Science.gov

Sample records for nadh-dependent glutamate dehydrogenase

  1. 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

  2. Characterization of a NADH-Dependent Glutamate Dehydrogenase Mutant of Arabidopsis Demonstrates the Key Role of this Enzyme in Root Carbon and Nitrogen Metabolism[W

    PubMed Central

    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

    2012-01-01

    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. PMID:23054470

  3. 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. PMID:23054470

  4. 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

  5. Optical isopropanol biosensor using NADH-dependent secondary alcohol dehydrogenase (S-ADH).

    PubMed

    Chien, Po-Jen; Ye, Ming; Suzuki, Takuma; Toma, Koji; Arakawa, Takahiro; Iwasaki, Yasuhiko; Mitsubayashi, Kohji

    2016-10-01

    Isopropanol (IPA) is an important solvent used in industrial activity often found in hospitals as antiseptic alcohol rub. Also, IPA may have the potential to be a biomarker of diabetic ketoacidosis. In this study, an optical biosensor using NADH-dependent secondary alcohol dehydrogenase (S-ADH) for IPA measurement was constructed and evaluated. An ultraviolet light emitting diode (UV-LED, λ=340nm) was employed as the excitation light to excite nicotinamide adenine dinucleotide (NADH). A photomultiplier tube (PMT) was connected to a two-way branch optical fiber for measuring the fluorescence emitted from the NADH. S-ADH was immobilized on the membrane to catalyze IPA to acetone and reduce NAD(+) to be NADH. This IPA biosensor shows highly sensitivity and selectivity, the calibration range is from 500 nmol L(-1) to 1mmolL(-1). The optimization of buffer pH, temperature, and the enzyme-immobilized method were also evaluated. The detection of IPA in nail related cosmetic using our IPA biosensor was also carried out. The results showed that large amounts of IPA were used in these kinds of cosmetics. This IPA biosensor comes with the advantages of rapid reaction, good reproducibility, and wide dynamic range, and is also expected to use for clinical IPA detections in serum or other medical and health related applications. PMID:27474326

  6. Methanoarchaeal sulfolactate dehydrogenase: prototype of a new family of NADH-dependent enzymes

    PubMed Central

    Irimia, Adriana; Madern, Dominique; Zaccaï, Giuseppe; Vellieux, Frédéric MD

    2004-01-01

    The crystal structure of the sulfolactate dehydrogenase from the hyperthermophilic and methanogenic archaeon Methanocaldococcus jannaschii was solved at 2.5 Å resolution (PDB id. 1RFM). The asymmetric unit contains a tetramer of tight dimers. This structure, complexed with NADH, does not contain a cofactor-binding domain with ‘Rossmann-fold' topology. Instead, the tertiary and quaternary structures indicate a novel fold. The NADH is bound in an extended conformation in each active site, in a manner that explains the pro-S specificity. Cofactor binding involves residues belonging to both subunits within the tight dimers, which are therefore the smallest enzymatically active units. The protein was found to be a homodimer in solution by size-exclusion chromatography, analytical ultracentrifugation and small-angle neutron scattering. Various compounds were tested as putative substrates. The results indicate the existence of a substrate discrimination mechanism, which involves electrostatic interactions. Based on sequence homology and phylogenetic analyses, several other enzymes were classified as belonging to this novel family of homologous (S)-2-hydroxyacid dehydrogenases. PMID:15014443

  7. Cloning and overexpression of an NADH-dependent alcohol dehydrogenase gene from Candida maris involved in (R)-selective reduction of 5-acetylfuro[2,3-c]pyridine.

    PubMed

    Kawano, Shigeru; Yano, Miho; Hasegawa, Junzo; Yasohara, Yoshihiko

    2011-01-01

    5-((R)-1-Hydroxyethyl)-furo[2,3-c]pyridine ((R)-FPH) is a useful chiral building block in the synthesis of pharmaceuticals. An NADH-dependent alcohol dehydrogenase (AFPDH) isolated from Candida maris catalyzed the reduction of 5-acetylfuro[2,3-c]pyridine (AFP) to (R)-FPH with 100% enantiomeric excess. The gene encoding AFPDH was cloned and sequenced. The AFPDH gene comprises 762 bp and encodes a polypeptide of 27,230 Da. The deduced amino acid sequence showed a high degree of similarity to those of other members of the short-chain alcohol dehydrogenase superfamily. The AFPDH gene was overexpressed in Escherichia coli under the control of the lac promoter. One L of the cultured broth of an E. coli transformant coexpressing AFPDH and the glucose dehydrogenase (GDH) gene reduced 250 g of AFP to (R)-FPH in an organic solvent two-phase system. Under coupling with NADH regeneration using 2-propanol, 1 L of the cultured broth of an E. coli transformant expressing the AFPDH gene reduced 150 g of AFP to (R)-FPH. The optical purity of the (R)-FPH formed was 100% enantiomeric excess under both reaction conditions. PMID:22056439

  8. 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

  9. 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. PMID:26221781

  10. Isocitrate Dehydrogenase and Glutamate Synthesis in Acetobacter suboxydans1

    PubMed Central

    Greenfield, Seymour; Claus, G. W.

    1969-01-01

    Acetobacter suboxydans is an obligate aerobe for which an operative tricarboxylic acid cycle has not been demonstrated. Glutamate synthesis has been reported to occur by mechanisms other than those utilizing isocitrate dehydrogenase, a tricarboxylic acid cycle enzyme not previously detected in this organism. We have recovered α-ketoglutarate and glutamate from a system containing citrate, nicotinamide adenine dinucleotide (NAD), a divalent cation, pyridoxal phosphate, an amino donor, and dialyzed, cell-free extract. Aconitase activity was readily detected in these extracts, but isocitrate dehydrogenase activity, measured by NAD reduction, was masked by a cyanide-resistant, particulate, reduced NAD oxidase. Isocitrate dehydrogenase activity could be demonstrated after centrifuging the extracts at 150,000 × g for 3 hr and treating the supernatant fluid with 2-heptyl-4-hydroxyquinoline N-oxide. It is concluded that A. suboxydans can utilize the conventional tricarboxylic acid cycle enzymes to convert citrate to α-ketoglutarate which can then undergo a transamination to glutamate. Images PMID:5361215

  11. 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

  12. 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

  13. Green tea polyphenols modulate insulin secretion by inhibiting glutamate dehydrogenase.

    PubMed

    Li, Changhong; Allen, Aron; Kwagh, Jae; Doliba, Nicolai M; Qin, Wei; Najafi, Habiba; Collins, Heather W; Matschinsky, Franz M; Stanley, Charles A; Smith, Thomas J

    2006-04-14

    Insulin secretion by pancreatic beta-cells is stimulated by glucose, amino acids, and other metabolic fuels. Glutamate dehydrogenase (GDH) has been shown to play a regulatory role in this process. The importance of GDH was underscored by features of hyperinsulinemia/hyperammonemia syndrome, where a dominant mutation causes the loss of inhibition by GTP and ATP. Here we report the effects of green tea polyphenols on GDH and insulin secretion. Of the four compounds tested, epigallocatechin gallate (EGCG) and epicatechin gallate were found to inhibit GDH with nanomolar ED(50) values and were therefore found to be as potent as the physiologically important inhibitor GTP. Furthermore, we have demonstrated that EGCG inhibits BCH-stimulated insulin secretion, a process that is mediated by GDH, under conditions where GDH is no longer inhibited by high energy metabolites. EGCG does not affect glucose-stimulated insulin secretion under high energy conditions where GDH is probably fully inhibited. We have further shown that these compounds act in an allosteric manner independent of their antioxidant activity and that the beta-cell stimulatory effects are directly correlated with glutamine oxidation. These results demonstrate that EGCG, much like the activator of GDH (BCH), can facilitate dissecting the complex regulation of insulin secretion by pharmacologically modulating the effects of GDH. PMID:16476731

  14. Glutamate dehydrogenase: genetic mapping and isolation of regulatory mutants of Klebsiella aerogenes.

    PubMed Central

    Bender, R A; Macaluso, A; Magasanik, B

    1976-01-01

    The gene for glutamate dehydrogenase (gdhD) has been mapped in Klebsiella aerogenes by P1 transduction. It is linked to pyrF and trp with the order pyrF-trp-gdh. Complementation analysis using F' episomes from Escherichia coli suggests an analogous location in E. coli. Two mutants able to produce glutamate dehydrogenase in the presence of high levels of glutamine synthetase have been isolated. One, tightly linked to gdhD, shows normal repression control by glutamine synthetase but produces four times as much glutamate dehydrogenase activity as does the wild type under all conditions tested. The other revertant is not linked to gdhD or glnA. PMID:6429

  15. 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

  16. The Role of Glutamine Oxoglutarate Aminotransferase and Glutamate Dehydrogenase in Nitrogen Metabolism in Mycobacterium bovis BCG

    PubMed Central

    Viljoen, Albertus J.; Kirsten, Catriona J.; Baker, Bienyameen; van Helden, Paul D.; Wiid, Ian J. F.

    2013-01-01

    Recent evidence suggests that the regulation of intracellular glutamate levels could play an important role in the ability of pathogenic slow-growing mycobacteria to grow in vivo. However, little is known about the in vitro requirement for the enzymes which catalyse glutamate production and degradation in the slow-growing mycobacteria, namely; glutamine oxoglutarate aminotransferase (GOGAT) and glutamate dehydrogenase (GDH), respectively. We report that allelic replacement of the Mycobacterium bovis BCG gltBD-operon encoding for the large (gltB) and small (gltD) subunits of GOGAT with a hygromycin resistance cassette resulted in glutamate auxotrophy and that deletion of the GDH encoding-gene (gdh) led to a marked growth deficiency in the presence of L-glutamate as a sole nitrogen source as well as reduction in growth when cultured in an excess of L-asparagine. PMID:24367660

  17. Glutamate Synthase: Properties of the Reduced Nicotinamide Adenine Dinucleotide-Dependent Enzyme from Saccharomyces cerevisiae

    PubMed Central

    Roon, Robert J.; Even, Harvey L.; Larimore, Fred

    1974-01-01

    A reduced nicotinamide adenine dinucleotide (NADH)-dependent glutamate synthase has been detected and partially purified from crude extracts of Saccharomyces cerevisiae. The enzyme is specific for NADH, glutamine, and α-ketoglutarate (Km values of 2.6 μM, 1.0 mM, and 140 μM, respectively) and has a pH optimum between 7.1 and 7.7. The stoichiometry of the reaction has been determined as 2 mol of glutamate synthesized per mol of glutamine consumed. Glutamate synthase can be distinguished from either of the glutamate dehydrogenases of yeast on the basis of its substrate requirements and behavior during agarose gel and ion exchange chromatography. Variations in the specific activity of glutamate synthase, which occur in response to changes in the growth medium, are similar in character to those observed with the nicotinamide adenine dinucleotide phosphate-dependent (anabolic) glutamate dehydrogenase. PMID:4362465

  18. 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

  19. Glutamate dehydrogenase in brain mitochondria: do lipid modifications and transient metabolon formation influence enzyme activity?

    PubMed Central

    McKenna, Mary C.

    2011-01-01

    Metabolism of glutamate, the primary excitatory neurotransmitter in brain, is complex and of paramount importance to overall brain function. Thus, understanding the regulation of enzymes involved in formation and disposal of glutamate and related metabolites is crucial to understanding glutamate metabolism. Glutamate dehydrogenase (GDH) is a pivotal enzyme that links amino acid metabolism and TCA cycle activity in brain and other tissues. The allosteric regulation of GDH has been extensively studied and characterized. Less is known about the influence of lipid modifications on GDH activity, and the participation of GDH in transient heteroenzyme complexes (metabolons) that can greatly influence metabolism by altering kinetic parameters and lead to channeling of metabolites. This review summarizes evidence for palmitoylation and acylation of GDH, information on protein binding, and information regarding the participation of GDH in transient heteroenzyme complexes. Recent studies suggest that a number of other proteins can bind to GDH altering activity and overall metabolism. It is likely that these modifications and interactions contribute additional levels of regulation of GDH activity and glutamate metabolism. PMID:21771624

  20. 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.

  1. The unique kinetic behavior of the very large NAD-dependent glutamate dehydrogenase from Janthinobacterium lividum.

    PubMed

    Kawakami, Ryushi; Oyama, Masaki; Sakuraba, Haruhiko; Ohshima, Toshihisa

    2010-01-01

    The kinetics of a very large NAD-dependent glutamate dehydrogenase from Janthinobacterium lividum showed positive cooperativity toward alpha-ketoglutarate and NADH, and the Michaelis-Menten type toward ammonium chloride in the absence of the catalytic activator, L-aspartate. An increase in the maximum activity accompanied the decrease in the S(0.5) values for alpha-ketoglutarate and NADH with the addition of L-aspartate, and the kinetic response for alpha-ketoglutarate changed completely to a typical Michaelis-Menten type in the presence of 10 mM L-aspartate. PMID:20378971

  2. Glutamate dehydrogenase 1 signals through antioxidant glutathione peroxidase 1 to regulate redox homeostasis and tumor growth

    PubMed Central

    Jin, Lingtao; Li, Dan; Alesi, Gina N.; Fan, Jun; Kang, Hee-Bum; Lu, Zhou; Boggon, Titus J.; Jin, Peng; Yi, Hong; Wright, Elizabeth R.; Duong, Duc; Seyfried, Nicholas T.; Egnatchik, Robert; DeBerardinis, Ralph J.; Magliocca, Kelly R.; He, Chuan; Arellano, Martha L.; Khoury, Hanna J.; Shin, Dong M.; Khuri, Fadlo R.; Kang, Sumin

    2015-01-01

    SUMMARY How mitochondrial glutaminolysis contributes to redox homeostasis in cancer cells remains unclear. Here we report that the mitochondrial enzyme glutamate dehydrogenase 1 (GDH1) is commonly upregulated in human cancers. GDH1 is important for redox homeostasis in cancer cells by controlling the intracellular levels of its product alpha-ketoglutarate (α-KG) and subsequent metabolite fumarate. Mechanistically, fumarate binds to and activates a ROS scavenging enzyme glutathione peroxidase 1 (GPx1). Targeting GDH1 by shRNA or a small molecule inhibitor R162 resulted in imbalanced redox homeostasis, leading to attenuated cancer cell proliferation and tumor growth. PMID:25670081

  3. The Role of Glutamine Synthetase and Glutamate Dehydrogenase in Cerebral Ammonia Homeostasis

    PubMed Central

    Cooper, Arthur J. L.

    2012-01-01

    In the brain, glutamine synthetase (GS), which is located predominantly in astrocytes, is largely responsible for the removal of both blood-derived and metabolically generated ammonia. Thus, studies with [13N]ammonia have shown that about 25% of blood-derived ammonia is removed in a single pass through the rat brain and that this ammonia is incorporated primarily into glutamine (amide) in astrocytes. Major pathways for cerebral ammonia generation include the glutaminase reaction and the glutamate dehydrogenase (GDH) reaction. The equilibrium position of the GDH-catalyzed reaction in vitro favors reductive amination of α-ketoglutarate at pH 7.4. Nevertheless, only a small amount of label derived from [13N]ammonia in rat brain is incorporated into glutamate and the α-amine of glutamine in vivo. Most likely the cerebral GDH reaction is drawn normally in the direction of glutamate oxidation (ammonia production) by rapid removal of ammonia as glutamine. Linkage of glutamate/α-ketoglutarate-utilizing aminotransferases with the GDH reaction channels excess amino acid nitrogen toward ammonia for glutamine synthesis. At high ammonia levels and/or when GS is inhibited the GDH reaction coupled with glutamate/α-ketoglutarate-linked aminotransferases may, however, promote the flow of ammonia nitrogen toward synthesis of amino acids. Preliminary evidence suggests an important role for the purine nucleotide cycle (PNC) as an additional source of ammonia in neurons (Net reaction: L-Aspartate + GTP + H2O → Fumarate + GDP + Pi + NH3) and in the beat cycle of ependyma cilia. The link of the PNC to aminotransferases and GDH/GS and its role in cerebral nitrogen metabolism under both normal and pathological (e.g. hyperammonemic encephalopathy) conditions should be a productive area for future research. PMID:22618691

  4. 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

  5. [Implication of glutamate, isocitrate and malate deshydrogenases in nitrogen assimilation in the cadmium-stressed tomato].

    PubMed

    Chaffei, Chiraz; Suzuki, Akira; Masclaux-Daubresse, Céline; Ghorbel, Mohamed Habib; Gouia, Houda

    2006-10-01

    Tomato seedlings grown on nitric medium and treated with various cadmium concentrations (0 to 50 microM) were used. Results obtained show that cadmium remains predominantly located in the roots, which then seem to play the role of trap-organs. Increasing cadmium concentration in the medium leads particularly to a decrease in NO3- accumulation, together with a decrease in the activity of glutamine synthetase and in the quantity of plastidic isoform ARNm (GS2), and, on the contrary, to an increase of the cytosolic isoform ARNm (GS1). On the other hand, stimulations were observed for NADH-dependent glutamate synthase, NADH-dependent glutamate dehydrogenase, ARNm quantity of this enzyme, ammonium accumulation, and protease activity. In parallel, stimulations were observed for NAD+ and NADP+-dependent malate dehydrogenase and NADP+-dependent isocitrate dehydrogenase. These results were discussed in relation to the hypothesis attributing to the dehydrogenase enzymes (GDH, MDH, ICDH) an important role in the plant defence processes against cadmium-induced stresses. PMID:17027640

  6. 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

  7. 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

  8. 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.

  9. Clostridium difficile glutamate dehydrogenase is a secreted enzyme that confers resistance to H2O2

    PubMed Central

    Girinathan, Brintha Prasummanna; Braun, Sterling E.

    2014-01-01

    Clostridium difficile produces an NAD-specific glutamate dehydrogenase (GDH), which converts l-glutamate into α-ketoglutarate through an irreversible reaction. The enzyme GDH is detected in the stool samples of patients with C. difficile-associated disease and serves as one of the diagnostic tools to detect C. difficile infection (CDI). We demonstrate here that supernatant fluids of C. difficile cultures contain GDH. To understand the role of GDH in the physiology of C. difficile, an isogenic insertional mutant of gluD was created in strain JIR8094. The mutant failed to produce and secrete GDH as shown by Western blot analysis. Various phenotypic assays were performed to understand the importance of GDH in C. difficile physiology. In TY (tryptose yeast extract) medium, the gluD mutant grew slower than the parent strain. Complementation of the gluD mutant with the functional gluD gene reversed the growth defect in TY medium. The presence of extracellular GDH may have a functional role in the pathogenesis of CDI. In support of this assumption we found higher sensitivity to H2O2 in the gluD mutant as compared to the parent strain. Complementation of the gluD mutant with the functional gluD gene reversed the H2O2 sensitivity. PMID:24145018

  10. 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

  11. 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.

  12. 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

  13. 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

  14. Determination of Glutamate Dehydrogenase Activity and Its Kinetics in Mouse Tissues using Metabolic Mapping (Quantitative Enzyme Histochemistry)

    PubMed Central

    Botman, Dennis; Tigchelaar, Wikky

    2014-01-01

    Glutamate dehydrogenase (GDH) catalyses the reversible conversion of glutamate into α-ketoglutarate with the concomitant reduction of NAD(P)+ to NAD(P)H or vice versa. GDH activity is subject to complex allosteric regulation including substrate inhibition. To determine GDH kinetics in situ, we assessed the effects of various glutamate concentrations in combination with either the coenzyme NAD+ or NADP+ on GDH activity in mouse liver cryostat sections using metabolic mapping. NAD+-dependent GDH Vmax was 2.5-fold higher than NADP+-dependent Vmax, whereas the Km was similar, 1.92 mM versus 1.66 mM, when NAD+ or NADP+ was used, respectively. With either coenzyme, Vmax was determined at 10 mM glutamate and substrate inhibition was observed at higher glutamate concentrations with a Ki of 12.2 and 3.95 for NAD+ and NADP+ used as coenzyme, respectively. NAD+- and NADP+-dependent GDH activities were examined in various mouse tissues. GDH activity was highest in liver and much lower in other tissues. In all tissues, the highest activity was found when NAD+ was used as a coenzyme. In conclusion, GDH activity in mice is highest in the liver with NAD+ as a coenzyme and highest GDH activity was determined at a glutamate concentration of 10 mM. PMID:25124006

  15. Unexpected Histone H3 Tail-clipping Activity of Glutamate Dehydrogenase*

    PubMed Central

    Mandal, Papita; Verma, Naveen; Chauhan, Sakshi; Tomar, Raghuvir S.

    2013-01-01

    Clipping of histone tails has been reported in several organisms. However, the significance and regulation of histone tail clipping largely remains unclear. According to recent discoveries H3 clipping has been found to be involved in regulation of gene expression and chromatin dynamics. Earlier we had provided evidence of tissue-specific proteolytic processing of histone H3 in White Leghorn chicken liver nuclei. In this study we identify a novel activity of glutamate dehydrogenase (GDH) as a histone H3-specific protease in chicken liver tissue. This protease activity is regulated by divalent ions and thiol-disulfide conversion in vitro. GDH specifically clips H3 in its free as well as chromatin-bound form. Furthermore, we have found an inhibitor that inhibits the H3-clipping activity of GDH. Like previously reported proteases, GDH too may have the potential to regulate/modulate post-translational modifications of histone H3 by removing the N-terminal residues of the histone. In short, our findings identify an unexpected proteolytic activity of GDH specific to histone H3 that is regulated by redox state, ionic concentrations, and a cellular inhibitor in vitro. PMID:23673664

  16. 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

  17. 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.

  18. 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

    2016-01-01

    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, 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. PMID:27418431

  19. Biochemical characterization of two glutamate dehydrogenases with different cofactor specificities from a hyperthermophilic archaeon Pyrobaculum calidifontis.

