoresis and electrofocusing techniques. 3. Plots of Km versus temperature for the cold-adapted and warm-adapted enzyme variants are complex in nature with apparent maximal enzyme–substrate affinity corresponding to the temperature at which the trout is acclimatized. Both substrates, dl-isocitrate and NADP+, give similar curves although the magnitude of the Km change with temperature is much decreased in the case of NADP+. 4. Ea values of approx. 18kcal/mol were determined for both the cold-adapted and warm-adapted enzyme variants. 5. In an attempt to determine how velocities can be increased at low temperatures, cation, pH requirements, metabolite and enzyme concentrations were examined. 6. NAD–isocitrate dehydrogenase could not be detected in trout tissues. ImagesFig. 1. PMID:4399398

  15. Activation of group II metabotropic glutamate receptors underlies microglial reactivity and neurotoxicity following stimulation with chromogranin A, a peptide up-regulated in Alzheimer's disease.

    PubMed

    Taylor, D L; Diemel, L T; Cuzner, M L; Pocock, J M

    2002-09-01

    Regulation of microglial reactivity and neurotoxicity is critical for neuroprotection in neurodegenerative diseases. Here we report that microglia possess functional group II metabotropic glutamate receptors, expressing mRNA and receptor protein for mGlu2 and mGlu3, negatively coupled to adenylate cyclase. Two different agonists of these receptors were able to induce a neurotoxic microglial phenotype which was attenuated by a specific antagonist. Chromogranin A, a secretory peptide expressed in amyloid plaques in Alzheimer's disease, activates microglia to a reactive neurotoxic phenotype. Chromogranin A-induced microglial activation and subsequent neurotoxicity may also involve an underlying stimulation of group II metabotropic glutamate receptors since their inhibition reduced chromogranin A-induced microglial reactivity and neurotoxicity. These results show that selective inhibition of microglial group II metabotropic glutamate receptors has a positive impact on neuronal survival, and may prove a therapeutic target in Alzheimer's disease. PMID:12358765

  16. Effect of some anthelmintics on malate dehydrogenase activity and mortality in two avian nematodes Ascaridia galli and Heterakis gallinae.

    PubMed

    Sharma, R K; Singh, K; Saxena, R; Saxena, K K

    1986-08-01

    Cambendazole and tiabendazole at 10(-4) M concentrations caused mortality in both the parasites after 10 min and 20 min, respectively. H. gallinae was killed by 10(-4) M haloxon but A. galli remained alive even after 60 min exposure. The effect of these drugs was found to be irreversible since no resumption of activity was observed when the parasites were returned to normal saline solution. The ratio of oxaloacetate reduction to malate oxidation in the homogenates of A. galli and H. gallinae was 4.38:1 and 3.17:1 respectively. Cambendazole at 10(-3) M inhibited the enzymic activity in both directions by 100% in both A. galli and H. gallinae. 10(-3) M tiabendazole, however, inhibited the malate oxidation by 82.8 and 60.8% and oxaloacetate reduction by 76.6 and 92% in A. galli and H. gallinae, respectively. Haloxon had little effect on malate dehydrogenase activity of any of the parasite. Assay of malate dehydrogenase, following the in vitro drug treatment cambendazole and tiabendazole, exhibited moderate inhibition of the activity in both the parasites.

  17. Structural basis for regulation of stability and activity in glyceraldehyde-3-phosphate dehydrogenases. Differential scanning calorimetry and molecular dynamics.

    PubMed

    Makshakova, Olga N; Semenyuk, Pavel I; Kuravsky, Mikhail L; Ermakova, Elena A; Zuev, Yuriy F; Muronetz, Vladimir I

    2015-05-01

    Tissue specific isoforms of human glyceraldehyde-3-phosphate dehydrogenase, somatic (GAPD) and sperm-specific (GAPDS), have been reported to display different levels of both stability and catalytic activity. Here we apply MD simulations to investigate molecular basis of this phenomenon. The protein is a tetramer where each subunit consists of two domains - catalytic and NAD-binding one. We demonstrated key residues responsible for intersubunit and interdomain interactions. Effect of several residues was studied by point mutations. Overall we considered three mutations (Glu96Gln, Glu244Gln and Asp311Asn) disrupting GAPDS-specific salt bridges. Comparison of calculated interaction energies with calorimetric enthalpies confirmed that intersubunit interactions were responsible for enhanced thermostability of GAPDS whereas interdomain interactions had indirect influence on intersubunit contacts. Mutation Asp311Asn was around 10Å far from the active center and corresponded to the closest natural substitution in the isoenzymes. MD simulations revealed that this residue had slight interaction with catalytic residues but influenced the hydrogen bond net and dynamics in active site. These effects can be responsible for a strong influence of this residue on catalytic activity. Overall, our results provide new insight into glyceraldehyde-3-phosphate dehydrogenase structure-function relationships and can be used for the engineering of mutant proteins with modified properties and for development of new inhibitors with indirect influence on the catalytic site. PMID:25869789