    PubMed

    Wakamatsu, Taisuke; Higashi, Chisato; Ohmori, Taketo; Doi, Katsumi; Ohshima, Toshihisa

    2013-05-01

    Two putative glutamate dehydrogenase (GDH) genes (pcal_1031 and pcal_1606) were found in a sulfur-dependent hyperthermophilic archaeon, Pyrobaculum calidifontis. The two genes were then expressed in Escherichia coli, and both of the recombinant gene products showed GDH activity. The two enzymes were then purified to homogeneity and characterized in detail. Although both purified GDHs had a hexameric structure and neither exhibited allosteric regulation, they showed different coenzyme specificities: one was specific for NAD(+), the other for NADP(+) and different heat activation mechanisms. In addition, there was little difference in the kinetic constants, optimal temperature, thermal stability, optimal pH and pH stability between the two enzymes. The overall sequence identity between the two proteins was very high (81%), but was not high in the region recognizing the 2' position of the adenine ribose moiety, which is responsible for coenzyme specificity. This is the first report on the identification of two GDHs with different coenzyme specificities from a single hyperthermophilic archaeon and the definition of their basic in vitro properties. PMID:23508687

  20. 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

  1. 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

  2. Enzymic assay of creatinine in serum and urine with creatinine iminohydrolase and glutamate dehydrogenase.

    PubMed

    Tanganelli, E; Prencipe, L; Bassi, D; Cambiaghi, S; Murador, E

    1982-07-01

    We describe an assay for creatinine in which it is converted by creatinine iminohydrolase (EC 3.5.4.21) into ammonia and N-methylhydantoin. The ammonia is subsequently assayed by use of alpha-ketoglutarate and glutamate dehydrogenase (EC 1.4.1.3). Use of NADPH as coenzyme eliminates all interferences from endogenous reactions. Endogenous ammonia in the sample is eliminated during a preincubation. The reaction reaches the endpoint in 15 min at working temperatures of 20-37 degrees C. No sample blank or reagent blank is needed. The standard curve is linear at least to 884 mumol (100 mg) of creatinine per liter. Average analytical recovery of creatinine in serum and urine is 99%. Within-run and between-run CVs are less than or equal to 2% and less than or equal to 6% for creatinine values of 335 mumol/L (38 mg/L) and 80 mumol/L (0 mg/L), respectively. Results by the described method (y) compare well with those by Jaffé's kinetic test (y = 1.01x -- 12.8), Berthelot/AutoAnalyzer method after treatment with immobilized creatinine iminohydrolase (y = 0.987x -- 13.2), Jaffé's test run on the SMA 12/60 (y = 1.011x -- 5.8), the Wahlefeld method (y = 1.014x -- 0.88), and Jaffé's test after deproteinization and absorption on fuller's earth (y = 0.985x -- 3.08). The method may be suitable for discrete, including centrifugal, automation. PMID:7083556

  3. 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

  4. 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

  5. Resolving the Role of Plant Glutamate Dehydrogenase. I. in vivo Real Time Nuclear Magnetic Resonance Spectroscopy Experiments

    PubMed Central

    Labboun, Soraya; Tercé-Laforgue, Thérèse; Roscher, Albrecht; Bedu, Magali; Restivo, Francesco M.; Velanis, Christos N.; Skopelitis, Damianos S.; Moshou, Panagiotis N.; Roubelakis-Angelakis, Kalliopi A.; Suzuki, Akira; Hirel, Bertrand

    2009-01-01

    In higher plants the glutamate dehydrogenase (GDH) enzyme catalyzes the reversible amination of 2-oxoglutarate to form glutamate, using ammonium as a substrate. For a better understanding of the physiological function of GDH either in ammonium assimilation or in the supply of 2-oxoglutarate, we used transgenic tobacco (Nicotiana tabacum L.) plants overexpressing the two genes encoding the enzyme. An in vivo real time 15N-nuclear magnetic resonance (NMR) spectroscopy approach allowed the demonstration that, when the two GDH genes were overexpressed individually or simultaneously, the transgenic plant leaves did not synthesize glutamate in the presence of ammonium when glutamine synthetase (GS) was inhibited. In contrast we confirmed that the primary function of GDH is to deaminate Glu. When the two GDH unlabeled substrates ammonium and Glu were provided simultaneously with either [15N]Glu or 15NH4+ respectively, we found that the ammonium released from the deamination of Glu was reassimilated by the enzyme GS, suggesting the occurrence of a futile cycle recycling both ammonium and Glu. Taken together, these results strongly suggest that the GDH enzyme, in conjunction with NADH-GOGAT, contributes to the control of leaf Glu homeostasis, an amino acid that plays a central signaling and metabolic role at the interface of the carbon and nitrogen assimilatory pathways. Thus, in vivo NMR spectroscopy appears to be an attractive technique to follow the flux of metabolites in both normal and genetically modified plants. PMID:19690000

  6. 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

  7. Identification of catalytic residues of a very large NAD-glutamate dehydrogenase from Janthinobacterium lividum by site-directed mutagenesis.

    PubMed

    Kawakami, Ryushi; Sakuraba, Haruhiko; Ohshima, Toshihisa

    2014-01-01

    We previously found a very large NAD-dependent glutamate dehydrogenase with approximately 170 kDa subunit from Janthinobacterium lividum (Jl-GDH) and predicted that GDH reaction occurred in the central domain of the subunit. To gain further insights into the role of the central domain, several single point mutations were introduced. The enzyme activity was completely lost in all single mutants of R784A, K810A, K820A, D885A, and S1142A. Because, in sequence alignment analysis, these residues corresponded to the residues responsible for glutamate binding in well-known small GDH with approximately 50 kDa subunit, very large GDH and well-known small GDH may share the same catalytic mechanism. In addition, we demonstrated that C1141, one of the three cysteine residues in the central domain, was responsible for the inhibition of enzyme activity by HgCl2, and HgCl2 functioned as an activating compound for a C1141T mutant. At low concentrations, moreover, HgCl2 was found to function as an activating compound for a wild-type Jl-GDH. This suggests that the mechanism for the activation is entirely different from that for the inhibition. PMID:25126984

  8. 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. PMID:10076069

  9. Chitosan promotes immune responses, ameliorates glutamic oxaloacetic transaminase and glutamic pyruvic transaminase, but enhances lactate dehydrogenase levels in normal mice in vivo

    PubMed Central

    YEH, MING-YANG; SHIH, YUNG-LUEN; CHUNG, HSUEH-YU; CHOU, JASON; LU, HSU-FENG; LIU, CHIA-HUI; LIU, JIA-YOU; HUANG, WEN-WEN; PENG, SHU-FEN; WU, LUNG-YUAN; CHUNG, JING-GUNG

    2016-01-01

    Chitosan, a naturally derived polymer, has been shown to possess antimicrobial and anti-inflammatory properties; however, little is known about the effect of chitosan on the immune responses and glutamic oxaloacetic transaminase (GOT), glutamic pyruvic transaminase (GPT) and lactate dehydrogenase (LDH) activities in normal mice. The aim of the present study was to investigate whether chitosan has an effect on the immune responses and GOT, GPT and LDH activities in mice in vivo. BALB/c mice were divided into four groups. The negative control group was treated with a normal diet; the positive control group was treated with a normal diet plus orally administered acetic acid and two treatment groups were treated with a normal diet plus orally administered chitosan in acetic acid at doses of 5 and 20 mg/kg, respectively, every other day for 24 days. Mice were weighed during the treatment, and following the treatment, blood was collected, and liver and spleen samples were isolated and weighted. The blood samples were used for measurement of white blood cell markers, and the spleen samples were used for analysis of phagocytosis, natural killer (NK) cell activity and cell proliferation using flow cytometry. The results indicated that chitosan did not markedly affect the body, liver and spleen weights at either dose. Chitosan increased the percentages of CD3 (T-cell marker), CD19 (B-cell marker), CD11b (monocytes) and Mac-3 (macrophages) when compared with the control group. However, chitosan did not affect the phagocytic activity of macrophages in peripheral blood mononuclear cells, although it decreased it in the peritoneal cavity. Treatment with 20 mg/kg chitosan led to a reduction in the cytotoxic activity of NK cells at an effector to target ratio of 25:1. Chitosan did not significantly promote B-cell proliferation in lipopolysaccharide-pretreated cells, but significantly decreased T-cell proliferation in concanavalin A-pretreated cells, and decreased the activity of

  10. Growth, photosynthesis, nitrogen partitioning and responses to CO2 enrichment in barley mutants lacking NADH-dependent nitrate reductase activity

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We examined plant growth, photosynthesis and leaf constituents of both the wild type (WT) and two mutant lines of barley (Hordeum vulgare L. cv. Steptoe) with defects in NADH-dependent nitrate reductase (NADH-NAR) activity. The first mutant, nar1, had a lesion within the NAR structural gene and the...

  11. A nuclear gene with many introns encoding ammonium-inducible chloroplastic NADP-specific glutamate dehydrogenase(s) in Chlorella sorokiniana.

    PubMed

    Cock, J M; Kim, K D; Miller, P W; Hutson, R G; Schmidt, R R

    1991-11-01

    Chlorella sorokiniana possesses ammonium-inducible, chloroplastic, NADP-specific glutamate dehydrogenase (NADP-GDH) homo- or heterohexamers composed of alpha- and/or beta-subunits which were previously shown to derive from precursor protein(s) of identical size. From the present studies, data are consistent with these two subunits being encoded by a single nuclear gene. The NADP-GDH gene is greater than 7 kb in length due to the presence of at least 21 introns, an unusually large number for a eukaryotic microorganism. The exons, identified by comparison with sequences of NADP-GDH cDNA clones, include a region which is highly conserved among NADP-GDH genes. This region in the C. sorokiniana gene is 77% and 73% identical to the corresponding regions in the Escherichia coli and Neurospora crassa NADP-GDH genes, respectively. Seventeen independent NADP-GDH cDNA clones were analyzed by restriction mapping and partial sequencing, and no differences were detected among them. The longest cDNA was fused in frame with lacZ in a Bluescript vector and was expressed in E. coli as NADP-GDH antigen. During a 240 min induction period, under conditions in which both types of subunits were synthesized, only a single (2.2 kb) NADP-GDH mRNA band was detected on northern blots using cDNA probes from the highly conserved and 3'-untranslated regions. Collectively, these results are consistent with a single mRNA encoding a precursor-protein which is differentially processed to yield either an alpha- or beta-subunit. PMID:1718478

  12. Engineering activity and stability of Thermotoga maritima glutamate dehydrogenase. II: construction of a 16-residue ion-pair network at the subunit interface.

    PubMed

    Lebbink, J H; Knapp, S; van der Oost, J; Rice, D; Ladenstein, R; de Vos, W M

    1999-06-01

    The role of an 18-residue ion-pair network, that is present in the glutamate dehydrogenase from the hyperthermophilic archaeon Pyrococcus furiosus, in conferring stability to other, less stable homologous enzymes, has been studied by introducing four new charged amino acid residues into the subunit interface of glutamate dehydrogenase from the hyperthermophilic bacterium Thermotoga maritima. These two GDHs are 55 % identical in amino acid sequence, differ greatly in thermo-activity and stability and derive from microbes with different phylogenetic positions. Amino acid substitutions were introduced as single mutations as well as in several combinations. Elucidation of the crystal structure of the quadruple mutant S128R/T158E/N117R/S160E T. maritima glutamate dehydrogenase showed that all anticipated ion-pairs are formed and that a 16-residue ion-pair network is present. Enlargement of existing networks by single amino acid substitutions unexpectedly resulted in a decrease in resistance towards thermal inactivation and thermal denaturation. However, combination of destabilizing single mutations in most cases restored stability, indicating the need for balanced charges at subunit interfaces and high cooperativity between the different members of the network. Combination of the three destabilizing mutations in triple mutant S128R/T158E/N117R resulted in an enzyme with a 30 minutes longer half-life of inactivation at 85 degrees C, a 3 degrees C higher temperature optimum for catalysis, and a 0.5 degrees C higher apparent melting temperature than that of wild-type glutamate dehydrogenase. These findings confirm the hypothesis that large ion-pair networks do indeed stabilize enzymes from hyperthermophilic organisms. PMID:10366510

  13. Highly efficient synthesis of chiral alcohols with a novel NADH-dependent reductase from Streptomyces coelicolor.

    PubMed

    Wang, Li-Juan; Li, Chun-Xiu; Ni, Yan; Zhang, Jie; Liu, Xiang; Xu, Jian-He

    2011-07-01

    An NADH-dependent reductase (ScCR) from Streptomyces coelicolor was discovered by genome mining for carbonyl reductases. ScCR was overexpressed in Escherichia coli BL21, purified to homogeneity and its catalytic properties were studied. This enzyme catalyzed the asymmetric reduction of a broad range of prochiral ketones including aryl ketones, α- and β-ketoesters, with high activity and excellent enantioselectivity (>99% ee) towards β-ketoesters. Among them, ethyl 4-chloro-3-oxobutanoate (COBE) was efficiently converted to ethyl (S)-4-chloro-3-hydroxybutanoate ((S)-CHBE), an important pharmaceutical intermediate, in water/toluene biphasic system. As much as 600 g/L (3.6M) of COBE was asymmetrically reduced within 22 h using 2-propanol as a co-substrate for NADH regeneration, resulting in a yield of 93%, an enantioselectivity of >99% ee, and a total turnover number (TTN) of 12,100. These results indicate the potential of ScCR for the industrial production of valuable chiral alcohols. PMID:21570826

  14. Metabolism changes during aging in the hippocampus and striatum of glud1 (glutamate dehydrogenase 1) transgenic mice.

    PubMed

    Choi, In-Young; Lee, Phil; Wang, Wen-Tung; Hui, Dongwei; Wang, Xinkun; Brooks, William M; Michaelis, Elias K

    2014-01-01

    The decline in neuronal function during aging may result from increases in extracellular glutamate (Glu), Glu-induced neurotoxicity, and altered mitochondrial metabolism. To study metabolic responses to persistently high levels of Glu at synapses during aging, we used transgenic (Tg) mice that over-express the enzyme Glu dehydrogenase (GDH) in brain neurons and release excess Glu in synapses. Mitochondrial GDH is important in amino acid and carbohydrate metabolism and in anaplerotic reactions. We monitored changes in nineteen neurochemicals in the hippocampus and striatum of adult, middle aged, and aged Tg and wild type (wt) mice, in vivo, using proton ((1)H) magnetic resonance spectroscopy. Significant differences between adult Tg and wt were higher Glu, N-acetyl aspartate (NAA), and NAA + NAA-Glu (NAAG) levels, and lower lactate in the Tg hippocampus and striatum than those of wt. During aging, consistent changes in Tg and wt hippocampus and striatum included increases in myo-inositol and NAAG. The levels of glutamine (Gln), a key neurochemical in the Gln-Glu cycle between neurons and astroglia, increased during aging in both the striatum and hippocampus of Tg mice, but only in the striatum of the wt mice. Age-related increases of Glu were observed only in the striatum of the Tg mice. PMID:24442550

  15. 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. PMID:24122651

  16. Metabolism Changes During Aging in the Hippocampus and Striatum of Glud1 (Glutamate Dehydrogenase 1) Transgenic Mice

    PubMed Central

    Choi, In-Young; Lee, Phil; Wang, Wen-Tung; Hui, Dongwei; Wang, Xinkun; Brooks, William M.

    2014-01-01

    The decline in neuronal function during aging may result from increases in extracellular glutamate (Glu), Glu-induced neurotoxicity, and altered mitochondrial metabolism. To study metabolic responses to persistently high levels of Glu at synapses during aging, we used transgenic (Tg) mice that over-express the enzyme Glu dehydrogenase (GDH) in brain neurons and release excess Glu in synapses. Mitochondrial GDH is important in amino acid and carbohydrate metabolism and in anaplerotic reactions. We monitored changes in nineteen neurochemicals in the hippocampus and striatum of adult, middle aged, and aged Tg and wild type (wt) mice, in vivo, using proton (1H) magnetic resonance spectroscopy. Significant differences between adult Tg and wt were higher Glu, N-acetyl aspartate (NAA), and NAA + NAA−Glu (NAAG) levels, and lower lactate in the Tg hippocampus and striatum than those of wt. During aging, consistent changes in Tg and wt hippocampus and striatum included increases in myo-inositol and NAAG. The levels of glutamine (Gln), a key neurochemical in the Gln-Glu cycle between neurons and astroglia, increased during aging in both the striatum and hippocampus of Tg mice, but only in the striatum of the wt mice. Age-related increases of Glu were observed only in the striatum of the Tg mice. PMID:24442550

  17. A plastidial localization and origin of L-glutamate dehydrogenase in a soybean cell culture. [Glycine max

    SciTech Connect

    Bhadula, S.K.; Shargool, P.D. )

    1991-01-01

    The subcellular distribution of L-glutamate dehydrogenase (GDH, EC 1.4.1.3.) was studied in SB3 soybean (Glycine max) cells using subcellular fractionation techniques. Compounds that inhibit protein synthesis either on 80s or 70s ribosomes were also used to give a preliminary idea of which subcellular fraction is involved in GDH synthesis. It was found that whereas cycloheximide and puromycin considerably reduced the total amount of protein synthesized by the cells, they did not appear to inhibit the synthesis of GDH. In the presence of chloramphenicol, both GDH activity and protein level in the cells were considerably reduced, suggesting that this enzyme was synthesized in organelles and not in the cytosol. Streptomycin, which inhibits plastid protein synthesis, also inhibited synthesis of GDH, indicating that a fraction of GDH activity was plastidial in origin. This is supported by the data on subcellular distribution of the enzyme, which showed that a major fraction of GDH is found in the plastidial fraction, although some activity is found associated with the mitochondrial fraction also. Since a major fraction of GDH activity was found in the plastidial fraction, the authors studied protein synthesis using isolated plastids and {sup 35}S-methionine. Using antibodies raised against purified GDH, they identified a {sup 35}S-labeled 41-kilodalton polypeptide synthesized by plastids as GDH.

  18. Mechanism of pressure-induced thermostabilization of proteins: Studies of glutamate dehydrogenases from the hyperthermophile Thermococcus litoralis

    PubMed Central

    Sun, Michael M.C.; Caillot, Raphaele; Mak, Gary; Robb, Frank T.; Clark, Douglas S.

    2001-01-01

    In this study, we investigated the effect of pressure on protein structure and stability at high temperature. Thermoinactivation experiments at 5 and 500 atm were performed using the wild-type (WT) enzyme and two single mutants (D167T and T138E) of the glutamate dehydrogenase (GDH) from the hyperthermophile Thermococcus litoralis. All three GDHs were stabilized, although to different degrees, by the application of 500 atm. Interestingly, the degree of pressure stabilization correlated with GDH stability as well as the magnitude of electrostatic repulsion created by residues at positions 138 and 167. Thermoinactivation experiments also were performed in the presence of trehalose. Addition of the sugar stabilized all three GDHs; the degree of sugar-induced thermostabilization followed the same order as pressure stabilization. Previous studies suggested a mechanism whereby the enzyme adopts a more compact and rigid structure and volume fluctuations away from the native state are diminished under pressure. The present results on the three GDHs allowed us to further confirm and refine the proposed mechanism for pressure-induced thermostabilization. In particular, we propose that pressure stabilizes against thermoinactivation by shifting the equilibrium between conformational substates of the GDH hexamer, thus inhibiting irreversible aggregation. PMID:11514665

  19. Role of the glutamate dehydrogenase reaction in furnishing aspartate nitrogen for urea synthesis: studies in perfused rat liver with 15N.

    PubMed Central

    Nissim, Itzhak; Horyn, Oksana; Luhovyy, Bohdan; Lazarow, Adam; Daikhin, Yevgeny; Nissim, Ilana; Yudkoff, Marc

    2003-01-01

    The present study was designed to determine: (i) the role of the reductive amination of alpha-ketoglutarate via the glutamate dehydrogenase reaction in furnishing mitochondrial glutamate and its transamination into aspartate; (ii) the relative incorporation of perfusate 15NH4Cl, [2-15N]glutamine or [5-15N]glutamine into carbamoyl phosphate and aspartate-N and, thereby, [15N]urea isotopomers; and (iii) the extent to which perfusate [15N]aspartate is taken up by the liver and incorporated into [15N]urea. We used a liver-perfusion system containing a physiological mixture of amino acids and ammonia similar to concentrations in vivo, with 15N label only in glutamine, ammonia or aspartate. The results demonstrate that in perfusions with a physiological mixture of amino acids, approx. 45 and 30% of total urea-N output was derived from perfusate ammonia and glutamine-N respectively. Approximately two-thirds of the ammonia utilized for carbamoyl phosphate synthesis was derived from perfusate ammonia and one-third from glutamine. Perfusate [2-15N]glutamine, [5-15N]glutamine or [15N]aspartate provided 24, 10 and 10% respectively of the hepatic aspartate-N pool, whereas perfusate 15NH4Cl provided approx. 37% of aspartate-N utilized for urea synthesis, secondary to the net formation of [15N]glutamate via the glutamate dehydrogenase reaction. The results suggest that the mitochondrial glutamate formed via the reductive amination of alpha-ketoglutarate may have a key role in ammonia detoxification by the following processes: (i) furnishing aspartate-N for ureagenesis; (ii) serving as a scavenger for excess ammonia; and (iii) improving the availability of the mitochondrial [glutamate] for synthesis of N -acetylglutamate. In addition, the current findings suggest that the formation of aspartate via the mitochondrial aspartate aminotransferase reaction may play an important role in the synthesis of cytosolic argininosuccinate. PMID:12935293

  20. External mitochondrial NADH-dependent reductase of redox cyclers: VDAC1 or Cyb5R3?

    PubMed

    Nikiforova, Anna B; Saris, Nils-Erik L; Kruglov, Alexey G

    2014-09-01

    It was reported that VDAC1 possesses an NADH oxidoreductase activity and plays an important role in the activation of xenobiotics in the outer mitochondrial membrane. In the present work, we evaluated the participation of VDAC1 and Cyb5R3 in the NADH-dependent activation of various redox cyclers in mitochondria. We show that external NADH oxidoreductase caused the redox cycling of menadione ≫ lucigenin>nitrofurantoin. Paraquat was predominantly activated by internal mitochondria oxidoreductases. An increase in the ionic strength stimulated and suppressed the redox cycling of negatively and positively charged acceptors, as was expected for the Cyb5R3-mediated reduction. Antibodies against Cyb5R3 but not VDAC substantially inhibited the NADH-related oxidoreductase activities. The specific VDAC blockers G3139 and erastin, separately or in combination, in concentrations sufficient for the inhibition of substrate transport, exhibited minimal effects on the redox cycler-dependent NADH oxidation, ROS generation, and reduction of exogenous cytochrome c. In contrast, Cyb5R3 inhibitors (6-propyl-2-thiouracil, p-chloromercuriobenzoate, quercetin, mersalyl, and ebselen) showed similar patterns of inhibition of ROS generation and cytochrome c reduction. The analysis of the spectra of the endogenous cytochromes b5 and c in the presence of nitrofurantoin and the inhibitors of VDAC and Cyb5R3 demonstrated that the redox cycler can transfer electrons from Cyb5R3 to endogenous cytochrome c. This caused the oxidation of outer membrane-bound cytochrome b5, which is in redox balance with Cyb5R3. The data obtained argue against VDAC1 and in favor of Cyb5R3 involvement in the activation of redox cyclers in the outer mitochondrial membrane. PMID:24945955

  1. Biochemical characterization and substrate profiling of a new NADH-dependent enoate reductase from Lactobacillus casei.

    PubMed

    Gao, Xiuzhen; Ren, Jie; Wu, Qiaqing; Zhu, Dunming

    2012-06-10

    Carbon-carbon double bond of α,β-unsaturated carbonyl compounds can be reduced by enoate reductase (ER), which is an important reaction in fine chemical synthesis. A putative enoate reductase gene from Lactobacillus casei str. Zhang was cloned into pET-21a+ and expressed in Escherichia coli BL21 (DE3) host cells. The encoded enzyme (LacER) was purified by ammonium sulfate precipitation and treatment in an acidic buffer. This enzyme was identified as a NADH-dependent enoate reductase, which had a K(m) of 0.034 ± 0.006 mM and k(cat) of (3.2 ± 0.2) × 10³ s⁻¹ toward NADH using 2-cyclohexen-1-one as the substrate. Its K(m) and k(cat) toward substrate 2-cyclohexen-1-one were 1.94 ± 0.04 mM and (8.4 ± 0.2) × 10³ s⁻¹, respectively. The enzyme showed a maximum activity at pH 8.0-9.0. The optimum temperature of the enzyme was 50-55°C, and LacER was relatively stable below 60 °C. The enzyme was active toward aliphatic alkenyl aldehyde, ketones and some cyclic anhydrides. Substituted groups of cyclic α,β-unsaturated ketones and its ring size have positive or negative effects on activity. (R)-(-)-Carvone was reduced to (2R,5R)-dihydrocarvone with 99% conversion and 98% (diasteromeric excess: de) stereoselectivity, indicating a high synthetic potential of LacER in asymmetric synthesis. PMID:22579387

  2. NAD(+)-specific glutamate dehydrogenase (EC.1.4.1.2) in Streptomyces coelicolor; in vivo characterization and the implication for nutrient-dependent secondary metabolism.

    PubMed

    Kim, Songhee H; Kim, Byung-Gee

    2016-06-01

    While glutamate and glutamate-rich compounds are widely used for culturing Streptomyces sp., little is known regarding glutamate catabolism at molecular level. Noting the presence of two distinct putative glutamate dehydrogenases (GDH), we constructed knockout mutants of each gene with Streptomyces coelicolor M145 and examined the functionality related to antibiotic production. Out of the two, the sco2999 knockout (ΔgdhB, NAD(+)-specific) showed outstanding effects; it decreased the growth sevenfold but initiated the undecylprodigiosin (RED) production in complex Difco nutrient media which otherwise does not support the production from M145. With glucose supplementation, the growth difference by ΔgdhB disappeared but we could obtain significantly increased actinorhodin (ACT) and RED biosynthesis with the mutant by limiting the glucose content (0.5∼1.0 %, w/v). Complementing the gene to the knockout mutant inhibited the production, confirming its gene specificity. Along with the extended impacts on overall nitrogen metabolism based on the intracellular metabolite analysis and enzyme assays, GdhB and glutamate utilization were shown to interfere with N-acetylglucosamine metabolism and the activity of its associated global transcriptional regulator (DasR). Taken together, GdhB-subjected to the nutritional context-dependent regulation-is proposed as a key member of central nitrogen metabolism to control the secondary metabolism initiation in exploiting the organic nitrogen sources. PMID:26969038

  3. Over-expression of NADH-dependent oxidoreductase (fucO) for increasing furfural or 5-hydroxymethylfurfural tolerance

    DOEpatents

    Miller, Elliot N.; Zhang, Xueli; Yomano, Lorraine P.; Wang, Xuan; Shanmugam, Keelnatham T.; Ingram, Lonnie O'Neal

    2015-10-13

    The subject invention pertains to the discovery that the NADH-dependent propanediol oxidoreductase (FucO) can reduce furfural. This allows for a new approach to improve furfural tolerance in bacterial and/or yeast cells used to produce desired products. Thus, novel biocatalysts (bacterial, fungal or yeast cells) exhibiting increased tolerance to furfural and 5-hydroxymethylfurfural (5-HMF) are provided as are methods of making and using such biocatalysts for the production of a desired product.

  4. Comparisons of mammalian Giardia duodenalis assemblages based on the β-giardin, glutamate dehydrogenase and triose phosphate isomerase genes.

    PubMed

    Scorza, Andrea V; Ballweber, Lora R; Tangtrongsup, Sahatchai; Panuska, Carla; Lappin, Michael R

    2012-10-26

    The objective of this study was to determine and compare the assemblages of Giardia duodenalis isolated from mammalian fecal samples using the β-giardin (bg), glutamate dehydrogenase (gdh) and triosephosphate isomerase (tpi) genes. A total of 202 samples, either submitted to the Veterinary Diagnostic Laboratory (Parasitology) at Colorado State University or part of ongoing research studies, were typed. A subset of 50 dog samples were also assessed by the tpi-D-specific primers. Of these, 183 were from dogs, 13 were from cats, two were from llamas, and one each was from a calf, an alpaca, a sheep, and a horse. The majority of the dogs (171 of 183 isolates) in this study were infected with only dog-adapted Assemblage C or D. The tpi-D-specific primers confirmed that 28 of the samples that typed as Assemblage D by the bg and gdh genes were also Assemblage D by the tpi-D-specific primers. Only 12 isolates were Assemblage A alone or Assemblage A and Assemblage C or D. Of the 13 cat isolates, seven were Assemblage F, two were Assemblage D, three were Assemblage A and 1 contained both Assemblages C and D. The calf isolate was Assemblage E (gdh, tpi) and the alpaca (bg, gdh), llamas (gdh), sheep (bg, gdh, tpi) and horse (tpi) isolates were all Assemblage A. When the assemblage could be determined for more than one gene, 91 of 117 dog isolates gave consistent results and 8 of 9 cat isolates gave consistent results. PMID:22652427

  5. A high-frequency mutation in Bacillus subtilis: requirements for the decryptification of the gudB glutamate dehydrogenase gene.

    PubMed

    Gunka, Katrin; Tholen, Stefan; Gerwig, Jan; Herzberg, Christina; Stülke, Jörg; Commichau, Fabian M

    2012-03-01

    Common laboratory strains of Bacillus subtilis encode two glutamate dehydrogenases: the enzymatically active protein RocG and the cryptic enzyme GudB that is inactive due to a duplication of three amino acids in its active center. The inactivation of the rocG gene results in poor growth of the bacteria on complex media due to the accumulation of toxic intermediates. Therefore, rocG mutants readily acquire suppressor mutations that decryptify the gudB gene. This decryptification occurs by a precise deletion of one part of the 9-bp direct repeat that causes the amino acid duplication. This mutation occurs at the extremely high frequency of 10(-4). Mutations affecting the integrity of the direct repeat result in a strong reduction of the mutation frequency; however, the actual sequence of the repeat is not essential. The mutation frequency of gudB was not affected by the position of the gene on the chromosome. When the direct repeat was placed in the completely different context of an artificial promoter, the precise deletion of one part of the repeat was also observed, but the mutation frequency was reduced by 3 orders of magnitude. Thus, transcription of the gudB gene seems to be essential for the high frequency of the appearance of the gudB1 mutation. This idea is supported by the finding that the transcription-repair coupling factor Mfd is required for the decryptification of gudB. The Mfd-mediated coupling of transcription to mutagenesis might be a built-in precaution that facilitates the accumulation of mutations preferentially in transcribed genes. PMID:22178973

  6. A High-Frequency Mutation in Bacillus subtilis: Requirements for the Decryptification of the gudB Glutamate Dehydrogenase Gene

    PubMed Central

    Gunka, Katrin; Tholen, Stefan; Gerwig, Jan; Herzberg, Christina; Stülke, Jörg

    2012-01-01

    Common laboratory strains of Bacillus subtilis encode two glutamate dehydrogenases: the enzymatically active protein RocG and the cryptic enzyme GudB that is inactive due to a duplication of three amino acids in its active center. The inactivation of the rocG gene results in poor growth of the bacteria on complex media due to the accumulation of toxic intermediates. Therefore, rocG mutants readily acquire suppressor mutations that decryptify the gudB gene. This decryptification occurs by a precise deletion of one part of the 9-bp direct repeat that causes the amino acid duplication. This mutation occurs at the extremely high frequency of 10−4. Mutations affecting the integrity of the direct repeat result in a strong reduction of the mutation frequency; however, the actual sequence of the repeat is not essential. The mutation frequency of gudB was not affected by the position of the gene on the chromosome. When the direct repeat was placed in the completely different context of an artificial promoter, the precise deletion of one part of the repeat was also observed, but the mutation frequency was reduced by 3 orders of magnitude. Thus, transcription of the gudB gene seems to be essential for the high frequency of the appearance of the gudB1 mutation. This idea is supported by the finding that the transcription-repair coupling factor Mfd is required for the decryptification of gudB. The Mfd-mediated coupling of transcription to mutagenesis might be a built-in precaution that facilitates the accumulation of mutations preferentially in transcribed genes. PMID:22178973

  7. A novel mechanism of V-type zinc inhibition of glutamate dehydrogenase results from disruption of subunit interactions necessary for efficient catalysis.

    PubMed

    Bailey, Jaclyn; Powell, Lakeila; Sinanan, Leander; Neal, Jacob; Li, Ming; Smith, Thomas; Bell, Ellis

    2011-09-01

    Bovine glutamate dehydrogenase is potently inhibited by zinc and the major impact is on V(max) suggesting a V-type effect on catalysis or product release. Zinc inhibition decreases as glutamate concentrations decrease suggesting a role for subunit interactions. With the monocarboxylic amino acid norvaline, which gives no evidence of subunit interactions, zinc does not inhibit. Zinc significantly decreases the size of the pre-steady state burst in the reaction but does not affect NADPH binding in the enzyme-NADPH-glutamate complex that governs the steady state turnover, again suggesting that zinc disrupts subunit interactions required for catalytic competence. While differential scanning calorimetry suggests zinc binds and induces a slightly conformationally more rigid state of the protein, limited proteolysis indicates that regions in the vicinity of the antennae regions and the trimer-trimer interface become more flexible. The structures of glutamate dehydrogenase bound with zinc and europium show that zinc binds between the three dimers of subunits in the hexamer, a region shown to bind novel inhibitors that block catalytic turnover, which is consistent with the above findings. In contrast, europium binds to the base of the antenna region and appears to abrogate the inhibitory effect of zinc. Structures of various states of the enzyme have shown that both regions are heavily involved in the conformational changes associated with catalytic turnover. These results suggest that the V-type inhibition produced with glutamate as the substrate results from disruption of subunit interactions necessary for efficient catalysis rather than by a direct effect on the active site conformation. PMID:21749647

  8. Involvement of GDH3-encoded NADP+-dependent Glutamate Dehydrogenase in Yeast Cell Resistance to Stress-induced Apoptosis in Stationary Phase Cells*

    PubMed Central

    Lee, Yong Joo; Kim, Kyung Jin; Kang, Hong Yong; Kim, Hye-Rim; Maeng, Pil Jae

    2012-01-01

    Glutamate metabolism is linked to a number of fundamental metabolic pathways such as amino acid metabolism, the TCA cycle, and glutathione (GSH) synthesis. In the yeast Saccharomyces cerevisiae, glutamate is synthesized from α-ketoglutarate by two NADP+-dependent glutamate dehydrogenases (NADP-GDH) encoded by GDH1 and GDH3. Here, we report the relationship between the function of the NADP-GDH and stress-induced apoptosis. Gdh3-null cells showed accelerated chronological aging and hypersusceptibility to thermal and oxidative stress during stationary phase. Upon exposure to oxidative stress, Gdh3-null strains displayed a rapid loss in viability associated with typical apoptotic hallmarks, i.e. reactive oxygen species accumulation, nuclear fragmentation, DNA breakage, and phosphatidylserine translocation. In addition, Gdh3-null cells, but not Gdh1-null cells, had a higher tendency toward GSH depletion and subsequent reactive oxygen species accumulation than did WT cells. GSH depletion was rescued by exogenous GSH or glutamate. The hypersusceptibility of stationary phase Gdh3-null cells to stress-induced apoptosis was suppressed by deletion of GDH2. Promoter swapping and site-directed mutagenesis of GDH1 and GDH3 indicated that the necessity of GDH3 for the resistance to stress-induced apoptosis and chronological aging is due to the stationary phase-specific expression of GDH3 and concurrent degradation of Gdh1 in which the Lys-426 residue plays an essential role. PMID:23105103

  9. 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

  10. Metabolism of hydroxypyruvate in a mutant of barley lacking NADH-dependent hydroxypyruvate reductase, an important photorespiratory enzyme activity

    SciTech Connect

    Murray, A.J.S.; Blackwell, R.D.; Lea, P.J. )

    1989-09-01

    A mutant of barley (Hordeum vulgare L.), LaPr 88/29, deficient in NADH-dependent hydroxypyruvate reductase (HPR) activity has been isolated. The activities of both NADH (5%) and NADPH-dependent (19%) HPR were severely reduced in this mutant compared to the wild type. Although lacking an enzyme in the main carbon pathway of photorespiration, this mutant was capable of CO{sub 2} fixation rates equivalent to 75% of that of the wild type, in normal atmospheres and 50% O{sub 2}. There also appeared to be little disruption to the photorespiratory metabolism as ammonia release, CO{sub 2} efflux and {sup 14}CO{sub 2} release from L-(U-{sup 14}C)serine feeding were similar in both mutant and wild-type leaves. When leaves of LaPr 88/29 were fed either ({sup 14}C)serine or {sup 14}CO{sub 2}, the accumulation of radioactivity was in serine and not in hydroxypyruvate, although the mutant was still able to metabolize over 25% of the supplied ({sup 14}C)serine into sucrose. After 3 hours in air the soluble amino acid pool was almost totally dominated by serine and glycine. LaPr 88/29 has also been used to show that NADH-glyoxylate reductase and NADH-HPR are probably not catalyzed by the same enzyme in barley and that over 80% of the NADPH-dependent HPR activity is due to the NADH-dependent enzyme. We also suggest that the alternative NADPH activity can metabolize a proportion, but not all, of the hydroxypyruvate produced during photorespiration and may thus form a useful backup to the NADH-dependent enzyme under conditions of maximal photorespiration.

  11. 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

  12. Slow motion picture of protein inactivation during single-droplet drying: a study of inactivation kinetics of L-glutamate dehydrogenase dried in an acoustic levitator.

    PubMed

    Lorenzen, Elke; Lee, Geoffrey

    2012-06-01

    A novel technique is presented to allow measurement of the kinetics of protein inactivation during drying of an acoustically levitated single droplet. Droplets/particles are removed from the acoustic field after various times during drying, and the state of the protein within them is analyzed. The influence of drying air temperature, relative humidity, buffer concentration, and the presence of a substrate on the inactivation of glutamate dehydrogenase is described. The kinetics of inactivation showed three distinct phases. The first phase of constant drying rate demonstrated little protein inactivation in the solution droplet. After the critical point of drying, a second phase was distinguishable when the surface temperature has risen sharply, but there is still only little inactivation of the protein in the solid particle. An onset point of rapid inactivation of the protein marked the start of the third phase that proceeded with approximately first-order rate kinetics. In the case of L-glutamate dehydrogenase, the evidence suggests that the residual moisture content of the solid and not the temperature alone determines the point of onset of protein inactivation. PMID:22447570

  13. NADH-dependent biosensor in Saccharomyces cerevisiae: principle and validation at the single cell level

    PubMed Central

    2014-01-01

    A reporter system was constructed to measure perturbations in the NADH/NAD+ co-factor balance in yeast, by using the green fluorescent protein gene under the control of the GPD2 promoter that is induced under conditions of excess of NADH. High fluorescence levels were obtained in a glycerol 3-phosphate dehydrogenase double deletion strain (gpd1Δgpd2Δ), which is deficient in the ability to regenerate NAD+ via glycerol formation. The responsiveness of the reporter system to externally induced perturbations in NADH oxidation was also evaluated in the gpd1Δgpd2Δ strain background by addition of acetoin, as well as by introduction of a set of heterologous xylose reductases (XRs) having different selectivities for NADH. Addition of acetoin during cell proliferation under oxygen-limited conditions resulted in a more than 2-fold decrease in mean fluorescence intensity as compared to the control experiment. Strains carrying XRs with different selectivities for NADH could be distinguished at the single cell level, so that the XR with the highest selectivity for NADH displayed the lowest fluorescence. In conclusion, the designed system successfully allowed for monitoring perturbations in the cellular redox metabolism caused by environmental changes, or by heterologous gene expression. The reporter system displayed high resolution in distinguishing cytosolic NADH oxidation capacity and hence has potential to be used for high-throughput screening based on the fluorescence of single cells. PMID:25401080

  14. [Cooperative inactivation of glutamate dehydrogenase of 2,2,6,6- tetramethyl-4-oxopiperidine-1-oxyl. Interpretation of results within the scope of a hexamer model with equivalent subunit orientation].

    PubMed

    Karabashian, L V; Agadzhanian, S A

    1988-01-01

    It was shown that the blockage of epsilon-amino group of Lis-126 residue by 2,2,6,6-tetramethyl-4-oxo-piperidine-1-oxyl (TMPO) leads to the cooperative inactivation of glutamate dehydrogenase (L-glutamate-NAD(P)-oxidoreductase, EC 1.4.1.3). The data concerning cooperative inactivation of the enzyme are interpreted by the model of hexamer with identical orientation of subunits. It was shown that the modification of any of enzyme subunits is accompanied by an inactivation of the hexamer's fragment which is a dimer, with subunits interacting reciprocally by means of isological contacts. PMID:3252150

  15. Insights into Flavin-based Electron Bifurcation via the NADH-dependent Reduced Ferredoxin:NADP Oxidoreductase Structure*

    PubMed Central

    Demmer, Julius K.; Huang, Haiyan; Wang, Shuning; Demmer, Ulrike; Thauer, Rudolf K.; Ermler, Ulrich

    2015-01-01

    NADH-dependent reduced ferredoxin:NADP oxidoreductase (NfnAB) is found in the cytoplasm of various anaerobic bacteria and archaea. The enzyme reversibly catalyzes the endergonic reduction of ferredoxin with NADPH driven by the exergonic transhydrogenation from NADPH onto NAD+. Coupling is most probably accomplished via the mechanism of flavin-based electron bifurcation. To understand this process on a structural basis, we heterologously produced the NfnAB complex of Thermotoga maritima in Escherichia coli, provided kinetic evidence for its bifurcating behavior, and determined its x-ray structure in the absence and presence of NADH. The structure of NfnAB reveals an electron transfer route including the FAD (a-FAD), the [2Fe-2S] cluster of NfnA and the FAD (b-FAD), and the two [4Fe-4S] clusters of NfnB. Ferredoxin is presumably docked onto NfnB close to the [4Fe-4S] cluster distal to b-FAD. NAD(H) binds to a-FAD and NADP(H) consequently to b-FAD, which is positioned in the center of the NfnAB complex and the site of electron bifurcation. Arg187 is hydrogen-bonded to N5 and O4 of the bifurcating b-FAD and might play a key role in adjusting a low redox potential of the FADH•/FAD pair required for ferredoxin reduction. A mechanism of FAD-coupled electron bifurcation by NfnAB is proposed. PMID:26139605

  16. Paraquat Toxicity Induced by Voltage-dependent Anion Channel 1 Acts as an NADH-dependent Oxidoreductase*

    PubMed Central

    Shimada, Hiroki; Hirai, Kei-Ichi; Simamura, Eriko; Hatta, Toshihisa; Iwakiri, Hiroki; Mizuki, Keiji; Hatta, Taizo; Sawasaki, Tatsuya; Matsunaga, Satoko; Endo, Yaeta; Shimizu, Shigeomi

    2009-01-01

    Paraquat (PQ), a herbicide used worldwide, causes fatal injury to organs upon high dose ingestion. Treatments for PQ poisoning are unreliable, and numerous deaths have been attributed inappropriate usage of the agent. It is generally speculated that a microsomal drug-metabolizing enzyme system is responsible for PQ toxicity. However, recent studies have demonstrated cytotoxicity via mitochondria, and therefore, the cytotoxic mechanism remains controversial. Here, we demonstrated that mitochondrial NADH-dependent PQ reductase containing a voltage-dependent anion channel 1 (VDAC1) is responsible for PQ cytotoxicity. When mitochondria were incubated with NADH and PQ, superoxide anion (O2˙̄) was produced, and the mitochondria ruptured. Outer membrane extract oxidized NADH in a PQ dose-dependent manner, and oxidation was suppressed by VDAC inhibitors. Zymographic analysis revealed the presence of VDAC1 protein in the oxidoreductase, and the direct binding of PQ to VDAC1 was demonstrated using biotinylated PQ. VDAC1-overexpressing cells showed increased O2˙̄ production and cytotoxicity, both of which were suppressed in VDAC1 knockdown cells. These results indicated that a VDAC1-containing mitochondrial system is involved in PQ poisoning. These insights into the mechanism of PQ poisoning not only demonstrated novel physiological functions of VDAC protein, but they may facilitate the development of new therapeutic approaches. PMID:19717555

  17. Gene cloning and characterization of the very large NAD-dependent l-glutamate dehydrogenase from the psychrophile Janthinobacterium lividum, isolated from cold soil.

    PubMed

    Kawakami, Ryushi; Sakuraba, Haruhiko; Ohshima, Toshihisa

    2007-08-01

    NAD-dependent l-glutamate dehydrogenase (NAD-GDH) activity was detected in cell extract from the psychrophile Janthinobacterium lividum UTB1302, which was isolated from cold soil and purified to homogeneity. The native enzyme (1,065 kDa, determined by gel filtration) is a homohexamer composed of 170-kDa subunits (determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis). Consistent with these findings, gene cloning and sequencing enabled deduction of the amino acid sequence of the subunit, which proved to be comprised of 1,575 amino acids with a combined molecular mass of 169,360 Da. The enzyme from this psychrophile thus appears to belong to the GDH family characterized by very large subunits, like those expressed by Streptomyces clavuligerus and Pseudomonas aeruginosa (about 180 kDa). The entire amino acid sequence of the J. lividum enzyme showed about 40% identity with the sequences from S. clavuligerus and P. aeruginosa enzymes, but the central domains showed higher homology (about 65%). Within the central domain, the residues related to substrate and NAD binding were highly conserved, suggesting that this is the enzyme's catalytic domain. In the presence of NAD, but not in the presence of NADP, this GDH exclusively catalyzed the oxidative deamination of l-glutamate. The stereospecificity of the hydride transfer to NAD was pro-S, which is the same as that of the other known GDHs. Surprisingly, NAD-GDH activity was markedly enhanced by the addition of various amino acids, such as l-aspartate (1,735%) and l-arginine (936%), which strongly suggests that the N- and/or C-terminal domains play regulatory roles and are involved in the activation of the enzyme by these amino acids. PMID:17526698

  18. The asymmetric distribution of enzymic activity between the six subunits of bovine liver glutamate dehydrogenase. Use of D- and L-glutamyl alpha-chloromethyl ketones (4-amino-6-chloro-5-oxohexanoic acid.

    PubMed Central

    Rasool, C G; Nicolaidis, S; Akhtar, M

    1976-01-01

    A method for the preparation of D- and L-glutamyl alpha-chloromethyl ketones (4-amino-6-chloro-5-oxohexanoic acid) is described. These chloromethyl ketones irreversibly inactivated bovine glutamate dehydrogenase, whereas several other related compounds had no adverse effect on the activity of the enzyme. The inactivation process was shown to be due to the modification of lysine-126. The time-courses for the inactivation and the incorporation of radioactivity from tritiated L-glutamyl alpha-chloromethyl ketone into the glutamate dehydrogenase were biphasic. The results were interpreted to suggest the involvement of 'negative co-operative' interactions in the reactivity of lysine-126. From the cumulative evidence it is argued that the first subunit of the enzyme, which takes part in catalysis, makes the largest, and the last the smallest, contribution to the overall catalysis. It is emphasized that three of the six subunits of the enzyme may possess as much as 80% of the total activity of bovine glutamate dehydrogenase. PMID:10889

  19. Identification of genotypes of Giardia intestinalis isolates from dogs in Japan by direct sequencing of the PCR amplified glutamate dehydrogenase gene.