  18. [Activity of NADP-dependent glyceraldehyde-phosphate dehydrogenase and phosphoenolpyruvate carboxylase in wheat leaves under water stress].

    PubMed

    Cherniad'ev, I I; Monakhova, O F

    2006-01-01

    The activities of NADP: glyceraldehyde-phosphate dehydrogenase (GAPDH), an enzyme complex comprising of phosphoglycerate kinase (EC 2.7.2.3) and glyceraldehyde-phosphate dehydrogenase (EC 1.2.1.13), and phosphoenolpyruvate carboxylase (PEPK; EC 4.1.1.31) in seedlings and leaves of wheat (Triticum aestivum L.) plants of the cultivars Mironovskaya 808 and Lutescens 758 have been compared under conditions of normal water supply, water deficiency, and subsequent rehydration. GAPDH activity, which determines the carbohydrate route of photosynthetic metabolism at the initial stages, is decreased by water stress to a greater extent than that of PEPK, on the activity of which non-carbohydrate metabolic pathways depend. Pretreatment of seedlings and mature plants with natural (6-benzylaminopurine) and synthetic (tidiazuron, kartolin-2, and kartolin-4) cytokinins attenuates the loss of enzyme activities during drought and facilitates their recovery within the period of rehydration; both effects are underlain by augmentation of reparation processes. The relative intensification of non-carbohydrate pathways of photosynthetic metabolism, observed under conditions of water deficiency, is accompanied by an increase in the osmotic pressure of cell sap. Possible mechanisms of this protector effect of cytokinin preparations are discussed. PMID:16878554

  19. Purification and Characterization of Glucose 6-Phosphate Dehydrogenase, 6-Phosphogluconate Dehydrogenase, and Glutathione Reductase from Rat Heart and Inhibition Effects of Furosemide, Digoxin, and Dopamine on the Enzymes Activities.

    PubMed

    Adem, Sevki; Ciftci, Mehmet

    2016-06-01

    The present study was aimed to investigate characterization and purification of glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, and glutathione reductase from rat heart and the inhibitory effect of three drugs. The purification of the enzymes was performed using 2',5'-ADP sepharose 4B affinity material. The subunit and the natural molecular weights were analyzed by SDS-PAGE and gel filtration. Biochemical characteristics such as the optimum temperature, pH, stable pH, and salt concentration were examined for each enzyme. Types of product inhibition and Ki values with Km and Vmax values of the substrates and coenzymes were determined. According to the obtained Ki and IC50 values, furosemide, digoxin, and dopamine showed inhibitory effect on the enzyme activities at low millimolar concentrations in vitro conditions. Dopamine inhibited the activity of these enzymes as competitive, whereas furosemide and digoxin inhibited the activity of the enzyme as noncompetitive.

  20. Metabotropic glutamate receptor 3 activation is required for long-term depression in medial prefrontal cortex and fear extinction.

    PubMed

    Walker, Adam G; Wenthur, Cody J; Xiang, Zixiu; Rook, Jerri M; Emmitte, Kyle A; Niswender, Colleen M; Lindsley, Craig W; Conn, P Jeffrey

    2015-01-27

    Clinical studies have revealed that genetic variations in metabotropic glutamate receptor 3 (mGlu3) affect performance on cognitive tasks dependent upon the prefrontal cortex (PFC) and may be linked to psychiatric conditions such as schizophrenia, bipolar disorder, and addiction. We have performed a series of studies aimed at understanding how mGlu3 influences PFC function and cognitive behaviors. In the present study, we found that activation of mGlu3 can induce long-term depression in the mouse medial PFC (mPFC) in vitro. Furthermore, in vivo administration of a selective mGlu3 negative allosteric modulator impaired learning in the mPFC-dependent fear extinction task. The results of these studies implicate mGlu3 as a major regulator of PFC function and cognition. Additionally, potentiators of mGlu3 may be useful in alleviating prefrontal impairments associated with several CNS disorders. PMID:25583490