    PubMed

    Abe, Niichiro; Kimata, Isao; Iseki, Motohiro

    2003-01-01

    Giardia has been detected in domestic dogs in Japan, but the genotype of isolates has remained unclear because identification has relied on conventional microscopy. Here we tried to identify the genotypes of four isolates from dogs in Japan by direct sequencing of the PCR amplified Giardia glutamate dehydrogenase (GDH) gene. The primer pair GDHF3 and GDHB5, targeting the GDH gene, was designed to prime a region of the GDH gene sequence conserved in the strains found to have the dog-specific genotype. The specific PCR product (approximately 220 bp), amplified with this primer pair, was only observed when Giardia DNA was used as the template. The sequences of the diagnostic fragments were identical among the isolates from dogs, and were differed by 15 bp or 1 bp from the strains, which were found to be the dog-specific genotypes, Assemblage C or D respectively. To verify the identity of the amplified DNA, a phylogenetic analysis was performed. Consequently, the sequence of the isolates from dogs clearly clustered with the strain found to be Assemblage D with neighbor-joining analyses. Therefore, all the isolates from dogs examined were identified as the dog-specific genotype, Assemblage D. In the present study, we revealed the genotype of Giardia isolates in Japan, and showed that direct sequencing of the PCR product amplified with the primer pair GDHF3 and GDHB5 was a useful tool for distinguishing between the zoonotic and dog-specific genotypes. PMID:12576701

  20. 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.

  1. Purification and Properties of NADH-Dependent 5,10-Methylenetetrahydrofolate Reductase (MetF) from Escherichia coli

    PubMed Central

    Sheppard, Christal A.; Trimmer, Elizabeth E.; Matthews, Rowena G.

    1999-01-01

    A K-12 strain of Escherichia coli that overproduces methylenetetrahydrofolate reductase (MetF) has been constructed, and the enzyme has been purified to apparent homogeneity. A plasmid specifying MetF with six histidine residues added to the C terminus has been used to purify histidine-tagged MetF to homogeneity in a single step by affinity chromatography on nickel-agarose, yielding a preparation with specific activity comparable to that of the unmodified enzyme. The native protein comprises four identical 33-kDa subunits, each of which contains a molecule of noncovalently bound flavin adenine dinucleotide (FAD). No additional cofactors or metals have been detected. The purified enzyme catalyzes the reduction of methylenetetrahydrofolate to methyltetrahydrofolate, using NADH as the reductant. Kinetic parameters have been determined at 15°C and pH 7.2 in a stopped-flow spectrophotometer; the Km for NADH is 13 μM, the Km for CH2-H4folate is 0.8 μM, and the turnover number under Vmax conditions estimated for the reaction is 1,800 mol of NADH oxidized min−1 (mol of enzyme-bound FAD)−1. NADPH also serves as a reductant, but exhibits a much higher Km. MetF also catalyzes the oxidation of methyltetrahydrofolate to methylenetetrahydrofolate in the presence of menadione, which serves as an electron acceptor. The properties of MetF from E. coli differ from those of the ferredoxin-dependent methylenetetrahydrofolate reductase isolated from the homoacetogen Clostridium formicoaceticum and more closely resemble those of the NADH-dependent enzyme from Peptostreptococcus productus and the NADPH-dependent enzymes from eukaryotes. PMID:9922232

  2. Importance of glutamate 87 and the substrate α-amine for the reaction catalyzed by D-arginine dehydrogenase.

    PubMed

    Ball, Jacob; Bui, Quan V V; Gannavaram, Swathi; Gadda, Giovanni

    2015-02-15

    Pseudomonas aeruginosa D-arginine dehydrogenase (PaDADH) catalyzes the oxidation of D-arginine to iminoarginine, which is non-enzymatically hydrolyzed to 2-ketoarginine and ammonia. Here, site-directed mutagenesis and pH effects were used to investigate binding and catalysis of zwitterionic and cationic substrates for the enzyme. An unprotonated group with apparent pKa value ⩾7.9 is required for binding D-arginine or D-lysine, but not D-methionine or D-leucine. This group is E87, as suggested by its replacement with leucine. An unprotonated group with pKa of 9.5, which persists in the H48F and E87L variants, is required for amine oxidation with all substrates. Since Y53 and Y249 were previously ruled out, the pKa is assigned to the substrate α-NH3(+) group, which previous QM/MM and Kd pH-profile demonstrated to be protonated for preferred binding to the enzyme. Lack of pH effects on the (D)kred with D-leucine established 9.5 as the intrinsic pKa, and D-leucine as a non-sticky substrate. D-Arginine, D-lysine and D-methionine and their corresponding iminoproducts were significantly stickier than D-leucine, as indicated by apparent pKa values <9.5 in both kcat/Km and kcat. Restricted proton movements in catalysis were established from hollowed kcat pH profiles in wild-type PaDADH with D-lysine and in the H48F and E87L enzymes with D-arginine. PMID:25637657

  3. Disorders of glutamate metabolism.

    PubMed

    Kelly, A; Stanley, C A

    2001-01-01

    The significant role the amino acid glutamate assumes in a number of fundamental metabolic pathways is becoming better understood. As a central junction for interchange of amino nitrogen, glutamate facilitates both amino acid synthesis and degradation. In the liver, glutamate is the terminus for release of ammonia from amino acids, and the intrahepatic concentration of glutamate modulates the rate of ammonia detoxification into urea. In pancreatic beta-cells, oxidation of glutamate mediates amino acid-stimulated insulin secretion. In the central nervous system, glutamate serves as an excitatory neurotransmittor. Glutamate is also the precursor of the inhibitory neurotransmittor GABA, as well as glutamine, a potential mediator of hyperammonemic neurotoxicity. The recent identification of a novel form of congenital hyperinsulinism associated with asymptomatic hyperammonemia assigns glutamate oxidation by glutamate dehydrogenase a more important role than previously recognized in beta-cell insulin secretion and hepatic and CNS ammonia detoxification. Disruptions of glutamate metabolism have been implicated in other clinical disorders, such as pyridoxine-dependent seizures, confirming the importance of intact glutamate metabolism. This article will review glutamate metabolism and clinical disorders associated with disrupted glutamate metabolism. PMID:11754524

  4. Pivotal Enzyme in Glutamate Metabolism of Poly-γ-Glutamate-Producing Microbes

    PubMed Central

    Ashiuchi, Makoto; Yamamoto, Takashi; Kamei, Tohru

    2013-01-01

    The extremely halophilic archaeon Natrialba aegyptiaca secretes the L-homo type of poly-γ-glutamate (PGA) as an extremolyte. We examined the enzymes involved in glutamate metabolism and verified the presence of L-glutamate dehydrogenases, L-aspartate aminotransferase, and L-glutamate synthase. However, neither glutamate racemase nor D-amino acid aminotransferase activity was detected, suggesting the absence of sources of D-glutamate. In contrast, D-glutamate-rich PGA producers mostly possess such intracellular sources of D-glutamate. The results of our present study indicate that the D-glutamate-anabolic enzyme “glutamate racemase” is pivotal in the biosynthesis of PGA. PMID:25371338

  5. Evolution of glutamate dehydrogenase genes: evidence for two paralogous protein families and unusual branching patterns of the archaebacteria in the universal tree of life.

    PubMed

    Benachenhou-Lahfa, N; Forterre, P; Labedan, B

    1993-04-01

    The existence of two families of genes coding for hexameric glutamate dehydrogenases has been deduced from the alignment of 21 primary sequences and the determination of the percentages of similarity between each pair of proteins. Each family could also be characterized by specific motifs. One family (Family I) was composed of gdh genes from six eubacteria and six lower eukaryotes (the primitive protozoan Giardia lamblia, the green alga Chlorella sorokiniana, and several fungi and yeasts). The other one (Family II) was composed of gdh genes from two eubacteria, two archaebacteria, and five higher eukaryotes (vertebrates). Reconstruction of phylogenetic trees using several parsimony and distance methods confirmed the existence of these two families. Therefore, these results reinforced our previously proposed hypothesis that two close but already different gdh genes were present in the last common ancestor to the three Ur-kingdoms (eubacteria, archaebacteria, and eukaryotes). The branching order of the different species of Family I was found to be the same whatever the method of tree reconstruction although it varied slightly according the region analyzed. Similarly, the topological positions of eubacteria and eukaryotes of Family II were independent of the method used. However, the branching of the two archaebacteria in Family II appeared to be unexpected: (1) the thermoacidophilic Sulfolobus solfataricus was found clustered with the two eubacteria of this family both in parsimony and distance trees, a situation not predicted by either one of the contradictory trees recently proposed; and (2) the branching of the halophilic Halobacterium salinarium varied according to the method of tree construction: it was closer to the eubacteria in the maximum parsimony tree and to eukaryotes in distance trees. Therefore, whatever the actual position of the halophilic species, archaebacteria did not appear to be monophyletic in these gdh gene trees. This result questions the

  6. 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

  7. 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.

  8. Glutamate release from platelets: exocytosis versus glutamate transporter reversal.

    PubMed

    Kasatkina, Ludmila A; Borisova, Tatiana A

    2013-11-01

    Platelets express neuronal and glial glutamate transporters EAAT 1-3 in the plasma membrane and vesicular glutamate transporters VGLUT 1,2 in the membrane of secretory granules. This study is focused on the assessment of non-exocytotic glutamate release, that is, the unstimulated release, heteroexchange and glutamate transporter reversal in platelets. Using the glutamate dehydrogenase assay, the absence of unstimulated release of endogenous glutamate from platelets was demonstrated, even after inhibition of glutamate transporters and cytoplasmic enzyme glutamine synthetase by dl-threo-β-benzyloxyaspartate and methionine sulfoximine, respectively. Depolarization of the plasma membrane by exposure to elevated [K(+)] did not induce the release of glutamate from platelets that was shown using the glutamate dehydrogenase assay and radiolabeled l-[(14)C]glutamate. Glutamate efflux by means of heteroexchange with transportable inhibitor of glutamate transporters dl-threo-β-hydroxyaspartate (dl-THA) was not observed. Furthermore, the protonophore cyanide-p-trifluoromethoxyphenyl-hydrazon (FCCP) and inhibitor of V-type H(+)-ATPase bafilomycin A1 also failed to stimulate the release of glutamate from platelets. However, exocytotic release of glutamate from secretory granules in response to thrombin stimulation was not prevented by elevated [K(+)], dl-THA, FCCP and bafilomycin A1. In contrast to nerve terminals, platelets cannot release glutamate in a non-exocytotic manner. Heteroexchange, transporter-mediated and unstimulated release of glutamate are not inherent to platelets. Therefore, platelets may be used as a peripheral marker/model for the analysis of glutamate uptake by brain nerve terminals only (direct function of transporters), whereas the mechanisms of glutamate release are different in platelets and nerve terminals. Glutamate is released by platelets exclusively by means of exocytosis. Also, reverse function of vesicular glutamate transporters of platelets is

  9. Molecular cloning, characterization and regulation of two different NADH-glutamate synthase cDNAs in bean nodules

    Technology Transfer Automated Retrieval System (TEKTRAN)

    NADH-dependent glutamate synthase (NADH-GOGAT; EC 1.4.1.14) is a key enzyme in primary ammonia assimilation in bean (Phaseolus vulgaris L.) nodules. Two different types of cDNA clones of PvNADH-GOGAT were isolated from two independent nodule cDNA libraries. The full-length cDNA clones of PvNADH-GOGA...

  10. ANTISENSE INHIBITION OF NADH-GLUTAMATE SYNTHASE IMPAIRS CARBON/NITROGEN ASSIMILATION IN NODULES OF ALFALFA (MEDICAGO SATIVA)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Legumes acquire significant amounts of nitrogen for growth from symbiotic nitrogen fixation in root nodules. The glutamine synthetase (GS)/NADH-dependent glutamate synthase (NADH-GOGAT) cycle catalyzes initial nitrogen assimilation. This report describes the impacts of specific reduction on nodule N...

  11. The odyssey of a young gene: structure-function studies in human glutamate dehydrogenases reveal evolutionary-acquired complex allosteric regulation mechanisms.

    PubMed

    Zaganas, Ioannis V; Kanavouras, Konstantinos; Borompokas, Nikolas; Arianoglou, Giovanna; Dimovasili, Christina; Latsoudis, Helen; Vlassi, Metaxia; Mastorodemos, Vasileios

    2014-01-01

    Mammalian glutamate dehydrogenase (GDH) catalyzes the reversible inter-conversion of glutamate to α-ketoglutarate and ammonia, interconnecting carbon skeleton and nitrogen metabolism. In addition, it functions as an energy switch by its ability to fuel the Krebs cycle depending on the energy status of the cell. As GDH lies at the intersection of several metabolic pathways, its activity is tightly regulated by several allosteric compounds that are metabolic intermediates. In contrast to other mammals that have a single GDH-encoding gene, humans and great apes possess two isoforms of GDH (hGDH1 and hGDH2, encoded by the GLUD1 and GLUD2 genes, respectively) with distinct regulation pattern, but remarkable sequence similarity (they differ, in their mature form, in only 15 of their 505 amino-acids). The GLUD2 gene is considered a very young gene, emerging from the GLUD1 gene through retro-position only recently (<23 million years ago). The new hGDH2 iso-enzyme, through random mutations and natural selection, is thought to have conferred an evolutionary advantage that helped its persistence through primate evolution. The properties of the two highly homologous human GDHs have been studied using purified recombinant hGDH1 and hGDH2 proteins obtained by expression of the corresponding cDNAs in Sf21 cells. According to these studies, in contrast to hGDH1 that maintains basal activity at 35-40 % of its maximal, hGDH2 displays low basal activity that is highly responsive to activation by rising levels of ADP and/or L-leucine which can also act synergistically. While hGDH1 is inhibited potently by GTP, hGDH2 shows remarkable GTP resistance. Furthermore, the two iso-enzymes are differentially inhibited by estrogens, polyamines and neuroleptics, and also differ in heat-lability. To elucidate the molecular mechanisms that underlie these different regulation patterns of the two iso-enzymes (and consequently the evolutionary adaptation of hGDH2 to a new functional role), we have

  12. Experimental evidence that overexpression of NR2B glutamate receptor subunit is associated with brain vacuolation in adult glutaryl-CoA dehydrogenase deficient mice: A potential role for glutamatergic-induced excitotoxicity in GA I neuropathology.

    PubMed

    Rodrigues, Marília Danyelle Nunes; Seminotti, Bianca; Amaral, Alexandre Umpierrez; Leipnitz, Guilhian; Goodman, Stephen Irwin; Woontner, Michael; de Souza, Diogo Onofre Gomes; Wajner, Moacir

    2015-12-15

    Glutaric aciduria type I (GA I) is biochemically characterized by accumulation of glutaric and 3-hydroxyglutaric acids in body fluids and tissues, particularly in the brain. Affected patients show progressive cortical leukoencephalopathy and chronic degeneration of the basal ganglia whose pathogenesis is still unclear. In the present work we investigated parameters of bioenergetics and redox homeostasis in various cerebral structures (cerebral cortex, striatum and hippocampus) and heart of adult wild type (Gcdh(+/+)) and glutaryl-CoA dehydrogenase deficient knockout (Gcdh(-/-)) mice fed a baseline chow. Oxidative stress parameters were also measured after acute lysine overload. Finally, mRNA expression of NMDA subunits and GLT1 transporter was determined in cerebral cortex and striatum of these animals fed a baseline or high lysine (4.7%) chow. No significant alterations of bioenergetics or redox status were observed in these mice. In contrast, mRNA expression of the NR2B glutamate receptor subunit and of the GLT1 glutamate transporter was higher in cerebral cortex of Gcdh(-/-) mice. Furthermore, NR2B expression was markedly elevated in striatum of Gcdh(-/-) animals receiving chronic Lys overload. These data indicate higher susceptibility of Gcdh(-/-) mice to excitotoxic damage, implying that this pathomechanism may contribute to the cortical and striatum alterations observed in GA I patients. PMID:26671102

  13. Increased furfural tolerance due to overexpression of NADH-dependent oxidoreductase FucO in Escherichia coli strains engineered for the production of ethanol and lactate.

    PubMed

    Wang, X; Miller, E N; Yomano, L P; Zhang, X; Shanmugam, K T; Ingram, L O

    2011-08-01

    Furfural is an important fermentation inhibitor in hemicellulose sugar syrups derived from woody biomass. The metabolism of furfural by NADPH-dependent oxidoreductases, such as YqhD (low K(m) for NADPH), is proposed to inhibit the growth and fermentation of xylose in Escherichia coli by competing with biosynthesis for NADPH. The discovery that the NADH-dependent propanediol oxidoreductase (FucO) can reduce furfural provided a new approach to improve furfural tolerance. Strains that produced ethanol or lactate efficiently as primary products from xylose were developed. These strains included chromosomal mutations in yqhD expression that permitted the fermentation of xylose broths containing up to 10 mM furfural. Expression of fucO from plasmids was shown to increase furfural tolerance by 50% and to permit the fermentation of 15 mM furfural. Product yields with 15 mM furfural were equivalent to those of control strains without added furfural (85% to 90% of the theoretical maximum). These two defined genetic traits can be readily transferred to enteric biocatalysts designed to produce other products. A similar strategy that minimizes the depletion of NADPH pools by native detoxification enzymes may be generally useful for other inhibitory compounds in lignocellulosic sugar streams and with other organisms. PMID:21685167

  14. Nitrogen and oxygen regulation of Bacillus subtilis nasDEF encoding NADH-dependent nitrite reductase by TnrA and ResDE.

    PubMed

    Nakano, M M; Hoffmann, T; Zhu, Y; Jahn, D

    1998-10-01

    The nitrate and nitrite reductases of Bacillus subtilis have two different physiological functions. Under conditions of nitrogen limitation, these enzymes catalyze the reduction of nitrate via nitrite to ammonia for the anabolic incorporation of nitrogen into biomolecules. They also function catabolically in anaerobic respiration, which involves the use of nitrate and nitrite as terminal electron acceptors. Two distinct nitrate reductases, encoded by narGHI and nasBC, function in anabolic and catabolic nitrogen metabolism, respectively. However, as reported herein, a single NADH-dependent, soluble nitrite reductase encoded by the nasDE genes is required for both catabolic and anabolic processes. The nasDE genes, together with nasBC (encoding assimilatory nitrate reductase) and nasF (required for nitrite reductase siroheme cofactor formation), constitute the nas operon. Data presented show that transcription of nasDEF is driven not only by the previously characterized nas operon promoter but also from an internal promoter residing between the nasC and nasD genes. Transcription from both promoters is activated by nitrogen limitation during aerobic growth by the nitrogen regulator, TnrA. However, under conditions of oxygen limitation, nasDEF expression and nitrite reductase activity were significantly induced. Anaerobic induction of nasDEF required the ResDE two-component regulatory system and the presence of nitrite, indicating partial coregulation of NasDEF with the respiratory nitrate reductase NarGHI during nitrate respiration. PMID:9765565

  15. Nitrogen and Oxygen Regulation of Bacillus subtilis nasDEF Encoding NADH-Dependent Nitrite Reductase by TnrA and ResDE

    PubMed Central

    Nakano, Michiko M.; Hoffmann, Tamara; Zhu, Yi; Jahn, Dieter

    1998-01-01

    The nitrate and nitrite reductases of Bacillus subtilis have two different physiological functions. Under conditions of nitrogen limitation, these enzymes catalyze the reduction of nitrate via nitrite to ammonia for the anabolic incorporation of nitrogen into biomolecules. They also function catabolically in anaerobic respiration, which involves the use of nitrate and nitrite as terminal electron acceptors. Two distinct nitrate reductases, encoded by narGHI and nasBC, function in anabolic and catabolic nitrogen metabolism, respectively. However, as reported herein, a single NADH-dependent, soluble nitrite reductase encoded by the nasDE genes is required for both catabolic and anabolic processes. The nasDE genes, together with nasBC (encoding assimilatory nitrate reductase) and nasF (required for nitrite reductase siroheme cofactor formation), constitute the nas operon. Data presented show that transcription of nasDEF is driven not only by the previously characterized nas operon promoter but also from an internal promoter residing between the nasC and nasD genes. Transcription from both promoters is activated by nitrogen limitation during aerobic growth by the nitrogen regulator, TnrA. However, under conditions of oxygen limitation, nasDEF expression and nitrite reductase activity were significantly induced. Anaerobic induction of nasDEF required the ResDE two-component regulatory system and the presence of nitrite, indicating partial coregulation of NasDEF with the respiratory nitrate reductase NarGHI during nitrate respiration. PMID:9765565

  16. The real-time resolution of proton-related transient-state steps in an enzymatic reaction. The early steps in the oxidative deamination reaction of bovine liver glutamate dehydrogenase.

    PubMed

    Singh, N; Maniscalco, S J; Fisher, H F

    1993-01-01

    We introduce a novel transient-state kinetic approach which can resolve proton and product time courses into a series of individual steps that comprise the reaction path. We have applied this approach to the oxidative deamination reaction catalyzed by bovine liver glutamate dehydrogenase, measuring both the product (NADPH) and proton time courses at various pH values. The global treatment (over all pH values) resolves the very early portion of this reaction quantitatively and provides a continuous time course for each of the six protonic species. We propose the following mechanism: L-glutamate binds to an open conformation of the enzyme-NADP complex, forming salt bridges between its alpha- and gamma-carboxyl groups and the protonated forms of enzyme lysine residues 114 and 90, respectively. In this position, the alpha-H atom of the substrate is too far from the nicotinamide ring for hydride transfer to occur. In the next step, three events occur in a concerted manner: lysine 126 loses a proton and acquires a single water molecule; the active site cleft closes; bulk water is expelled; the substrate and coenzyme are forced closer together and remain in a nonaqueous environment during the ensuing chemical events, returning to an open conformation only in time to allow the product release steps to occur. Thus, substrate binding accomplishes a number of important tasks which are themselves an integral part of the catalytic mechanism. Combining the novel transient state approach developed here with steady-state kinetic information can produce a detailed mechanistic resolution of otherwise hidden steps. PMID:8093240

  17. The NAD(P)H-utilizing glutamate dehydrogenase of Bacteroides thetaiotaomicron belongs to enzyme family I, and its activity is affected by trans-acting gene(s) positioned downstream of gdhA.