  1. Metabotropic glutamate receptor 3 activation is required for long-term depression in medial prefrontal cortex and fear extinction

    PubMed Central

    Walker, Adam G.; Wenthur, Cody J.; Xiang, Zixiu; Rook, Jerri M.; Emmitte, Kyle A.; Niswender, Colleen M.; Lindsley, Craig W.; Conn, P. Jeffrey

    2015-01-01

    Clinical studies have revealed that genetic variations in metabotropic glutamate receptor 3 (mGlu3) affect performance on cognitive tasks dependent upon the prefrontal cortex (PFC) and may be linked to psychiatric conditions such as schizophrenia, bipolar disorder, and addiction. We have performed a series of studies aimed at understanding how mGlu3 influences PFC function and cognitive behaviors. In the present study, we found that activation of mGlu3 can induce long-term depression in the mouse medial PFC (mPFC) in vitro. Furthermore, in vivo administration of a selective mGlu3 negative allosteric modulator impaired learning in the mPFC-dependent fear extinction task. The results of these studies implicate mGlu3 as a major regulator of PFC function and cognition. Additionally, potentiators of mGlu3 may be useful in alleviating prefrontal impairments associated with several CNS disorders. PMID:25583490

  2. Fluvoxamine increased glutamate release by activating both 5-HT(3) and sigma-1 receptors in prelimbic cortex of chronic restraint stress C57BL/6 mice.

    PubMed

    Fu, Yingmei; Yu, Shunying; Guo, Xiaoyun; Li, Xia; Li, Ting; Li, Huafang; Dong, Yi

    2012-04-01

    Emerging evidence from therapeutic trials in humans and animal models suggests that in the treatment of depression, antidepressants play a role by targeting the glutamatergic system. Fluvoxamine is one of the widely used SSRIs which has been considered to target monoamine neurotransmitter reuptake mechanisms. However, whether fluvoxamine has an effect on the glutamate release is still unclear. The present experiment studied the effect of fluvoxamine on presynaptic glutamate release in prelimbic cortex, both in control C57BL/6 mice and chronic restraint stress C57BL/6 mice, and further investigated the mechanism underlying this effect by using patch clamp, on-line fluorimetry, pharmacological approaches combined with other techniques. The results showed that fluvoxamine increased the glutamate release in the depression model mice but it had no effect on the glutamate release in the control mice. The mechanism underlying these effects in depression model mice was that, fluvoxamine firstly activated presynaptic 5-HT(3) receptors, which transiently increased the Ca(2+) concentration. The increase of Ca(2+) concentration via 5-HT(3) receptors caused the activation of sigma-1 receptors, which were activated by fluvoxamine. The activation of sigma-1 receptors increased the intrasynaptosomal Ca(2+) concentration significantly through the outflow of endoplasmic reticulum calcium and finally activated PKC. These results suggested that fluvoxamine may have a selective effect and different mechanism based on the condition of animal. PMID:22306004

  3. Curcumin attenuates glutamate neurotoxicity in the hippocampus by suppression of ER stress-associated TXNIP/NLRP3 inflammasome activation in a manner dependent on AMPK.

    PubMed

    Li, Ying; Li, Jia; Li, Shanshan; Li, Yi; Wang, Xiangxiang; Liu, Baolin; Fu, Qiang; Ma, Shiping

    2015-07-01

    Curcumin is a natural polyphenolic compound in Curcuma longa with beneficial effects on neuronal protection. This study aims to investigate the action of curcumin in the hippocampus subjected to glutamate neurotoxicity. Glutamate stimulation induced reactive oxygen species (ROS), endoplasmic reticulum stress (ER stress) and TXNIP/NLRP3 inflammasome activation, leading to damage in the hippocampus. Curcumin treatment in the hippocampus or SH-SY5Y cells inhibited IRE1α and PERK phosphorylation with suppression of intracellular ROS production. Curcumin increased AMPK activity and knockdown of AMPKα with specific siRNA abrogated its inhibitory effects on IRE1α and PERK phosphorylation, indicating that AMPK activity was essential for the suppression of ER stress. As a result, curcumin reduced TXNIP expression and inhibited NLRP3 inflammasome activation by downregulation of NLRP3 and cleaved caspase-1 induction, and thus reduced IL-1β secretion. Specific fluorescent probe and flow cytometry analysis showed that curcumin prevented mitochondrial malfunction and protected cell survival from glutamate neurotoxicity. Moreover, oral administration of curcumin reduced brain infarct volume and attenuated neuronal damage in rats subjected to middle cerebral artery occlusion. Immunohistochemistry showed that curcumin inhibited p-IRE1α, p-PERK and NLRP3 expression in hippocampus CA1 region. Together, these results showed that curcumin attenuated glutamate neurotoxicity by inhibiting ER stress-associated TXNIP/NLRP3 inflammasome activation via the regulation of AMPK, and thereby protected the hippocampus from ischemic insult. PMID:25791922