    PubMed Central

    Baggio, L; Morrison, M

    1996-01-01

    Previous studies have suggested that regulation of the enzymes of ammonia assimilation in human colonic Bacteroides species is coordinated differently than in other eubacteria. The gene encoding an NAD(P)H-dependent glutamate dehydrogenase (gdhA) in Bacteroides thetaiotaomicron was cloned and expressed in Escherichia coli by mutant complementation from the recombinant plasmid pANS100. Examination of the predicted GdhA amino acid sequence revealed that this enzyme possesses motifs typical of the family I-type hexameric GDH proteins. Northern blot analysis with a gdhA-specific probe indicated that a single transcript with an electrophoretic mobility of approximately 1.6 kb was produced in both B. thetaiotaomicron and E. coli gdhA+ transformants. Although gdhA transcription was unaffected, no GdhA enzyme activity could be detected in E. coli transformants when smaller DNA fragments from pANS100, which contained the entire gdhA gene, were analyzed. Enzyme activity was restored if these E. coli strains were cotransformed with a second plasmid, which contained a 3-kb segment of DNA located downstream of the gdhA coding region. Frameshift mutagenesis within the DNA downstream of gdhA in pANS100 also resulted in the loss of GdhA enzyme activity. Collectively, these results are interpreted as evidence for the role of an additional gene product(s) in modulating the activity of GDH enzyme activity. Insertional mutagenesis experiments which led to disruption of the gdhA gene on the B. thetaiotaomicron chromosome indicated that gdhA mutants were not glutamate auxotrophs, but attempts to isolate similar mutants with insertion mutations in the region downstream of the gdhA gene were unsuccessful. PMID:8955404

  18. 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

  19. Biochemical and Structural Studies of NADH-Dependent FabG Used To Increase the Bacterial Production of Fatty Acids under Anaerobic Conditions

    PubMed Central

    Javidpour, Pouya; Pereira, Jose H.; Goh, Ee-Been; McAndrew, Ryan P.; Ma, Suzanne M.; Friedland, Gregory D.; Keasling, Jay D.; Chhabra, Swapnil R.; Adams, Paul D.

    2014-01-01

    Major efforts in bioenergy research have focused on producing fuels that can directly replace petroleum-derived gasoline and diesel fuel through metabolic engineering of microbial fatty acid biosynthetic pathways. Typically, growth and pathway induction are conducted under aerobic conditions, but for operational efficiency in an industrial context, anaerobic culture conditions would be preferred to obviate the need to maintain specific dissolved oxygen concentrations and to maximize the proportion of reducing equivalents directed to biofuel biosynthesis rather than ATP production. A major concern with fermentative growth conditions is elevated NADH levels, which can adversely affect cell physiology. The purpose of this study was to identify homologs of Escherichia coli FabG, an essential reductase involved in fatty acid biosynthesis, that display a higher preference for NADH than for NADPH as a cofactor. Four potential NADH-dependent FabG variants were identified through bioinformatic analyses supported by crystallographic structure determination (1.3- to 2.0-Å resolution). In vitro assays of cofactor (NADH/NADPH) preference in the four variants showed up to ∼35-fold preference for NADH, which was observed with the Cupriavidus taiwanensis FabG variant. In addition, FabG homologs were overexpressed in fatty acid- and methyl ketone-overproducing E. coli host strains under anaerobic conditions, and the C. taiwanensis variant led to a 60% higher free fatty acid titer and 75% higher methyl ketone titer relative to the titers of the control strains. With further engineering, this work could serve as a starting point for establishing a microbial host strain for production of fatty acid-derived biofuels (e.g., methyl ketones) under anaerobic conditions. PMID:24212572

  20. Biochemical and structural studies of NADH-dependent FabG used to increase the bacterial production of fatty acids under anaerobic conditions.

    PubMed

    Javidpour, Pouya; Pereira, Jose H; Goh, Ee-Been; McAndrew, Ryan P; Ma, Suzanne M; Friedland, Gregory D; Keasling, Jay D; Chhabra, Swapnil R; Adams, Paul D; Beller, Harry R

    2014-01-01

    Major efforts in bioenergy research have focused on producing fuels that can directly replace petroleum-derived gasoline and diesel fuel through metabolic engineering of microbial fatty acid biosynthetic pathways. Typically, growth and pathway induction are conducted under aerobic conditions, but for operational efficiency in an industrial context, anaerobic culture conditions would be preferred to obviate the need to maintain specific dissolved oxygen concentrations and to maximize the proportion of reducing equivalents directed to biofuel biosynthesis rather than ATP production. A major concern with fermentative growth conditions is elevated NADH levels, which can adversely affect cell physiology. The purpose of this study was to identify homologs of Escherichia coli FabG, an essential reductase involved in fatty acid biosynthesis, that display a higher preference for NADH than for NADPH as a cofactor. Four potential NADH-dependent FabG variants were identified through bioinformatic analyses supported by crystallographic structure determination (1.3- to 2.0-Å resolution). In vitro assays of cofactor (NADH/NADPH) preference in the four variants showed up to ≈ 35-fold preference for NADH, which was observed with the Cupriavidus taiwanensis FabG variant. In addition, FabG homologs were overexpressed in fatty acid- and methyl ketone-overproducing E. coli host strains under anaerobic conditions, and the C. taiwanensis variant led to a 60% higher free fatty acid titer and 75% higher methyl ketone titer relative to the titers of the control strains. With further engineering, this work could serve as a starting point for establishing a microbial host strain for production of fatty acid-derived biofuels (e.g., methyl ketones) under anaerobic conditions. PMID:24212572

  1. 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.

  2. Crystallization and preliminary X-ray analysis of the electron-transfer complex of Rieske-type [2Fe–2S] ferredoxin and NADH-dependent ferredoxin reductase derived from Acidovorax sp. strain KKS102

    SciTech Connect

    Senda, Miki; Kishigami, Shinya; Kimura, Shigenobu; Senda, Toshiya

    2007-06-01

    The electron-transfer complex of BphA3, a Rieske-type [2Fe–2S] ferredoxin, and BphA4, a NADH-dependent ferredoxin reductase, was crystallized by the sitting-drop vapour-diffusion method under anaerobic conditions. The electron-transfer complex of BphA3, a Rieske-type [2Fe–2S] ferredoxin, and BphA4, a NADH-dependent ferredoxin reductase, was crystallized using the sitting-drop vapour-diffusion method under anaerobic conditions. The obtained crystals were analyzed by SDS–PAGE, which showed that they contained both BphA3 and BphA4. The crystals belong to space group P2{sub 1}, with unit-cell parameters a = 60.60, b = 173.72, c = 60.98 Å, β = 115.8°, and diffracted to a resolution of 1.9 Å.

  3. 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 Central

    McGowan, Karin L.

    2012-01-01

    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. PMID:22259201

  4. 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

  5. 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. PMID:22259201

  6. 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

  7. 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

  8. 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

  9. Use of the anti-Prelog stereospecific alcohol dehydrogenase from Leifsonia and Pseudomonas for producing chiral alcohols.

    PubMed

    Itoh, Nobuya

    2014-05-01

    The asymmetric reduction of ketones is one of the most promising processes for producing chiral alcohols. However, dehydrogenases or reductases that can catalyze the reduction of ketones to give anti-Prelog chiral alcohols have been limited to some NADP(+)/NADPH-dependent enzymes. Recently, we reported a novel NAD(+)/NADH-dependent alcohol dehydrogenase (ADH) from Leifsonia sp. and Pseudomonas ADH homologs from soil metagenomes. Moreover, we have established an efficient hydrogen-transfer bioreduction process with 2-propanol as a hydrogen donor using Leifsonia ADH. This review focuses on the recent development of novel ADHs for producing industrially useful anti-Prelog chiral alcohols from various ketones. PMID:24615386

  10. Substitutions at the cofactor phosphate-binding site of a clostridial alcohol dehydrogenase lead to unexpected changes in substrate specificity

    PubMed Central

    Maddock, Danielle J.; Patrick, Wayne M.; Gerth, Monica L.

    2015-01-01

    Changing the cofactor specificity of an enzyme from nicotinamide adenine dinucleotide 2′-phosphate (NADPH) to the more abundant NADH is a common strategy for increasing overall enzyme efficiency in microbial metabolic engineering. The aim of this study was to switch the cofactor specificity of the primary–secondary alcohol dehydrogenase from Clostridium autoethanogenum, a bacterium with considerable promise for the bio-manufacturing of fuels and other petrochemicals, from strictly NADPH-dependent to NADH-dependent. We used insights from a homology model to build a site-saturation library focussed on residue S199, the position deemed most likely to disrupt binding of the 2′-phosphate of NADPH. Although the CaADH(S199X) library did not yield any NADH-dependent enzymes, it did reveal that substitutions at the cofactor phosphate-binding site can cause unanticipated changes in the substrate specificity of the enzyme. Using consensus-guided site-directed mutagenesis, we were able to create an enzyme that was stringently NADH-dependent, albeit with a concomitant reduction in activity. This study highlights the role that distal residues play in substrate specificity and the complexity of enzyme–cofactor interactions. PMID:26034298

  11. Substitutions at the cofactor phosphate-binding site of a clostridial alcohol dehydrogenase lead to unexpected changes in substrate specificity.

    PubMed

    Maddock, Danielle J; Patrick, Wayne M; Gerth, Monica L

    2015-08-01

    Changing the cofactor specificity of an enzyme from nicotinamide adenine dinucleotide 2'-phosphate (NADPH) to the more abundant NADH is a common strategy for increasing overall enzyme efficiency in microbial metabolic engineering. The aim of this study was to switch the cofactor specificity of the primary-secondary alcohol dehydrogenase from Clostridium autoethanogenum, a bacterium with considerable promise for the bio-manufacturing of fuels and other petrochemicals, from strictly NADPH-dependent to NADH-dependent. We used insights from a homology model to build a site-saturation library focussed on residue S199, the position deemed most likely to disrupt binding of the 2'-phosphate of NADPH. Although the CaADH(S199X) library did not yield any NADH-dependent enzymes, it did reveal that substitutions at the cofactor phosphate-binding site can cause unanticipated changes in the substrate specificity of the enzyme. Using consensus-guided site-directed mutagenesis, we were able to create an enzyme that was stringently NADH-dependent, albeit with a concomitant reduction in activity. This study highlights the role that distal residues play in substrate specificity and the complexity of enzyme-cofactor interactions. PMID:26034298

  12. Exciting Times for Pancreatic Islets: Glutamate Signaling in Endocrine Cells.

    PubMed

    Otter, Silke; Lammert, Eckhard

    2016-03-01

    Glutamate represents a key excitatory neurotransmitter in the central nervous system, and also modulates the function and viability of endocrine cells in pancreatic islets. In insulin-secreting beta cells, glutamate acts as an intracellular messenger, and its transport into secretory granules promotes glucose- and incretin-stimulated insulin secretion. Mitochondrial degradation of glutamate also contributes to insulin release when glutamate dehydrogenase is allosterically activated. It also signals extracellularly via glutamate receptors (AMPA and NMDA receptors) to modulate glucagon, insulin and somatostatin secretion, and islet cell survival. Its degradation products, GABA and γ-hydroxybutyrate, are released and also influence islet cell behavior. Thus, islet glutamate receptors, such as the NMDA receptors, might serve as possible drug targets to develop new medications for adjunct treatment of diabetes. PMID:26740469

  13. d-Xylose Metabolism in Hypocrea jecorina: Loss of the Xylitol Dehydrogenase Step Can Be Partially Compensated for by lad1-Encoded l-Arabinitol-4-Dehydrogenase

    PubMed Central

    Seiboth, Bernhard; Hartl, Lukas; Pail, Manuela; Kubicek, Christian P.

    2003-01-01

    With the goal of the genetic characterization of the d-xylose pathway in Hypocrea jecorina (anamorph: Trichoderma reesei), we cloned the xdh1 gene, encoding NAD-xylitol dehydrogenase, which catalyzes the second step of fungal d-xylose catabolism. This gene encodes a 363-amino-acid protein which has a mass of 38 kDa, belongs to the zinc-containing alcohol dehydrogenase family, exhibits high sequence identity to the published sequences of xylitol dehydrogenases from yeast origins, but contains a second, additional binding site for Zn2+. The enzyme catalyzed the NAD-dependent oxidation of xylitol and d-sorbitol and the NADH-dependent reduction of d-xylulose and d-fructose. No activity was observed with NADP, l-arabinose, or l-arabinitol. A single 1.4-kb transcript was formed during growth on xylan, d-xylose, l-arabinose, l-arabinitol and, at a lower abundance, xylitol, d-galactose, galactitol, and lactose but not on d-glucose and glycerol. xdh1 deletion mutants exhibited 50% reduced growth rates on d-xylose, whereas growth rates on xylitol remained unaltered. These mutants contained 30% of the xylitol dehydrogenase activity of the parent strain, indicating the presence of a second xylitol dehydrogenase. This activity was shown to be due to lad1-encoded l-arabinitol-4-dehydrogenase, because H. jecorina xdh1 lad1 double-deletion strains failed to grow on d-xylose or xylitol. In contrast, lad1 deletion strains of H. jecorina grew normally on these carbon sources. These results show that H. jecorina contains a single xylitol dehydrogenase which is encoded by xdh1 and is involved in the metabolism of d-xylose and that lad1-encoded l-arabinitol-4-dehydrogenase can compensate for it partially in mutants with a loss of xdh1 function. PMID:14555469

  14. 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

  15. Monosodium glutamate-induced oxidative kidney damage and possible mechanisms: a mini-review.

    PubMed

    Sharma, Amod

    2015-01-01

    Animal studies suggest that chronic monosodium glutamate (MSG) intake induces kidney damage by oxidative stress. However, the underlying mechanisms are still unclear, despite the growing evidence and consensus that α-ketoglutarate dehydrogenase, glutamate receptors and cystine-glutamate antiporter play an important role in up-regulation of oxidative stress in MSG-induced renal toxicity. This review summaries evidence from studies into MSG-induced renal oxidative damage, possible mechanisms and their importance from a toxicological viewpoint. PMID:26493866

  16. Central Role of Glutamate Metabolism in the Maintenance of Nitrogen Homeostasis in Normal and Hyperammonemic Brain.

    PubMed

    Cooper, Arthur J L; Jeitner, Thomas M

    2016-01-01

    Glutamate is present in the brain at an average concentration-typically 10-12 mM-far in excess of those of other amino acids. In glutamate-containing vesicles in the brain, the concentration of glutamate may even exceed 100 mM. Yet because glutamate is a major excitatory neurotransmitter, the concentration of this amino acid in the cerebral extracellular fluid must be kept low-typically µM. The remarkable gradient of glutamate in the different cerebral compartments: vesicles > cytosol/mitochondria > extracellular fluid attests to the extraordinary effectiveness of glutamate transporters and the strict control of enzymes of glutamate catabolism and synthesis in well-defined cellular and subcellular compartments in the brain. A major route for glutamate and ammonia removal is via the glutamine synthetase (glutamate ammonia ligase) reaction. Glutamate is also removed by conversion to the inhibitory neurotransmitter γ-aminobutyrate (GABA) via the action of glutamate decarboxylase. On the other hand, cerebral glutamate levels are maintained by the action of glutaminase and by various α-ketoglutarate-linked aminotransferases (especially aspartate aminotransferase and the mitochondrial and cytosolic forms of the branched-chain aminotransferases). Although the glutamate dehydrogenase reaction is freely reversible, owing to rapid removal of ammonia as glutamine amide, the direction of the glutamate dehydrogenase reaction in the brain in vivo is mainly toward glutamate catabolism rather than toward the net synthesis of glutamate, even under hyperammonemia conditions. During hyperammonemia, there is a large increase in cerebral glutamine content, but only small changes in the levels of glutamate and α-ketoglutarate. Thus, the channeling of glutamate toward glutamine during hyperammonemia results in the net synthesis of 5-carbon units. This increase in 5-carbon units is accomplished in part by the ammonia-induced stimulation of the anaplerotic enzyme pyruvate carboxylase

  17. Central Role of Glutamate Metabolism in the Maintenance of Nitrogen Homeostasis in Normal and Hyperammonemic Brain

    PubMed Central

    Cooper, Arthur J. L.; Jeitner, Thomas M.

    2016-01-01

    Glutamate is present in the brain at an average concentration—typically 10–12 mM—far in excess of those of other amino acids. In glutamate-containing vesicles in the brain, the concentration of glutamate may even exceed 100 mM. Yet because glutamate is a major excitatory neurotransmitter, the concentration of this amino acid in the cerebral extracellular fluid must be kept low—typically µM. The remarkable gradient of glutamate in the different cerebral compartments: vesicles > cytosol/mitochondria > extracellular fluid attests to the extraordinary effectiveness of glutamate transporters and the strict control of enzymes of glutamate catabolism and synthesis in well-defined cellular and subcellular compartments in the brain. A major route for glutamate and ammonia removal is via the glutamine synthetase (glutamate ammonia ligase) reaction. Glutamate is also removed by conversion to the inhibitory neurotransmitter γ-aminobutyrate (GABA) via the action of glutamate decarboxylase. On the other hand, cerebral glutamate levels are maintained by the action of glutaminase and by various α-ketoglutarate-linked aminotransferases (especially aspartate aminotransferase and the mitochondrial and cytosolic forms of the branched-chain aminotransferases). Although the glutamate dehydrogenase reaction is freely reversible, owing to rapid removal of ammonia as glutamine amide, the direction of the glutamate dehydrogenase reaction in the brain in vivo is mainly toward glutamate catabolism rather than toward the net synthesis of glutamate, even under hyperammonemia conditions. During hyperammonemia, there is a large increase in cerebral glutamine content, but only small changes in the levels of glutamate and α-ketoglutarate. Thus, the channeling of glutamate toward glutamine during hyperammonemia results in the net synthesis of 5-carbon units. This increase in 5-carbon units is accomplished in part by the ammonia-induced stimulation of the anaplerotic enzyme pyruvate

  18. Mutant alcohol dehydrogenase leads to improved ethanol tolerance in Clostridium thermocellum

    SciTech Connect

    Brown, Steven D; Guss, Adam M; Karpinets, Tatiana V; Parks, Jerry M; Smolin, Nikolai; Yang, Shihui; Land, Miriam L; Klingeman, Dawn Marie; Bhandiwad, Ashwini; Rodriguez, Jr., Miguel; Raman, Babu; Shao, Xiongjun; Mielenz, Jonathan R; Smith, Jeremy C; Keller, Martin; Lynd, Lee R

    2011-01-01

    Clostridium thermocellum is a thermophilic, obligately anaerobic, Gram-positive bacterium that is a candidate microorganism for converting cellulosic biomass into ethanol through consolidated bioprocessing. Ethanol intolerance is an important metric in terms of process economics, and tolerance has often been described as a complex and likely multigenic trait for which complex gene interactions come into play. Here, we resequence the genome of an ethanol-tolerant mutant, show that the tolerant phenotype is primarily due to a mutated bifunctional acetaldehyde-CoA/alcohol dehydrogenase gene (adhE), hypothesize based on structural analysis that cofactor specificity may be affected, and confirm this hypothesis using enzyme assays. Biochemical assays confirm a complete loss of NADH-dependent activity with concomitant acquisition of NADPH-dependent activity, which likely affects electron flow in the mutant. The simplicity of the genetic basis for the ethanol-tolerant phenotype observed here informs rational engineering of mutant microbial strains for cellulosic ethanol production.

  19. The NAD(P)H-dependent glutamate dehydrogenase activities of Prevotella ruminicola B(1)4 can be attributed to one enzyme (GdhA), and gdhA expression is regulated in response to the nitrogen source available for growth.

    PubMed Central

    Wen, Z; Morrison, M

    1996-01-01

    Prevotella ruminicola B(1)4 possesses both NADPH- and NADH-linked glutamate dehydrogenase (GDH) activities, with the greatest specific activity being measured from ammonia-limited cultures. Relative to cells grown in the presence of 1 mM ammonium chloride, the NADPH-dependent activity was decreased approximately 10-fold when peptides were provided as a nitrogen source. Nondenaturing polyacrylamide gel electrophoresis (PAGE) was used to visualize the GDH protein(s) in cell extracts of P. ruminicola. For all growth conditions tested, only one GDH protein was detectable, and its relative abundance, as well as its reactivity with either NAD(P)+ or NAD(P)H, correlated well with the specific activities measured from whole-cell assays. Consistent with the findings from enzyme assays and PAGE activity gels, Northern (RNA) blot analysis revealed that expression of a gene encoding NAD(P)H-GDH activity was greatest in ammonia-grown cultures and that GDH activity is regulated in response to nitrogen source (ammonia versus peptides), probably at the level of transcription. A gene encoding the NAD(P)H-utilizing GDH activity (gdhA) was cloned, and its nucleotide sequence was determined and shown to contain an open reading frame of 1,332 bp which would encode a polypeptide of 48.8 kDa. The deduced amino acid sequence possesses three highly conserved motifs typical of family I GDHs, but several unique amino acid substitutions within these motifs were evident. These results are discussed within the context of ruminal nitrogen metabolism and the growth efficiency of succinate- and propionate-producing anaerobic bacteria. PMID:8837439

  20. Purification and partial kinetic and physical characterization of two NADP-specific glutamate dehydrogenase isoenzymes and their protein precursors, and measurement of the patterns of accumulation and rates of degradation of their nonidentical subunits in synchronized cells of Chlorella cultured in different concentrations of ammonia

    SciTech Connect

    Bascomb, N.F.

    1986-01-01

    Two ammonium-inducible, chloroplast-localized, NADP-specific glutamate dehydrogenases were purified from Chlorella sorokiniana. They were homopolymers of either alpha or beta subunits with molecular weights of 55,500 and 53,000, respectively. These isoenzymes were separated by their differential binding to the substrate affinity column. Peptide mapping of purified alpha and beta subunits showed them to have a high degree of sequence homology. By use of SDS slab-gel electrophoresis and a Western blot/immunodetection procedure, patterns of accumulation of alpha and beta subunits (in their holoenzyme) were measured in cells cultured in media, containing different concentrations of ammonia. Pulse-chase experiments with (/sup 35/S)sulfate were performed to measured the rates of degradation of the two isoenzymes. When the culture medium contained 2 mM ammonia or lower, cells accumulated only the alpha holoenzyme. Above 2 mM ammonia, cells contained both enzymes; however, their patterns of accumulation and rates of degradation were very different. The physiological role of alpha and beta holoenzymes appears to be ammonia assimilation at low and high external ammonia concentrations, respectively. From in vitro-translation studies with total cellular poly(A)/sup +/RNA, isolated from cells engaged in synthesis of alpha or beta holoenzymes or both, it was concluded that alpha and beta subunits have protein precursor(s) or identical molecular weight (M/sub r/ = 58,500). When the putative protein-precursor(s) were incubated in vitro, with cell-free extracts from Chlorella cells, they were processed to proteins the size of alpha and beta subunits.

  1. Enzyme-Doped Thin Films and Optical Fiber Sensors for Glutamate

    NASA Astrophysics Data System (ADS)

    Rickus, Jenna L.; Tobin, Allan J.; Zink, Jeffrey I.; Dunn, Bruce S.