  4. Aspartate 46, a second sphere ligand to the catalytic zinc, is essential for activity of yeast alcohol dehydrogenase

    SciTech Connect

    Ganzhorn, A.J.; Plapp, B.V.

    1987-05-01

    The crystal structure of horse liver alcohol dehydrogenase (ADH) shows a hydrogen bond between the imidazole of His-67, a ligand to the active site zinc, and the carboxylate of Asp-49. Both residues are conserved in alcohol dehydrogenases. Directed mutagenesis was used to replace the homologous Asp-46 in ADH I from S. cerevisiae with asparagine. The substitution did not alter the overall structure of the enzyme, as judged by CD measurements, but the removal of a negative charge was evident in electrophoresis, and in the absorption and fluorescence spectra. The mutant and wild-type enzymes had similar zinc contents as determined by atomic absorption spectroscopy. Active site titration and steady state kinetics indicated that binding of coenzymes, substrates and substrate analogs is 4-24 fold weaker in the asparagine enzyme. The turnover numbers were reduced by a factor of 70 for ethanol oxidation and 30 for acetaldehyde reduction at pH 7.3, 30/sup 0/C. Dead end inhibition studies and the kinetic isotope effect showed that NAD and ethanol binding follow a rapid equilibrium random mechanism as opposed to the ordered mechanism found for ADH I. They conclude that the carboxyl group of Asp-46 is essential for the electrostatic environment near the active site zinc. Amidation may affect the geometry and/or coordination of the metal complex.

  5. Purification of xylitol dehydrogenase and improved production of xylitol by increasing XDH activity and NADH supply in Gluconobacter oxydans.

    PubMed

    Zhang, Jinliang; Li, Sha; Xu, Hong; Zhou, Peng; Zhang, Lujia; Ouyang, Pingkai

    2013-03-20

    Gluconobacter oxydans is known to be a suitable candidate for producing xylitol from d-arabitol. In this study, the enzyme responsible for reducing d-xylulose to xylitol was purified from G. oxydans NH-10 and characterized as xylitol dehydrogenase. It has been reported that XDH depends exclusively on NAD(+)/NADH as cofactors with a relatively low activity, which was proposed to be the direct reason for its limiting the overall conversion process. To better produce xylitol, an engineered G. oxydans PXPG was constructed to coexpress the XDH gene and a cofactor regeneration enzyme (glucose dehydrogenase) gene from Bacillus subtilis. Activities for both enzymes were more than twofold higher in the G. oxydans PXPG than in the wild strain. Approximately 12.23 g/L xylitol was obtained from 30 g/L d-arabitol by resting cells of the engineered strain with a conversion yield of 40.8%, whereas only 7.56 g/L xylitol was produced by the wild strain with a yield of 25.2%. These results demonstrated that increasing the XDH activity and the cofactor NADH supply could improve the xylitol productivity notably.

  6. Autodisplay of active sorbitol dehydrogenase (SDH) yields a whole cell biocatalyst for the synthesis of rare sugars.

    PubMed

    Jose, Joachim; von Schwichow, Steffen

    2004-04-01

    Whole cell biocatalysts are attractive technological tools for the regio- and enantioselective synthesis of products, especially from substrates with several identical reactive groups. In the present study, a whole cell biocatalyst for the synthesis of rare sugars from polyalcohols was constructed. For this purpose, sorbitol dehydrogenase (SDH) from Rhodobacter sphaeroides, a member of the short-chain dehydrogenase/reductase (SDR) family, was expressed on the surface of Escherichia coli using Autodisplay. Autodisplay is an efficient surface display system for Gram-negative bacteria and is based on the autotransporter secretion pathway. Transport of SDH to the outer membrane was monitored by SDS-PAGE and Western blotting of different cell fractions. The surface exposure of the enzyme could be verified by immunofluorescence microscopy and fluorescence activated cell sorting (FACS). The activity of whole cells displaying SDH at the surface was determined in an optical test. Specific activities were found to be 12 mU per 3.3 x 10(8) cells for the conversion of D-glucitol (sorbitol) to D-fructose, 7 mU for the conversion D-galactitol to D-tagatose, and 17 mU for the conversion of L-arabitol to L-ribulose. The whole cell biocatalyst obtained by surface display of SDH could also produce D-glucitol from D-fructose (29 mU per 3.3 x 10(8) cells).