    2002-10-01

    Biomolecules encapsulated in porous silicate glass using the sol-gel process form optically transparent materials capable of biorecognition. We are working to design biosensors from these materials for the detection of glutamate, the major excitatory neurotransmitter in the central nervous system. Previously we demonstrated the ability of glutamate dehydrogenase (GDH)-doped sol-gel bulk materials to measure glutamate at varying concentrations. Here we show that GDH can be encapsulated in a thin film while retaining its enzymatic activity. The films are likely to be reaction limited rather than diffusion limited, as the reaction rate at saturating glutamate concentrations varies linearly with enzyme loading. At a given enzyme loading, the film reaction rate increases with increasing glutamate concentration, demonstrating its potential as a glutamate sensor material. In addition we have shown that the enzyme-doped sol-gel glass can be deposited onto the tip of an optical fiber. The fiber is active and responds to the presence of glutamate.

  2. Metabotropic glutamate receptors inhibit microglial glutamate release

    PubMed Central

    McMullan, Stephen M; Phanavanh, Bounleut; Guo Li, Gary; Barger, Steven W

    2012-01-01

    Pro-inflammatory stimuli evoke an export of glutamate from microglia that is sufficient to contribute to excitotoxicity in neighbouring neurons. Since microglia also express various glutamate receptors themselves, we were interested in the potential feedback of glutamate on this system. Several agonists of mGluRs (metabotropic glutamate receptors) were applied to primary rat microglia, and the export of glutamate into their culture medium was evoked by LPS (lipopolysaccharide). Agonists of group-II and -III mGluR ACPD [(1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid] and L-AP4 [L-(+)-2-amino-4-phosphonobutyric acid] were both capable of completely blocking the glutamate export without interfering with the production of NO (nitric oxide); the group-I agonist tADA (trans-azetidine-2,4-dicarboxylic acid) was ineffective. Consistent with the possibility of feedback, inhibition of mGluR by MSPG [(R,S)-α-2-methyl-4sulfonophenylglycine] potentiated glutamate export. As the group-II and -III mGluR are coupled to Gαi-containing G-proteins and the inhibition of adenylate cyclase, we explored the role of cAMP in this effect. Inhibition of cAMP-dependent protein kinase [also known as protein kinase A (PKA)] by H89 mimicked the effect of ACPD, and the mGluR agonist had its actions reversed by artificially sustaining cAMP through the PDE (phosphodiesterase) inhibitor IBMX (isobutylmethylxanthine) or the cAMP mimetic dbcAMP (dibutyryl cAMP). These data indicate that mGluR activation attenuates a potentially neurotoxic export of glutamate from activated microglia and implicate cAMP as a contributor to this aspect of microglial action. PMID:22770428

  3. Effect of biotin on transcription levels of key enzymes and glutamate efflux in glutamate fermentation by Corynebacterium glutamicum.

    PubMed

    Cao, Yan; Duan, Zuoying; Shi, Zhongping

    2014-02-01

    Biotin is an important factor affecting the performance of glutamate fermentation by biotin auxotrophic Corynebacterium glutamicum and glutamate is over-produced only when initial biotin content is controlled at suitable levels or initial biotin is excessive but with Tween 40 addition during fermentation. The transcription levels of key enzymes at pyruvate, isocitrate and α-ketoglutarate metabolic nodes, as well as transport protein (TP) of glutamate were investigated under the conditions of varied biotin contents and Tween 40 supplementation. When biotin was insufficient, the genes encoding key enzymes and TP were down-regulated in the early production phase, in particular, the transcription level of isocitrate dehydrogenase (ICDH) which was only 2% of that of control. Although the cells' morphology transformation and TP level were not affected, low transcription level of ICDH led to lower final glutamate concentration (64 g/L). When biotin was excessive, the transcription levels of key enzymes were at comparable levels as those of control with ICDH as an exception, which was only 3-22% of control level throughout production phase. In this case, little intracellular glutamate accumulation (1.5 mg/g DCW) and impermeable membrane resulted in non glutamate secretion into broth, even though the quantity of TP was more than 10-folds of control level. Addition of Tween 40 when biotin was excessive stimulated the expression of all key enzymes and TP, intracellular glutamate content was much higher (10-12 mg/g DCW), and final glutamate concentration reached control level (75-80 g/L). Hence, the membrane alteration and TP were indispensable in glutamate secretion. Biotin and Tween 40 influenced the expression level of ICDH and glutamate efflux, thereby influencing glutamate production. PMID:23990041

  4. Differential Glutamate Metabolism in Proliferating and Quiescent Mammary Epithelial Cells.

    PubMed

    Coloff, Jonathan L; Murphy, J Patrick; Braun, Craig R; Harris, Isaac S; Shelton, Laura M; Kami, Kenjiro; Gygi, Steven P; Selfors, Laura M; Brugge, Joan S

    2016-05-10

    Mammary epithelial cells transition between periods of proliferation and quiescence during development, menstrual cycles, and pregnancy, and as a result of oncogenic transformation. Utilizing an organotypic 3D tissue culture model coupled with quantitative metabolomics and proteomics, we identified significant differences in glutamate utilization between proliferating and quiescent cells. Relative to quiescent cells, proliferating cells catabolized more glutamate via transaminases to couple non-essential amino acid (NEAA) synthesis to α-ketoglutarate generation and tricarboxylic acid (TCA) cycle anaplerosis. As cells transitioned to quiescence, glutamine consumption and transaminase expression were reduced, while glutamate dehydrogenase (GLUD) was induced, leading to decreased NEAA synthesis. Highly proliferative human tumors display high transaminase and low GLUD expression, suggesting that proliferating cancer cells couple glutamine consumption to NEAA synthesis to promote biosynthesis. These findings describe a competitive and partially redundant relationship between transaminases and GLUD, and they reveal how coupling of glutamate-derived carbon and nitrogen metabolism can be regulated to support cell proliferation. PMID:27133130

  5. SLC1 Glutamate Transporters

    PubMed Central

    Grewer, Christof; Gameiro, Armanda; Rauen, Thomas

    2014-01-01

    The plasma membrane transporters for the neurotransmitter glutamate belong to the solute carrier 1 (SLC1) family. They are secondary active transporters, taking up glutamate into the cell against a substantial concentration gradient. The driving force for concentrative uptake is provided by the cotransport of Na+ ions and the countertransport of one K+ in a step independent of the glutamate translocation step. Due to eletrogenicity of transport, the transmembrane potential can also act as a driving force. Glutamate transporters are expressed in many tissues, but are of particular importance in the brain, where they contribute to the termination of excitatory neurotransmission. Glutamate transporters can also run in reverse, resulting in glutamate release from cells. Due to these important physiological functions, glutamate transporter expression and, therefore, the transport rate, are tightly regulated. This review summarizes recent literature on the functional and biophysical properties, structure-function relationships, regulation, physiological significance, and pharmacology of glutamate transporters. Particular emphasis is on the insight from rapid kinetic and electrophysiological studies, transcriptional regulation of transporter expression, and reverse transport and its importance for pathophysiological glutamate release under ischemic conditions. PMID:24240778

  6. On the mechanism underlying tellurite reduction by Aeromonas caviae ST dihydrolipoamide dehydrogenase.

    PubMed

    Arenas, F A; Leal, C A; Pinto, C A; Arenas-Salinas, M A; Morales, W A; Cornejo, F A; Díaz-Vásquez, W A; Vásquez, C C

    2014-07-01

    The dihydrolipoamide dehydrogenase (LpdA) from the tellurite-resistant bacterium Aeromonas caviae ST reduces tellurite to elemental tellurium. To characterize this NADH-dependent activity, the A. caviae lpdA gene was subjected to site-directed mutagenesis and genes containing C45A, H322Y and E354K substitutions were individually transformed into Escherichia coli Δlpd. Cells expressing the modified genes exhibited decreased pyruvate dehydrogenase, dihydrolipoamide dehydrogenase and TR activity regarding that observed with the wild type A. caviae lpdA gene. In addition, cells expressing the altered lpdA genes showed increased oxidative stress levels and tellurite sensitivity than those carrying the wild type counterpart. The involvement of Cys residues in LpdA's TR activity was analyzed using specific inhibitors that interact with catalytic cysteines and/or disulfide bridges such as aurothiomalate, zinc or nickel. TR activity of purified LpdA was drastically affected by these compounds. Since LpdA belongs to the flavoprotein family, the involvement of the FAD/NAD(P)(+)-binding domain in TR activity was determined. FAD removal from purified LpdA results in loss of TR activity, which was restored with exogenously added FAD. Substitutions in E354, involved in FAD/NADH binding, resulted in low TR activity because of flavin loss. Finally, changing H322 (involved in NAD(+)/NADH binding) by tyrosine also resulted in altered TR activity. PMID:24680738

  7. Glucose-6-phosphate dehydrogenase

    MedlinePlus

    ... this page: //medlineplus.gov/ency/article/003671.htm Glucose-6-phosphate dehydrogenase test To use the sharing features on this page, please enable JavaScript. Glucose-6-phosphate dehydrogenase (G6PD) is a type of ...

  8. Polysaccharides from wolfberry antagonizes glutamate excitotoxicity in rat cortical neurons.

    PubMed

    Ho, Yuen-Shan; Yu, Man-Shan; Yik, Suet-Yi; So, Kwok-Fai; Yuen, Wai-Hung; Chang, Raymond Chuen-Chung

    2009-12-01

    Glutamate excitotoxicity is involved in many neurodegenerative diseases including Alzheimer's disease (AD). Attenuation of glutamate toxicity is one of the therapeutic strategies for AD. Wolfberry (Lycium barbarum) is a common ingredient in oriental cuisines. A number of studies suggest that wolfberry has anti-aging properties. In recent years, there is a trend of using dried Wolfberry as food supplement and health product in UK and North America. Previously, we have demonstrated that a fraction of polysaccharide from Wolfberry (LBA) provided remarkable neuroprotective effects against beta-amyloid peptide-induced cytotoxicity in primary cultures of rat cortical neurons. To investigate whether LBA can protect neurons from other pathological factors such as glutamate found in Alzheimer brain, we examined whether it can prevent neurotoxicity elicited by glutamate in primary cultured neurons. The glutamate-induced cell death as detected by lactate dehydrogenase assay and caspase-3-like activity assay was significantly reduced by LBA at concentrations ranging from 10 to 500 microg/ml. Protective effects of LBA were comparable to memantine, a non-competitive NMDA receptor antagonist. LBA provided neuroprotection even 1 h after exposure to glutamate. In addition to glutamate, LBA attenuated N-methyl-D-aspartate (NMDA)-induced neuronal damage. To further explore whether LBA might function as antioxidant, we used hydrogen peroxide (H(2)O(2)) as oxidative stress inducer in this study. LBA could not attenuate the toxicity of H(2)O(2). Furthermore, LBA did not attenuate glutamate-induced oxidation by using NBT assay. Western blot analysis indicated that glutamate-induced phosphorylation of c-jun N-terminal kinase (JNK) was reduced by treatment with LBA. Taken together, LBA exerted significant neuroprotective effects on cultured cortical neurons exposed to glutamate. PMID:19499323

  9. Glutamate and Neurodegenerative Disease

    NASA Astrophysics Data System (ADS)

    Schaeffer, Eric; Duplantier, Allen

    As the main excitatory neurotransmitter in the mammalian central nervous system, glutamate is critically involved in most aspects of CNS function. Given this critical role, it is not surprising that glutamatergic dysfunction is associated with many CNS disorders. In this chapter, we review the literature that links aberrant glutamate neurotransmission with CNS pathology, with a focus on neurodegenerative diseases. The biology and pharmacology of the various glutamate receptor families are discussed, along with data which links these receptors with neurodegenerative conditions. In addition, we review progress that has been made in developing small molecule modulators of glutamate receptors and transporters, and describe how these compounds have helped us understand the complex pharmacology of glutamate in normal CNS function, as well as their potential for the treatment of neurodegenerative diseases.

  10. Deletion of the structural gene for the NADH-dehydrogenase subunit 4 of Synechocystis 6803 alters respiratory properties.

    PubMed Central

    Dzelzkalns, V A; Obinger, C; Regelsberger, G; Niederhauser, H; Kamensek, M; Peschek, G A; Bogorad, L

    1994-01-01

    Chloroplasts and cyanobacteria contain genes encoding polypeptides homologous to some subunits of the mitochondrial respiratory NADH-ubiquinol oxidoreductase complex (NADH dehydrogenase). Nothing is known of the role of the NADH dehydrogenase complex in photosynthesis, respiration, or other functions in chloroplasts, and little is known about the specific roles of the perhaps 42 subunits of this complex in the mitochondrion. Inactivation of a gene for subunit 4 (ndhD-2, ndh4) of this complex in the cyanobacterium Synechocystis 6803 has no effect on photosynthesis, judging from the rate of photoautotrophic growth of mutant cells, but the mutant's respiratory rate is about 6 times greater than that of wild-type cells. Respiratory electron transport activity in cyanobacteria is associated both with photosynthetic thylakoid membranes and with the outer cytoplasmic membrane of the cell. Cytoplasmic membranes of mutant cells have much greater NADH-dependent cytochrome reductase activity than preparations from wild-type cells; this activity remains at wild-type levels in isolated thylakoid membranes. It is suggested that the 56.6-kD product of ndhD-2 is not essential for the activity of a cytoplasmic membrane-bound NADH dehydrogenase but that it regulates the rate of electron flow through the complex, establishing a link between this ndh gene and respiration. The activity of the molecularly distinct thylakoid-bound NADH dehydrogenase is apparently unaffected by the loss of ndhD-2. PMID:7846157

  11. Changes in NAD(P)H fluorescence and membrane current produced by glutamate uptake into salamander Müller cells.

    PubMed Central

    Barbour, B; Magnus, C; Szatkowski, M; Gray, P T; Attwell, D

    1993-01-01

    1. Glutamate uptake into isolated, whole-cell patch-clamped glial cells was studied by monitoring the increase of cell fluorescence generated as glutamate and NAD(P) were converted into alpha-ketoglutarate and NAD(P)H by glutamate dehydrogenase. The current generated by the glutamate uptake carrier was recorded simultaneously. 2. L-Glutamate evoked an increase of cell fluorescence and an inward uptake current. L- and D-aspartate generated an uptake current but no fluorescence response, consistent with the amino acid specificity of glutamate dehydrogenase. 3. In the absence of external sodium the glutamate-evoked fluorescence response and uptake current were abolished, showing that there is no sodium-independent glutamate uptake across the cell membrane. 4. Varying the glutamate concentration altered both the fluorescence response and the uptake current. The fluorescence response saturated at a lower glutamate concentration than the uptake current, and depended in a Michaelis-Menten fashion on the uptake current. 5. The fluorescence response and the uptake current were reduced by membrane depolarization, and also by removal of intracellular potassium. 6. The dependence of the fluorescence response on uptake current when membrane potential was altered or intracellular potassium was removed was the same as that seen when the external glutamate concentration was altered. 7. These fluorescence studies show that glutamate uptake is inhibited by depolarization and by removal of intracellular potassium, consistent with the conclusion of earlier work in which uptake was monitored solely as a membrane current. The data are consistent with high-affinity electrogenic sodium- and potassium-dependent glutamate uptake with fixed stoichiometry being the only significant influx route for glutamate. Other possible interpretations of the data are also discussed. PMID:8105078

  12. In situ Regeneration of NADH via Lipoamide Dehydrogenase-catalyzed Electron Transfer Reaction Evidenced by Spectroelectrochemistry

    SciTech Connect

    Tam, Tsz Kin; Chen, Baowei; Lei, Chenghong; Liu, Jun

    2012-08-01

    NAD/NADH is a coenzyme found in all living cells, carrying electrons from one reaction to another. We report on characterizations of in situ regeneration of NADH via lipoamide dehydrogenase (LD)-catalyzed electron transfer reaction to regenerate NADH using UV-vis spectroelectrochemistry. The Michaelis-Menten constant (Km) and maximum velocity (Vmax) of NADH regeneration were measured as 0.80 {+-} 0.15 mM and 1.91 {+-} 0.09 {micro}M s-1 in a 1-mm thin-layer spectroelectrochemical cell using gold gauze as the working electrode at the applied potential -0.75 V (vs. Ag/AgCl). The electrocatalytic reduction of the NAD system was further coupled with the enzymatic conversion of pyruvate to lactate by lactate dehydrogenase to examine the coenzymatic activity of the regenerated NADH. Although the reproducible electrocatalytic reduction of NAD into NADH is known to be difficult compared to the electrocatalytic oxidation of NADH, our spectroelectrochemical results indicate that the in situ regeneration of NADH via LD-catalyzed electron transfer reaction is fast and sustainable and can be potentially applied to many NAD/NADH-dependent enzyme systems.

  13. 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

    2016-01-01

    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

  14. Neuronal pyruvate carboxylation supports formation of transmitter glutamate.

    PubMed

    Hassel, B; Brâthe, A

    2000-02-15

    Release of transmitter glutamate implies a drain of alpha-ketoglutarate from neurons, because glutamate, which is formed from alpha-ketoglutarate, is taken up by astrocytes. It is generally believed that this drain is compensated by uptake of glutamine from astrocytes, because neurons are considered incapable of de novo synthesis of tricarboxylic acid cycle intermediates, which requires pyruvate carboxylation. Here we show that cultured cerebellar granule neurons form releasable [(14)C]glutamate from H(14)CO(3)(-) and [1-(14)C]pyruvate via pyruvate carboxylation, probably mediated by malic enzyme. The activity of pyruvate carboxylation was calculated to be approximately one-third of the pyruvate dehydrogenase activity in neurons. Furthermore, intrastriatal injection of NaH(14)CO(3) or [1-(14)C]pyruvate labeled glutamate better than glutamine, showing that pyruvate carboxylation occurs in neurons in vivo. This means that neurons themselves to a large extent may support their release of glutamate, and thus entails a revision of the current view of glial-neuronal interactions and the importance of the glutamine cycle. PMID:10662824

  15. 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

  16. Effect of an Oxygen-Tolerant Bifurcating Butyryl Coenzyme A Dehydrogenase/Electron-Transferring Flavoprotein Complex from Clostridium difficile on Butyrate Production in Escherichia coli

    PubMed Central

    Aboulnaga, El-Hussiny; Pinkenburg, Olaf; Schiffels, Johannes; El-Refai, Ahmed; Buckel, Wolfgang

    2013-01-01

    The butyrogenic genes from Clostridium difficile DSM 1296T have been cloned and expressed in Escherichia coli. The enzymes acetyl-coenzyme A (CoA) C-acetyltransferase, 3-hydroxybutyryl-CoA dehydrogenase, crotonase, phosphate butyryltransferase, and butyrate kinase and the butyryl-CoA dehydrogenase complex composed of the dehydrogenase and two electron-transferring flavoprotein subunits were individually produced in E. coli and kinetically characterized in vitro. While most of these enzymes were measured using well-established test systems, novel methods to determine butyrate kinase and butyryl-CoA dehydrogenase activities with respect to physiological function were developed. Subsequently, the individual genes were combined to form a single plasmid-encoded operon in a plasmid vector, which was successfully used to confer butyrate-forming capability to the host. In vitro and in vivo studies demonstrated that C. difficile possesses a bifurcating butyryl-CoA dehydrogenase which catalyzes the NADH-dependent reduction of ferredoxin coupled to the reduction of crotonyl-CoA also by NADH. Since the reoxidation of ferredoxin by a membrane-bound ferredoxin:NAD+-oxidoreductase enables electron transport phosphorylation, additional ATP is formed. The butyryl-CoA dehydrogenase from C. difficile is oxygen stable and apparently uses oxygen as a co-oxidant of NADH in the presence of air. These properties suggest that this enzyme complex might be well suited to provide butyryl-CoA for solventogenesis in recombinant strains. The central role of bifurcating butyryl-CoA dehydrogenases and membrane-bound ferredoxin:NAD oxidoreductases (Rhodobacter nitrogen fixation [RNF]), which affect the energy yield of butyrate fermentation in the clostridial metabolism, is discussed. PMID:23772070

  17. Plant Formate Dehydrogenase

    SciTech Connect

    John Markwell

    2005-01-10

    The research in this study identified formate dehydrogenase, an enzyme that plays a metabolic role on the periphery of one-carbon metabolism, has an unusual localization in Arabidopsis thaliana and that the enzyme has an unusual kinetic plasticity. These properties make it possible that this enzyme could be engineered to attempt to engineer plants with an improved photosynthetic efficiency. We have produced transgenic Arabidopsis and tobacco plants with increased expression of the formate dehydrogenase enzyme to initiate further studies.

  18. Requirement of de novo synthesis of the OdhI protein in penicillin-induced glutamate production by Corynebacterium glutamicum.

    PubMed

    Kim, Jongpill; Fukuda, Hirohisa; Hirasawa, Takashi; Nagahisa, Keisuke; Nagai, Kazuo; Wachi, Masaaki; Shimizu, Hiroshi

    2010-04-01

    We found that penicillin-induced glutamate production by Corynebacterium glutamicum is inhibited when a de novo protein synthesis inhibitor, chloramphenicol, is added simultaneously with penicillin. When chloramphenicol was added 4 h after penicillin addition, glutamate production was essentially unaffected. (3)H-Leucine incorporation experiments revealed that protein synthesis continued for 1 h after penicillin addition and then gradually decreased. These results suggest that de novo protein synthesis within 4 h of penicillin treatment is required for the induction of glutamate production. To identify the protein(s) necessary for penicillin-induced glutamate production, proteome analysis of penicillin-treated C. glutamicum cells was performed with two-dimensional gel electrophoresis. Of more than 500 proteins detected, the amount of 13 proteins, including OdhI (an inhibitory protein for 2-oxoglutarate dehydrogenase complex), significantly increased upon penicillin treatment. Artificial overexpression of the odhI gene resulted in the decreased specific activity of the 2-oxoglutarate dehydrogenase complex and increased glutamate production without any triggers. These results suggest that the de novo synthesis of OdhI is the necessary factor for penicillin-induced glutamate overproduction by C. glutamicum. Moreover, continuous glutamate production was achieved by overexpression of odhI without any triggers. Thus, the odhI-overexpressing strain of C. glutamicum can be useful for efficient glutamate production. PMID:19956942

  19. A novel point mutation in a 3{prime} splice site of the NADH-cytochrome b{sub 5} reductase gene results in immunologically undetectable enzyme and impaired NADH-dependent ascorbate regeneration in cultured fibroblasts of a patient with type II hereditary methemoglobinemia

    SciTech Connect

    Shirabe, Komie; Takeshita, Masazumi; Landi, M.T.

    1995-08-01

    Hereditary methemoglobinemia with generalized deficiency of NADH-cytochrome b{sub 5} reductase (b{sub 5}R) (type II) is a rare disease characterized by severe developmental abnormalities, which often lead to premature death. Although the molecular relationship between the symptoms of this condition and the enzyme deficit are not understood, it is thought that an important cause is the loss of the lipid metabolizing activities of the endoplasmic reticulum-located reductase. However, the functions of the form located on outer mitochondrial membranes have not been considered previously. In this study, we have analyzed the gene of an Italian patient and identified a novel G{r_arrow}T transversion at the splice-acceptor site of the 9th exon, which results in the complete absence of immunologically detectable b{sub 5}R in blood cells and skin fibroblasts. In cultured fibroblasts of the patient, NADH-dependent cytochrome c reductase, ferricyanide reductase, and semidehydroascorbate reductase activities were severely reduced. The latter activity is known to be due to b{sub 5}R located on outer mitochondrial membranes. Thus, our results demonstrate that the reductase in its two membrane locations, endoplasmic reticulum and outer mitochondrial membranes, is the product of the same gene and suggest that a defect in ascorbate regeneration may contribute to the phenotype of hereditary methemoglobinemia of generalized type. 37 refs., 5 figs., 2 tabs.

  20. Imaging extracellular waves of glutamate during calcium signaling in cultured astrocytes.

    PubMed

    Innocenti, B; Parpura, V; Haydon, P G

    2000-03-01

    A growing body of evidence proposes that glial cells have the potential to play a role as modulators of neuronal activity and synaptic transmission by releasing the neurotransmitter glutamate (Arague et al., 1999). We explore the spatial nature of glutamate release from astrocytes with an enzyme-linked assay system and CCD imaging technology. In the presence of glutamate, L-glutamic dehydrogenase (GDH) reduces NAD(+) to NADH, a product that fluoresces when excited with UV light. Theoretically, provided that GDH and NAD(+) are present in the bathing saline, the release of glutamate from stimulated astrocytes can be optically detected by monitoring the accumulation of NADH. Indeed, stimuli that induce a wave of elevated calcium among astrocytes produced a corresponding spread of extracellular NADH fluorescence. Treatment of cultures either with thapsigargin, to deplete internal calcium stores, or with the membrane-permeant calcium chelator BAPTA AM significantly decreased the accumulation of NADH, demonstrating that this fluorometric assay effectively monitors calcium-dependent glutamate release. With a temporal resolution of 500 msec and spatial resolution of approximately 20 micrometer, discrete regions of glutamate release were not reliably resolved. The wave of glutamate release that underlies the NADH fluorescence propagated at an average speed of approximately 26 micrometer/sec, correlating with the rate of calcium wave progression (10-30 micrometer/sec), and caused a localized accumulation of glutamate in the range of 1-100 microM. Further analysis of the fluorescence accumulation clearly demonstrated that glutamate is released in a regenerative manner, with subsequent cells that are involved in the calcium wave releasing additional glutamate. PMID:10684881

  1. Engineering a d-lactate dehydrogenase that can super-efficiently utilize NADPH and NADH as cofactors

    PubMed Central

    Meng, Hengkai; Liu, Pi; Sun, Hongbing; Cai, Zhen; Zhou, Jie; Lin, Jianping; Li, Yin

    2016-01-01

    Engineering the cofactor specificity of a natural enzyme often results in a significant decrease in its activity on original cofactor. Here we report that a NADH-dependent dehydrogenase (d-LDH) from Lactobacillus delbrueckii 11842 can be rationally engineered to efficiently use both NADH and NADPH as cofactors. Point mutations on three amino acids (D176S, I177R, F178T) predicted by computational analysis resulted in a modified enzyme designated as d-LDH*. The Kcat/Km of the purified d-LDH* on NADPH increased approximately 184-fold while the Kcat/Km on NADH also significantly increased, showing for the first time that a rationally engineered d-LDH could exhibit comparable activity on both NADPH and NADH. Further kinetic analysis revealed that the enhanced affinity with NADH or NADPH and the significant increased Kcat of d-LDH* resulted in the significant increase of d-LDH* activity on both NADPH and NADH. This study thus demonstrated that the cofactor specificity of dehydrogenase can be broadened by using targeted engineering approach, and the engineered enzyme can efficiently function in NADH-rich, or NADPH-rich, or NADH and NADPH-rich environment. PMID:27109778

  2. Two Pathways of Glutamate Fermentation by Anaerobic Bacteria

    PubMed Central

    Buckel, Wolfgang; Barker, H. A.

    1974-01-01

    Two pathways are involved in the fermentation of glutamate to acetate, butyrate, carbon dioxide, and ammonia—the methylaspartate and the hydroxyglutarate pathways which are used by Clostridium tetanomorphum and Peptococcus aerogenes, respectively. Although these pathways give rise to the same products, they are easily distinguished by different labeling patterns of the butyrate when [4-14C]glutamate is used as substrate. Schmidt degradation of the radioactive butyrate from C. tetanomorphum yielded equally labeled propionate and carbon dioxide, whereas nearly all the radioactivity of the butyrate from P. aerogenes was recovered in the corresponding propionate. This procedure was used as a test for the pathway of glutamate fermentation by 15 strains (9 species) of anaerobic bacteria. The labeling patterns of the butyrate indicate that glutamate is fermented via the methylaspartate pathway by C. tetani, C. cochlearium, and C. saccarobutyricum, and via the hydroxyglutarate pathway by Acidaminococcus fermentans, C. microsporum, Fusobacterium nucleatum, and F. fusiformis. Enzymes specific for each pathway were assayed in crude extracts of the above organisms. 3-Methylaspartase was found only in clostridia which use the methylaspartate pathway, including Clostridium SB4 and C. sticklandii, which probably degrade glutamate to acetate and carbon dioxide by using a second amino acid as hydrogen acceptor. High levels of 2-hydroxyglutarate dehydrogenase were found exclusively in organisms that use the hydroxyglutarate pathway. The data indicate that only two pathways are involved in the fermentation of glutamate by the bacteria analyzed. The methylaspartate pathway appears to be used only by species of Clostridium, whereas the hydroxyglutarate pathway is used by representatives of several genera. PMID:4813895

  3. Alcohol dehydrogenases from Scheffersomyces stipitis involved in the detoxification of aldehyde inhibitors derived from lignocellulosic biomass conversion.

    PubMed

    Ma, Menggen; Wang, Xu; Zhang, Xiaoping; Zhao, Xianxian

    2013-09-01

    Aldehyde inhibitors such as furfural and 5-hydroxymethylfurfural (HMF) are generated from biomass pretreatment. Scheffersomyces stipitis is able to reduce furfural and HMF to less toxic furanmethanol and furan-2,5-dimethanol; however, the enzymes involved in the reductive reaction still remain unknown. In this study, transcription responses of two known and five putative alcohol dehydrogenase genes from S. stipitis were analyzed under furfural and HMF stress conditions. All the seven alcohol dehydrogenase genes were also cloned and overexpressed for their activity analyses. Our results indicate that transcriptions of SsADH4 and SsADH6 were highly induced under furfural and HMF stress conditions, and the proteins encoded by them exhibited NADH- and/or NADPH-dependent activities for furfural and HMF reduction, respectively. For furfural reduction, NADH-dependent activity was also observed in SsAdh1p and NAD(P)H-dependent activities were also observed in SsAdh5p and SsAdh7p. For HMF reduction, NADPH-dependent activities were also observed in SsAdh5p and SsAdh7p. SsAdh4p displayed the highest NADPH-dependent specific activity and catalytic efficiency for reduction of both furfural and HMF among the seven alcohol dehydrogenases. Enzyme activities of all SsADH proteins were more stable under acidic condition. For most SsADH proteins, the optimum temperature for enzyme activities was 30 °C and more than 50 % enzyme activities remained at 60 °C. Reduction activities of formaldehyde, acetaldehyde, isovaleraldehyde, benzaldehyde, and phenylacetaldehyde were also observed in some SsADH proteins. Our results indicate that multiple alcohol dehydrogenases in S. stipitis are involved in the detoxification of aldehyde inhibitors derived from lignocellulosic biomass conversion. PMID:23912116

  4. Furfural reduction mechanism of a zinc-dependent alcohol dehydrogenase from Cupriavidus necator JMP134

    PubMed Central

    Kang, ChulHee; Hayes, Robert; Sanchez, Emiliano J.; Webb, Brian N.; Li, Qunrui; Hooper, Travis; Nissen, Mark S.; Xun, Luying

    2012-01-01

    Summary FurX is a tetrameric Zn-dependent alcohol dehydrogenase (ADH) from Cupriavidus necator JMP134. The enzyme rapidly reduces furfural with NADH as the reducing power. For the first time among characterized ADHs, the high-resolution structures of all reaction steps were obtained in a time-resolved manner, thereby illustrating the complete catalytic events of NADH-dependent reduction of furfural and the dynamic Zn2+ coordination among Glu66, water, substrate and product. In the fully closed conformation of the NADH complex, the catalytic turnover proved faster than observed for the partially closed conformation due to an effective proton transfer network. The domain motion triggered by NAD(H) association/dissociation appeared to facilitate dynamic interchanges in Zn2+ coordination with substrate and product molecules, ultimately increasing the enzymatic turnover rate. NAD+ dissociation appeared to be a slow process, involving multiple steps in concert with a domain opening and reconfiguration of Glu66. This agrees with the report that the cofactor is not dissociated from FurX during ethanol-dependent reduction of furfural, in which ethanol reduces NAD+ to NADH that is subsequently used for furfural reduction. PMID:22081946

  5. Regulation of glutamate metabolism by protein kinases in mycobacteria.

    PubMed

    O'Hare, Helen M; Durán, Rosario; Cerveñansky, Carlos; Bellinzoni, Marco; Wehenkel, Anne Marie; Pritsch, Otto; Obal, Gonzalo; Baumgartner, Jens; Vialaret, Jérome; Johnsson, Kai; Alzari, Pedro M

    2008-12-01

    Protein kinase G of Mycobacterium tuberculosis has been implicated in virulence and in regulation of glutamate metabolism. Here we show that this kinase undergoes a pattern of autophosphorylation that is distinct from that of other M. tuberculosis protein kinases characterized to date and we identify GarA as a substrate for phosphorylation by PknG. Autophosphorylation of PknG has little effect on kinase activity but promotes binding to GarA, an interaction that is also detected in living mycobacteria. PknG phosphorylates GarA at threonine 21, adjacent to the residue phosphorylated by PknB (T22), and these two phosphorylation events are mutually exclusive. Like the homologue OdhI from Corynebacterium glutamicum, the unphosphorylated form of GarA is shown to inhibit alpha-ketoglutarate decarboxylase in the TCA cycle. Additionally GarA is found to bind and modulate the activity of a large NAD(+)-specific glutamate dehydrogenase with an unusually low affinity for glutamate. Previous reports of a defect in glutamate metabolism caused by pknG deletion may thus be explained by the effect of unphosphorylated GarA on these two enzyme activities, which may also contribute to the attenuation of virulence. PMID:19019160

  6. Metabotropic Glutamate Receptors

    PubMed Central

    Dillon, James; Franks, Christopher J.; Murray, Caitriona; Edwards, Richard J.; Calahorro, Fernando; Ishihara, Takeshi; Katsura, Isao; Holden-Dye, Lindy; O'Connor, Vincent

    2015-01-01

    Glutamatergic neurotransmission is evolutionarily conserved across animal phyla. A major class of glutamate receptors consists of the metabotropic glutamate receptors (mGluRs). In C. elegans, three mGluR genes, mgl-1, mgl-2, and mgl-3, are organized into three subgroups, similar to their mammalian counterparts. Cellular reporters identified expression of the mgls in the nervous system of C. elegans and overlapping expression in the pharyngeal microcircuit that controls pharyngeal muscle activity and feeding behavior. The overlapping expression of mgls within this circuit allowed the investigation of receptor signaling per se and in the context of receptor interactions within a neural network that regulates feeding. We utilized the pharmacological manipulation of neuronally regulated pumping of the pharyngeal muscle in the wild-type and mutants to investigate MGL function. This defined a net mgl-1-dependent inhibition of pharyngeal pumping that is modulated by mgl-3 excitation. Optogenetic activation of the pharyngeal glutamatergic inputs combined with electrophysiological recordings from the isolated pharyngeal preparations provided further evidence for a presynaptic mgl-1-dependent regulation of pharyngeal activity. Analysis of mgl-1, mgl-2, and mgl-3 mutant feeding behavior in the intact organism after acute food removal identified a significant role for mgl-1 in the regulation of an adaptive feeding response. Our data describe the molecular and cellular organization of mgl-1, mgl-2, and mgl-3. Pharmacological analysis identified that, in these paradigms, mgl-1 and mgl-3, but not mgl-2, can modulate the pharyngeal microcircuit. Behavioral analysis identified mgl-1 as a significant determinant of the glutamate-dependent modulation of feeding, further highlighting the significance of mGluRs in complex C. elegans behavior. PMID:25869139

  7. Separation of dehydrogenases on polyaminomethylstyrene.

    PubMed

    Schöpp, W; Meinert, S; Thyfronitou, J; Aurich, H

    1975-01-29

    The binding of dehydrogenases, especially alcohol dehydrogenase, and other proteins to several ion exchangers and hydrophobic polymers was investigated. Quantitative parameters for the stability of the polymer-protein complexes (obtained form double reciprocal plots) indicate a high but different affinity of many proteins for polyaminomethylstyrene. The chromatography of a mixture of five dehydrogenases and human serum albumin on polyaminomethylstyrene is described. PMID:237012

  8. 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

  9. 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

  10. Concerted modulation of alanine and glutamate metabolism in young Medicago truncatula seedlings under hypoxic stress

    PubMed Central

    Limami, Anis M.; Glévarec, Gaëlle; Ricoult, Claudie; Cliquet, Jean-Bernard; Planchet, Elisabeth

    2008-01-01

    The modulation of primary nitrogen metabolism by hypoxic stress was studied in young Medicago truncatula seedlings. Hypoxic seedlings were characterized by the up-regulation of glutamate dehydrogenase 1 (GDH1) and mitochondrial alanine aminotransferase (mAlaAT), and down-regulation of glutamine synthetase 1b (GS1b), NADH-glutamate synthase (NADH-GOGAT), glutamate dehydrogenase 3 (GDH3), and isocitrate dehydrogenase (ICDH) gene expression. Hypoxic stress severely inhibited GS activity and stimulated NADH-GOGAT activity. GDH activity was lower in hypoxic seedlings than in the control, however, under either normoxia or hypoxia, the in vivo activity was directed towards glutamate deamination. 15NH4 labelling showed for the first time that the adaptive reaction of the plant to hypoxia consisted of a concerted modulation of nitrogen flux through the pathways of both alanine and glutamate synthesis. In hypoxic seedlings, newly synthesized 15N-alanine increased and accumulated as the major amino acid, asparagine synthesis was inhibited, while 15N-glutamate was synthesized at a similar rate to that in the control. A discrepancy between the up-regulation of GDH1 expression and the down-regulation of GDH activity by hypoxic stress highlighted for the first time the complex regulation of this enzyme by hypoxia. Higher rates of glycolysis and ethanol fermentation are known to cause the fast depletion of sugar stores and carbon stress. It is proposed that the expression of GDH1 was stimulated by hypoxia-induced carbon stress, while the enzyme protein might be involved during post-hypoxic stress contributing to the regeneration of 2-oxoglutarate via the GDH shunt. PMID:18508812

  11. 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. PMID:6144677

  12. Glutamate neurotoxicity in rat cerebellar granule cells: a major role for xanthine oxidase in oxygen radical formation.

    PubMed

    Atlante, A; Gagliardi, S; Minervini, G M; Ciotti, M T; Marra, E; Calissano, P

    1997-05-01

    To gain insight into the mechanism through which the neurotransmitter glutamate causally participates in several neurological diseases, in vitro cultured cerebellar granule cells were exposed to glutamate and oxygen radical production was investigated. To this aim, a novel procedure was developed to detect oxygen radicals; the fluorescent dye 2',7'-dichlorofluorescein was used to detect production of peroxides, and a specific search for the possible conversion of the enzyme xanthine dehydrogenase into xanthine oxidase after the excitotoxic glutamate pulse was undertaken. A 100 microM glutamate pulse administered to 7-day-old cerebellar granule cells is accompanied by the onset of neuronal death, the appearance of xanthine oxidase, and production of oxygen radicals. Xanthine oxidase activation and superoxide (O2.-) production are completely inhibited by concomitant incubation of glutamate with MK-801, a specific NMDA receptor antagonist, or by chelation of external calcium with EGTA. Partial inhibition of both cell death and parallel production of reactive oxygen species is achieved with allopurinol, a xanthine oxidase inhibitor, leupeptin, a protease inhibitor, reducing agents such as glutathione or dithiothreitol, antioxidants such as vitamin E and vitamin C, and externally added superoxide dismutase. It is concluded that glutamate-triggered, NMDA-mediated, massive Ca2+ influx induces rapid conversion of xanthine dehydrogenase into xanthine oxidase with subsequent production of reactive oxygen species that most probably have a causal involvement in the initial steps of the series of intracellular events leading to neuronal degeneration and death. PMID:9109530

  13. Glutamic acid as anticancer agent: An overview

    PubMed Central

    Dutta, Satyajit; Ray, Supratim; Nagarajan, K.

    2013-01-01

    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. PMID:24227952

  14. Computational Studies of Glutamate Transporters

    PubMed Central

    Setiadi, Jeffry; Heinzelmann, Germano; Kuyucak, Serdar

    2015-01-01

    Glutamate is the major excitatory neurotransmitter in the human brain whose binding to receptors on neurons excites them while excess glutamate are removed from synapses via transporter proteins. Determination of the crystal structures of bacterial aspartate transporters has paved the way for computational investigation of their function and dynamics at the molecular level. Here, we review molecular dynamics and free energy calculation methods used in these computational studies and discuss the recent applications to glutamate transporters. The focus of the review is on the insights gained on the transport mechanism through computational methods, which otherwise is not directly accessible by experimental probes. Recent efforts to model the mammalian glutamate and other amino acid transporters, whose crystal structures have not been solved yet, are included in the review. PMID:26569328

  15. Cyanobacterial NADPH dehydrogenase complexes

    SciTech Connect

    Ogawa, Teruo; Mi, Hualing

    2007-07-01

    Cyanobacteria possess functionally distinct multiple NADPH dehydrogenase (NDH-1) complexes that are essential to CO2 uptake, photosystem-1 cyclic electron transport and respiration. The unique nature of cyanobacterial NDH-1 complexes is the presence of subunits involved in CO2 uptake. Other than CO2 uptake, chloroplastic NDH-1 complex has similar role as cyanobacterial NDH-1 complexes in photosystem-1 cyclic electron transport and respiration (chlororespiration). In this mini-review we focus on the structure and function of cyanobacterial NDH-1 complexes and their phylogeny. The function of chloroplastic NDH-1 complex and characteristics of plants defective in NDH-1 are also described forcomparison.

  16. Glyceraldehyde-3-phosphate dehydrogenase-catalyzed chain oxidation of reduced nicotinamide adenine dinucleotide by perhydroxyl radicals

    SciTech Connect

    Chan, P.C.

    1980-02-10

    The chain oxidation of glyceraldehyde-3-phosphate dehydrogenase NADH by perhydroxyl radicals and propagated by molecular oxygen was studied by the xanthine-xanthine oxidase system, /sup 60/Co ..gamma..-ray, and pulse radiolysis. The chain length, amount of NADH oxidized per HO/sub 2/ generated, increases with increasing acidity of the medium and reaches a value of 73 at pH 5.0. The rate constant for the oxidation of the glyceraldehyde-3-phosphate dehydrogenase NADH complex by HO/sub 2/ was estimated to be 2 x 10/sup 7/ m/sup -1/s/sup -1/ at ambient temperatures (23-24/sup 0/C). Rate studies as a function of pH indicate that O/sub 2//sup -/ is unreactive toward the glyceraldehyde-3-phosphate dehydrogenase NADH complex. Other dehydrogenases (malate dehydrogenase, glutamate dehydrogenase, and isocitric dehydrogenase) studied showed no catalytic activity in the oxidation of NADH by HO/sub 2//O/sub 2//sup -/.

  17. Genotoxicity of monosodium glutamate.

    PubMed

    Ataseven, Nazmiye; Yüzbaşıoğlu, Deniz; Keskin, Ayten Çelebi; Ünal, Fatma

    2016-05-01

    Monosodium glutamate (MSG) is one of the most widely used flavor enhancers throughout the world. The aim of this study is to investigate the genotoxic potential of MSG by using chromosome aberrations (CAs), sister-chromatid exchanges (SCEs), cytokinesis-blocked micronucleus (CBMN), and random amplified polymorphic DNA-polimerase chain reaction (RAPD-PCR) in cultured human lymphocytes and alkaline comet assays in isolated human lymphocytes, which were incubated with six concentrations (250, 500, 1000, 2000, 4000 and 8000 μg/mL) of MSG. The result of this study indicated that MSG significantly and dose dependently increased the frequencies of CAs, SCE and MN in all treatments and times, compared with control. However, the replication (RI) and nuclear division indices (NDI) were not affected. In this paper, in vitro genotoxic effects of the MSG was also investigated on human peripheral lymphocytes by analysing the RAPD-PCR with arbitrary 10-mer primers. The changes occurring in RAPD profiles after MSG treatment include increase or decrease in band intensity and gain or loss of bands. In the comet assay, this additive caused DNA damage at all concentrations in isolated human lymphocytes after 1-h in vitro exposure. Our results demonstrate that MSG is genotoxic to the human peripheral blood lymphocytes in vitro. PMID:26929995

  18. 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 ...