  7. Glutamate Limitation, BvgAS Activation, and (p)ppGpp Regulate the Expression of the Bordetella pertussis Type 3 Secretion System

    PubMed Central

    Kamachi, Kazunari; Yonezawa, Hideo; Fukutomi, Toshiyuki; Kawakami, Hayato; Kamiya, Shigeru

    2015-01-01

    ABSTRACT Bordetella pertussis is a bacterium that is considered to be highly adapted to humans, and it has not been isolated from the environment. As this bacterium does not utilize sugars, the abundant supply of glutamate in Stainer Scholte (SS) medium enables B. pertussis to grow efficiently in liquid culture in vitro, and as such, SS medium is a popular choice for laboratory experiments. However, the concentration of glutamate in the in vivo niche of B. pertussis is quite low. We investigated the bacterial response to low concentrations of glutamate to elucidate bacterial physiology via the expression of the type 3 secretion system (T3SS), and we discuss its relationship to the Bvg mode in which the two-component regulator of pathogenesis (BvgAS) is activated. Glutamate limitation induced the expression of both the T3SS apparatus and effector genes at the transcriptional level. (p)ppGpp, a modulator of the stringent response, was necessary for maximum expression of the T3SS genes. These observations indicate that the expression of the T3SS is managed by nutrient starvation. In addition, the autoaggregation ability was high in the absence of glutamate and no autoaggregation was observed in glutamate-replete medium. Taken together, glutamate-limited conditions in Bvg+ mode elicit the high expression of T3SS genes in B. pertussis and promotes its sessile form. IMPORTANCE Bordetella pertussis is a highly contagious pathogen that causes respiratory infectious disease. In spite of the increasing use of vaccination, the number of patients with pertussis is increasing. The proteins produced in vivo often are different from the protein profile under laboratory conditions; therefore, the development of conditions reflecting the host environment is important to understand native bacterial behavior. In the present study, we examined the effect of glutamate limitation, as its concentration in vivo is much lower than that in the culture medium currently used for B. pertussis

  8. 3D-QSAR Studies on a Series of Dihydroorotate Dehydrogenase Inhibitors: Analogues of the Active Metabolite of Leflunomide

    PubMed Central

    Li, Shun-Lai; He, Mao-Yu; Du, Hong-Guang

    2011-01-01

    The active metabolite of the novel immunosuppressive agent leflunomide has been shown to inhibit the enzyme dihydroorotate dehydrogenase (DHODH). This enzyme catalyzes the fourth step in de novo pyrimidine biosynthesis. Self-organizing molecular field analysis (SOMFA), a simple three-dimensional quantitative structure-activity relationship (3D-QSAR) method is used to study the correlation between the molecular properties and the biological activities of a series of analogues of the active metabolite. The statistical results, cross-validated rCV2 (0.664) and non cross-validated r2 (0.687), show a good predictive ability. The final SOMFA model provides a better understanding of DHODH inhibitor-enzyme interactions, and may be useful for further modification and improvement of inhibitors of this important enzyme. PMID:21686163

  9. Tissue-type plasminogen activator-plasmin-BDNF modulate glutamate-induced phase-shifts of the mouse suprachiasmatic circadian clock in vitro.

    PubMed

    Mou, Xiang; Peterson, Cynthia B; Prosser, Rebecca A

    2009-10-01

    The mammalian circadian clock in the suprachiasmatic nucleus (SCN) maintains environmental synchrony through light signals transmitted by glutamate released from retinal ganglion terminals. Brain-derived neurotrophic factor (BDNF) is required for light/glutamate to reset the clock. In the hippocampus, BDNF is activated by the extracellular protease, plasmin, which is produced from plasminogen by tissue-type plasminogen activator (tPA). We provide data showing expression of proteins from the plasminogen activation cascade in the SCN and their involvement in circadian clock phase-resetting. Early night glutamate application to SCN-containing brain slices resets the circadian clock. Plasminogen activator inhibitor-1 (PAI-1) blocked these shifts in slices from wild-type mice but not mice lacking its stabilizing protein, vitronectin (VN). Plasmin, but not plasminogen, prevented inhibition by PAI-1. Both plasmin and active BDNF reversed alpha(2)-antiplasmin inhibition of glutamate-induced shifts. alpha(2)-Antiplasmin decreased the conversion of inactive to active BDNF in the SCN. Finally, both tPA and BDNF allowed daytime glutamate-induced phase-resetting. Together, these data are the first to demonstrate expression of these proteases in the SCN, their involvement in modulating photic phase-shifts, and their activation of BDNF in the SCN, a potential 'gating' mechanism for photic phase-resetting. These data also demonstrate a functional interaction between PAI-1 and VN in adult brain. Given the usual association of these proteins with the extracellular matrix, these data suggest new lines of investigation into the locations and processes modulating mammalian circadian clock phase-resetting.

  10. Functional magnetic resonance spectroscopy of glutamate in schizophrenia and major depressive disorder: anterior cingulate activity during a color-word Stroop task

    PubMed Central

    Taylor, Reggie; Neufeld, Richard W J; Schaefer, Betsy; Densmore, Maria; Rajakumar, Nagalingam; Osuch, Elizabeth A; Williamson, Peter C; Théberge, Jean

    2015-01-01

    Background: Glutamate abnormalities have been suggested to be associated with symptoms of schizophrenia. Using functional magnetic resonance spectroscopy (1H-fMRS), it is possible to monitor glutamate dynamically in the activated brain areas, which has yet to be reported in schizophrenia. It was hypothesized that subjects with schizophrenia would have weaker glutamatergic responses in the anterior cingulate to a color-word Stroop Task. AIMS: The aim of this study was to gain insight into the health of GLU neurotransmission and the GLU-GLN cycle in SZ using a 1H-fMRS protocol. Methods: Spectra were acquired from the anterior cingulate of 16 participants with schizophrenia, 16 healthy controls and 16 participants with major depressive disorder (MDD) while performing the Stroop task in a 7T magnetic resonance imaging scanner. 1H-fMRS spectra were acquired for 20 min in which there were three 4-min blocks of cross fixation interleaved with two 4-min blocks of the Stroop paradigm. Results: A repeated-measures analysis of variance revealed a main effect of time for glutamate concentrations of all groups (P<0.001). The healthy control group increased glutamate concentrations in the first run of the Stroop task (P=0.006) followed by a decrease in the recovery period (P=0.007). Neither the schizophrenia (P=0.107) nor MDD (P=0.081) groups had significant glutamate changes in the first run of the task, while the schizophrenia group had a significant increase in glutamine (P=0.005). The MDD group decreased glutamate concentrations in the second run of the task (P=0.003), as did all the groups combined (P=0.003). Conclusions: 1H-fMRS data were successfully acquired from psychiatric subjects with schizophrenia and mood disorder using a cognitive paradigm for the first time. Future study designs should further elucidate the glutamatergic response to functional activation in schizophrenia. PMID:27336037

  11. The peroxisome proliferator activated receptor gamma agonist pioglitazone increases functional expression of the glutamate transporter excitatory amino acid transporter 2 (EAAT2) in human glioblastoma cells

    PubMed Central

    Ching, Jared; Amiridis, Stephanie; Stylli, Stanley S.; Bjorksten, Andrew R.; Kountouri, Nicole; Zheng, Thomas; Paradiso, Lucy; Luwor, Rodney B.; Morokoff, Andrew P.; O'Brien, Terence J.; Kaye, Andrew H.

    2015-01-01

    Glioma cells release glutamate through expression of system xc−, which exchanges intracellular glutamate for extracellular cysteine. Lack of the excitatory amino acid transporter 2 (EAAT2) expression maintains high extracellular glutamate levels in the glioma microenvironment, causing excitotoxicity to surrounding parenchyma. Not only does this contribute to the survival and proliferation of glioma cells, but is involved in the pathophysiology of tumour-associated epilepsy (TAE). We investigated the role of the peroxisome proliferator activated receptor gamma (PPARγ) agonist pioglitazone in modulating EAAT2 expression in glioma cells. We found that EAAT2 expression was increased in a dose depen