  19. Metabolite Profiling Reveals YihU as a Novel Hydroxybutyrate Dehydrogenase for Alternative Succinic Semialdehyde Metabolism in Escherichia coli*

    PubMed Central

    Saito, Natsumi; Robert, Martin; Kochi, Hayataro; Matsuo, Goh; Kakazu, Yuji; Soga, Tomoyoshi; Tomita, Masaru

    2009-01-01

    The search for novel enzymes and enzymatic activities is important to map out all metabolic activities and reveal cellular metabolic processes in a more exhaustive manner. Here we present biochemical and physiological evidence for the function of the uncharacterized protein YihU in Escherichia coli using metabolite profiling by capillary electrophoresis time-of-flight mass spectrometry. To detect enzymatic activity and simultaneously identify possible substrates and products of the putative enzyme, we profiled a complex mixture of metabolites in the presence or absence of YihU. In this manner, succinic semialdehyde was identified as a substrate for YihU. The purified YihU protein catalyzed in vitro the NADH-dependent reduction of succinic semialdehyde to γ-hydroxybutyrate. Moreover, a yihU deletion mutant displayed reduced tolerance to the cytotoxic effects of exogenous addition of succinic semialdehyde. Profiling of intracellular metabolites following treatment of E. coli with succinic semialdehyde supports the existence of a YihU-catalyzed reduction of succinic semialdehyde to γ-hydroxybutyrate in addition to its known oxidation to succinate and through the tricarboxylic acid cycle. These findings suggest that YihU is a novel γ-hydroxybutyrate dehydrogenase involved in the metabolism of succinic semialdehyde, and other potentially toxic intermediates that may accumulate under stress conditions in E. coli. PMID:19372223

  20. 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

  1. From Alcohol Dehydrogenase to a “One-way” Carbonyl Reductase by Active-site Redesign

    PubMed Central

    Klimacek, Mario; Nidetzky, Bernd

    2010-01-01

    Directional preference in catalysis is often used to distinguish alcohol dehydrogenases from carbonyl reductases. However, the mechanistic basis underpinning this discrimination is weak. In mannitol 2-dehydrogenase from Pseudomonas fluorescens, stabilization of (partial) negative charge on the substrate oxyanion by the side chains of Asn-191 and Asn-300 is a key feature of catalysis in the direction of alcohol oxidation. We have disrupted this ability through individual and combined substitutions of the two asparagines by aspartic acid. Kinetic data and their thermodynamic analysis show that the internal equilibrium of enzyme-NADH-fructose and enzyme-NAD+-mannitol (Kint) was altered dramatically (104- to 105-fold) from being balanced in the wild-type enzyme (Kint ≈ 3) to favoring enzyme-NAD+-mannitol in the single site mutants, N191D and N300D. The change in Kint reflects a selective slowing down of the mannitol oxidation rate, resulting because Asn → Asp replacement (i) disfavors partial abstraction of alcohol proton by Lys-295 in a step preceding catalytic hydride transfer, and (ii) causes stabilization of a nonproductive enzyme-NAD+-mannitol complex. N191D and N300D appear to lose fructose binding affinity due to deprotonation of the respective Asp above apparent pK values of 5.3 ± 0.1 and 6.3 ± 0.2, respectively. The mutant incorporating both Asn→Asp substitutions behaved as a slow “fructose reductase” at pH 5.2, lacking measurable activity for mannitol oxidation in the pH range 6.8–10. A mechanism is suggested in which polarization of the substrate carbonyl by a doubly protonated diad of Asp and Lys-295 facilitates NADH-dependent reduction of fructose by N191D and N300D under optimum pH conditions. Creation of an effectively “one-way” reductase by active-site redesign of a parent dehydrogenase has not been previously reported and holds promise in the development of carbonyl reductases for application in organic synthesis. PMID:20639204

  2. Cloning, purification and crystallization of Thermus thermophilus proline dehydrogenase

    SciTech Connect

    White, Tommi A.; Tanner, John J.

    2005-08-01

    Cloning, purification and crystallization of T. thermophilus proline dehydrogenase is reported. The detergent n-octyl β-d-glucopyranoside was used to reduce polydispersity, which enabled crystallization. Nature recycles l-proline by converting it to l-glutamate. This four-electron oxidation process is catalyzed by the two enzymes: proline dehydrogenase (PRODH) and Δ{sup 1}-pyrroline-5-carboxylate dehydrogenase. This note reports the cloning, purification and crystallization of Thermus thermophilus PRODH, which is the prototype of a newly discovered superfamily of bacterial monofunctional PRODHs. The results presented here include production of a monodisperse protein solution through use of the detergent n-octyl β-d-glucopyranoside and the growth of native crystals that diffracted to 2.3 Å resolution at Advanced Light Source beamline 4.2.2. The space group is P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 82.2, b = 89.6, c = 94.3 Å. The asymmetric unit is predicted to contain two protein molecules and 46% solvent. Molecular-replacement trials using a fragment of the PRODH domain of the multifunctional Escherichia coli PutA protein as the search model (24% amino-acid sequence identity) did not produce a satisfactory solution. Therefore, the structure of T. thermophilus PRODH will be determined by multiwavelength anomalous dispersion phasing using a selenomethionyl derivative.

  3. Glutamate receptors at atomic resolution

    SciTech Connect

    Mayer, Mark L.

    2010-12-03

    At synapses throughout the brain and spinal cord, the amino-acid glutamate is the major excitatory neurotransmitter. During evolution, a family of glutamate-receptor ion channels seems to have been assembled from a kit consisting of discrete ligand-binding, ion-channel, modulatory and cytoplasmic domains. Crystallographic studies that exploit this unique architecture have greatly aided structural analysis of the ligand-binding core, but the results also pose a formidable challenge, namely that of resolving the allosteric mechanisms by which individual domains communicate and function in an intact receptor.

  4. Glutamate in peripheral organs: Biology and pharmacology.

    PubMed

    Du, Jie; Li, Xiao-Hui; Li, Yuan-Jian

    2016-08-01

    Glutamate is a versatile molecule existing in both the central nervous system and peripheral organs. Previous studies have mainly focussed on the biological effect of glutamate in the brain. Recently, abundant evidence has demonstrated that glutamate also participates in the regulation of physiopathological functions in peripheral tissues, including the lung, kidney, liver, heart, stomach and immune system, where the glutamate/glutamate receptor/glutamate transporter system plays an important role in the pathogenesis of certain diseases, such as myocardial ischaemia/reperfusion injury and acute gastric mucosa injury. All these findings provide new insight into the biology and pharmacology of glutamate and suggest a potential therapeutic role of glutamate in non-neurological diseases. PMID:27164423

  5. Cloning and expression of a putative alcohol dehydrogenase gene of Entamoeba histolytica and its application to immunological examination.

    PubMed Central

    Kimura, A; Hara, Y; Kimoto, T; Okuno, Y; Minekawa, Y; Nakabayashi, T

    1996-01-01

    To clone and express the genes encoding major antigens of Entamoeba histolytica, we constructed a lambda gt11 cDNA library for E. histolytica HM1:IMSS and screened it with pooled sera from patients with amoebiasis. A 1,223-bp cDNA was cloned (clone 1223), and its nucleotide sequence was determined. The amino acid sequence predicted to be encoded by the open reading frame of clone 1223 consisted of 396 residues and showed 32.5 and 32.3% homology to the NADH-dependent butanol dehydrogenases I and II (bdhA and bdhB) of Clostridium acetobutylicum, respectively. In addition, 29 of the 34 consensus positions of bdhA and bdhB were also well conserved in clone 1223. The recombinant protein expressed from clone 1223 had an estimated molecular mass of 43.5 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The antigenicity and specificity of the recombinant protein were evaluated by an enzyme-linked immunosorbent assay using sera obtained from two clinical groups of patients with amoebiasis and a group of healthy controls. The recombinant protein had potent and specific antigenicity. In all, 53 serum samples (88.3%) from 60 patients with amoebiasis were positive for immunoglobulin G antibody against the recombinant protein, with a mean optical density value of 0.42. In contrast, 53 of 54 healthy control serum samples were negative, with only 1 positive serum sample showing the lower optical density value. These results suggested that clone 1223 is promising in terms of providing a useful antigen for the accurate serodiagnosis of amoebiasis and that the gene encodes a putative alcohol dehydrogenase of E. histolytica. PMID:8705667

  6. Tissue Distribution of Glutamate Synthase and Glutamine Synthetase in Rice Leaves 1

    PubMed Central

    Yamaya, Tomoyuki; Hayakawa, Toshihiko; Tanasawa, Keisuke; Kamachi, Kazunari; Mae, Tadahiko; Ojima, Kunihiko

    1992-01-01

    To further explore the function of NADH-dependent glutamate synthase (GOGAT), the tissue distribution of NADH-GOGAT protein and activity was investigated in rice (Oryza sativa L.) leaves. The distributions of ferredoxin (Fd)-dependent GOGAT, plastidic glutamine synthetase, and cytosolic glutamine synthetase proteins were also determined in the same tissues. High levels of NADH-GOGAT protein (33.1 μg protein/g fresh weight) and activity were detected in the 10th leaf blade before emergence. The unexpanded, nongreen portion of the 9th leaf blade contained more than 50% of the NADH-GOGAT protein and activity per gram fresh weight when compared with the 10th leaf. The expanding, green portion of the 9th leaf blade outside of the sheath contained a slightly lower abundance of NADH-GOGAT protein than the nongreen portion of the 9th blade on a fresh weight basis. The fully expanded leaf blades at positions lower than the 9th leaf had decreased NADH-GOGAT levels as a function of increasing age, and the oldest, 5th blade contained only 4% of the NADH-GOGAT protein compared with the youngest 10th leaf blade. Fd-GOGAT protein, on the other hand, was the major form of GOGAT in the green tissues, and the highest amount of Fd-GOGAT protein (111 μg protein/g fresh weight) was detected in the 7th leaf blade. In the nongreen 10th leaf blade, the content of Fd-GOGAT protein was approximately 7% of that found in the 7th leaf blade. In addition, the content of NADH-GOGAT protein in the 10th leaf blade was about 4 times higher than that of Fd-GOGAT protein. The content of plastidic glutamine synthetase polypeptide was also the highest in the 7th leaf blade (429 μg/g fresh weight) and lowest in nongreen blades and sheaths. On the other hand, the relative abundance of the cytosolic glutamine synthetase polypeptide was the highest in the oldest leaf blade, decreasing to 10 to 20% of that value in young, nongreen leaves. These results suggest that NADH-GOGAT is important for the

  7. Lactate dehydrogenase-elevating virus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    This book chapter describes the taxonomic classification of Lactate dehydrogenase-elevating virus (LDV). Included are: host, genome, classification, morphology, physicochemical and physical properties, nucleic acid, proteins, lipids, carbohydrates, geographic range, phylogenetic properties, biologic...

  8. 21 CFR 182.1045 - Glutamic acid.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 3 2013-04-01 2013-04-01 false Glutamic acid. 182.1045 Section 182.1045 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) FOOD FOR HUMAN....1045 Glutamic acid. (a) Product. Glutamic acid. (b) (c) Limitations, restrictions, or explanation....

  9. 21 CFR 182.1045 - Glutamic acid.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 3 2011-04-01 2011-04-01 false Glutamic acid. 182.1045 Section 182.1045 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) FOOD FOR HUMAN....1045 Glutamic acid. (a) Product. Glutamic acid. (b) (c) Limitations, restrictions, or explanation....

  10. 21 CFR 182.1045 - Glutamic acid.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Glutamic acid. 182.1045 Section 182.1045 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) FOOD FOR HUMAN....1045 Glutamic acid. (a) Product. Glutamic acid. (b) (c) Limitations, restrictions, or explanation....

  11. 21 CFR 182.1045 - Glutamic acid.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 3 2012-04-01 2012-04-01 false Glutamic acid. 182.1045 Section 182.1045 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) FOOD FOR HUMAN....1045 Glutamic acid. (a) Product. Glutamic acid. (b) (c) Limitations, restrictions, or explanation....

  12. 21 CFR 182.1045 - Glutamic acid.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 3 2014-04-01 2014-04-01 false Glutamic acid. 182.1045 Section 182.1045 Food and... GENERALLY RECOGNIZED AS SAFE Multiple Purpose GRAS Food Substances § 182.1045 Glutamic acid. (a) Product. Glutamic acid. (b) (c) Limitations, restrictions, or explanation. This substance is generally recognized...

  13. Modes of glutamate receptor gating

    PubMed Central

    Popescu, Gabriela K

    2012-01-01

    Abstract The time course of excitatory synaptic currents, the major means of fast communication between neurons of the central nervous system, is encoded in the dynamic behaviour of post-synaptic glutamate-activated channels. First-pass attempts to explain the glutamate-elicited currents with mathematical models produced reaction mechanisms that included only the most basic functionally defined states: resting vs. liganded, closed vs. open, responsive vs. desensitized. In contrast, single-molecule observations afforded by the patch-clamp technique revealed an unanticipated kinetic multiplicity of transitions: from microseconds-lasting flickers to minutes-long modes. How these kinetically defined events impact the shape of the synaptic response, how they relate to rearrangements in receptor structure, and whether and how they are physiologically controlled represent currently active research directions. Modal gating, which refers to the slowest, least frequently observed ion-channel transitions, has been demonstrated for representatives of all ion channel families. However, reaction schemes have been largely confined to the short- and medium-range time scales. For glutamate receptors as well, modal gating has only recently come under rigorous scrutiny. This article reviews the evidence for modal gating of glutamate receptors and the still developing hypotheses about the mechanism(s) by which modal shifts occur and the ways in which they may impact the time course of synaptic transmission. PMID:22106181

  14. 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

  15. Alcohol Dehydrogenase from Methylobacterium organophilum

    PubMed Central

    Wolf, H. J.; Hanson, R. S.

    1978-01-01

    The alcohol dehydrogenase from Methylobacterium organophilum, a facultative methane-oxidizing bacterium, has been purified to homogeneity as indicated by sodium dodecyl sulfate-gel electrophoresis. It has several properties in common with the alcohol dehydrogenases from other methylotrophic bacteria. The active enzyme is a dimeric protein, both subunits having molecular weights of about 62,000. The enzyme exhibits broad substrate specificity for primary alcohols and catalyzes the two-step oxidation of methanol to formate. The apparent Michaelis constants of the enzyme are 2.9 × 10−5 M for methanol and 8.2 × 10−5 M for formaldehyde. Activity of the purified enzyme is dependent on phenazine methosulfate. Certain characteristics of this enzyme distinguish it from the other alcohol dehydrogenases of other methylotrophic bacteria. Ammonia is not required for, but stimulates the activity of newly purified enzyme. An absolute dependence on ammonia develops after storage of the purified enzyme. Activity is not inhibited by phosphate. The fluorescence spectrum of the enzyme indicates that it and the cofactor associated with it may be chemically different from the alcohol dehydrogenases from other methylotrophic bacteria. The alcohol dehydrogenases of Hyphomicrobium WC-65, Pseudomonas methanica, Methylosinus trichosporium, and several facultative methylotrophs are serologically related to the enzyme purified in this study. The enzymes of Rhodopseudomonas acidophila and of organisms of the Methylococcus group did not cross-react with the antiserum prepared against the alcohol dehydrogenase of M. organophilum. Images PMID:80974

  16. The glutamate aspartate transporter (GLAST) mediates L-glutamate-stimulated ascorbate-release via swelling-activated anion channels in cultured neonatal rodent astrocytes.

    PubMed

    Lane, Darius J R; Lawen, Alfons

    2013-03-01

    Vitamin C (ascorbate) plays important neuroprotective and neuromodulatory roles in the mammalian brain. Astrocytes are crucially involved in brain ascorbate homeostasis and may assist in regenerating extracellular ascorbate from its oxidised forms. Ascorbate accumulated by astrocytes can be released rapidly by a process that is stimulated by the excitatory amino acid, L-glutamate. This process is thought to be neuroprotective against excitotoxicity. Although of potential clinical interest, the mechanism of this stimulated ascorbate-release remains unknown. Here, we report that primary cultures of mouse and rat astrocytes release ascorbate following initial uptake of dehydroascorbate and accumulation of intracellular ascorbate. Ascorbate-release was not due to cellular lysis, as assessed by cellular release of the cytosolic enzyme lactate dehydrogenase, and was stimulated by L-glutamate and L-aspartate, but not the non-excitatory amino acid L-glutamine. This stimulation was due to glutamate-induced cellular swelling, as it was both attenuated by hypertonic and emulated by hypotonic media. Glutamate-stimulated ascorbate-release was also sensitive to inhibitors of volume-sensitive anion channels, suggesting that the latter may provide the conduit for ascorbate efflux. Glutamate-stimulated ascorbate-release was not recapitulated by selective agonists of either ionotropic or group I metabotropic glutamate receptors, but was completely blocked by either of two compounds, TFB-TBOA and UCPH-101, which non-selectively and selectively inhibit the glial Na(+)-dependent excitatory amino acid transporter, GLAST, respectively. These results suggest that an impairment of astrocytic ascorbate-release may exacerbate neuronal dysfunction in neurodegenerative disorders and acute brain injury in which excitotoxicity and/or GLAST deregulation have been implicated. PMID:22886112

  17. 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

  18. Nonvesicular Release of Glutamate by Glial xCT Transporters Suppresses Glutamate Receptor Clustering In Vivo

    PubMed Central

    Augustin, Hrvoje; Grosjean, Yael; Chen, Kaiyun; Sheng, Qi; Featherstone, David E.

    2008-01-01

    We hypothesized that cystine/glutamate transporters (xCTs) might be critical regulators of ambient extracellular glutamate levels in the nervous system and that misregulation of this glutamate pool might have important neurophysiological and/or behavioral consequences. To test this idea, we identified and functionally characterized a novel Drosophila xCT gene, which we subsequently named “genderblind” (gb). Genderblind is expressed in a previously overlooked subset of peripheral and central glia. Genetic elimination of gb causes a 50% reduction in extracellular glutamate concentration, demonstrating that xCT transporters are important regulators of extracellular glutamate. Consistent with previous studies showing that extracellular glutamate regulates postsynaptic glutamate receptor clustering, gb mutants show a large (200–300%) increase in the number of postsynaptic glutamate receptors. This increase in postsynaptic receptor abundance is not accompanied by other obvious synaptic changes and is completely rescued when synapses are cultured in wild-type levels of glutamate. Additional in situ pharmacology suggests that glutamate-mediated suppression of glutamate receptor clustering depends on receptor desensitization. Together, our results suggest that (1) xCT transporters are critical for regulation of ambient extracellular glutamate in vivo; (2) ambient extracellular glutamate maintains some receptors constitutively desensitized in vivo; and (3) constitutive desensitization of ionotropic glutamate receptors suppresses their ability to cluster at synapses. PMID:17202478

  19. Ligands for Ionotropic Glutamate Receptors

    NASA Astrophysics Data System (ADS)

    Swanson, Geoffrey T.; Sakai, Ryuichi

    Marine-derived small molecules and peptides have played a central role in elaborating pharmacological specificities and neuronal functions of mammalian ionotropic glutamate receptors (iGluRs), the primary mediators of excitatory syn-aptic transmission in the central nervous system (CNS). As well, the pathological sequelae elicited by one class of compounds (the kainoids) constitute a widely-used animal model for human mesial temporal lobe epilepsy (mTLE). New and existing molecules could prove useful as lead compounds for the development of therapeutics for neuropathologies that have aberrant glutamatergic signaling as a central component. In this chapter we discuss natural source origins and pharmacological activities of those marine compounds that target ionotropic glutamate receptors.

  20. Michael hydratase alcohol dehydrogenase or just alcohol dehydrogenase?

    PubMed Central

    2014-01-01

    The Michael hydratase – alcohol dehydrogenase (MhyADH) from Alicycliphilus denitrificans was previously identified as a bi-functional enzyme performing a hydration of α,β-unsaturated ketones and subsequent oxidation of the formed alcohols. The investigations of the bi-functionality were based on a spectrophotometric assay and an activity staining in a native gel of the dehydrogenase. New insights in the recently discovered organocatalytic Michael addition of water led to the conclusion that the previously performed experiments to identify MhyADH as a bi-functional enzyme and their results need to be reconsidered and the reliability of the methodology used needs to be critically evaluated. PMID:24949265

  1. Downregualtion of dynamin-related protein 1 attenuates glutamate-induced excitotoxicity via regulating mitochondrial function in a calcium dependent manner in HT22 cells

    SciTech Connect

    Zhang, Chi; Yuan, Xian-rui; Li, Hao-yu; Zhao, Zi-jin; Liao, Yi-wei; Wang, Xiang-yu; Su, Jun; Sang, Shu-shan; Liu, Qing

    2014-01-03

    Highlights: •Downregulation of Drp-1 attenuates glutamate-induced excitotoxicity. •Downregulation of Drp-1 inhibits glutamate-induced apoptosis. •Downregulation of Drp-1 reduces glutamate-induced mitochondrial dysfunction. •Downregulation of Drp-1 preserves intracellular calcium homeostasis. -- Abstract: Glutamate-mediated excitotoxicity is involved in many acute and chronic brain diseases. Dynamin related protein 1 (Drp-1), one of the GTPase family of proteins that regulate mitochondrial fission and fusion balance, is associated with apoptotic cell death in cancer and neurodegenerative diseases. Here we investigated the effect of downregulating Drp-1 on glutamate excitotoxicity-induced neuronal injury in HT22 cells. We found that downregulation of Drp-1 with specific small interfering RNA (siRNA) increased cell viability and inhibited lactate dehydrogenase (LDH) release after glutamate treatment. Downregulation of Drp-1 also inhibited an increase in the Bax/Bcl-2 ratio and cleavage of caspase-9 and caspase-3. Drp-1 siRNA transfection preserved the mitochondrial membrane potential (MMP), reduced cytochrome c release, enhanced ATP production, and partly prevented mitochondrial swelling. In addition, Drp-1 knockdown attenuated glutamate-induced increases of cytoplasmic and mitochondrial Ca{sup 2+}, and preserved the mitochondrial Ca{sup 2+} buffering capacity after excitotoxicity. Taken together, these results suggest that downregulation of Drp-1 protects HT22 cells against glutamate-induced excitatory damage, and this neuroprotection may be dependent at least in part on the preservation of mitochondrial function through regulating intracellular calcium homeostasis.

  2. Evidence for Hysteretic Substrate Channeling in the Proline Dehydrogenase and Δ1-Pyrroline-5-carboxylate Dehydrogenase Coupled Reaction of Proline Utilization A (PutA)*

    PubMed Central

    Moxley, Michael A.; Sanyal, Nikhilesh; Krishnan, Navasona; Tanner, John J.; Becker, Donald F.

    2014-01-01

    PutA (proline utilization A) is a large bifunctional flavoenzyme with proline dehydrogenase (PRODH) and Δ1-pyrroline-5-carboxylate dehydrogenase (P5CDH) domains that catalyze the oxidation of l-proline to l-glutamate in two successive reactions. In the PRODH active site, proline undergoes a two-electron oxidation to Δ1-pyrroline-5-carboxlylate, and the FAD cofactor is reduced. In the P5CDH active site, l-glutamate-γ-semialdehyde (the hydrolyzed form of Δ1-pyrroline-5-carboxylate) undergoes a two-electron oxidation in which a hydride is transferred to NAD+-producing NADH and glutamate. Here we report the first kinetic model for the overall PRODH-P5CDH reaction of a PutA enzyme. Global analysis of steady-state and transient kinetic data for the PRODH, P5CDH, and coupled PRODH-P5CDH reactions was used to test various models describing the conversion of proline to glutamate by Escherichia coli PutA. The coupled PRODH-P5CDH activity of PutA is best described by a mechanism in which the intermediate is not released into the bulk medium, i.e., substrate channeling. Unexpectedly, single-turnover kinetic experiments of the coupled PRODH-P5CDH reaction revealed that the rate of NADH formation is 20-fold slower than the steady-state turnover number for the overall reaction, implying that catalytic cycling speeds up throughput. We show that the limiting rate constant observed for NADH formation in the first turnover increases by almost 40-fold after multiple turnovers, achieving half of the steady-state value after 15 turnovers. These results suggest that EcPutA achieves an activated channeling state during the approach to steady state and is thus a new example of a hysteretic enzyme. Potential underlying causes of activation of channeling are discussed. PMID:24352662

  3. The Involvement of Glutamate Metabolism in the Resistance to Thermal, Nutritional, and Oxidative Stress in Trypanosoma cruzi

    PubMed Central

    Magdaleno, Anahí; Suárez Mantilla, Brian; Rocha, Sandra C.; Pral, Elizabeth M. F.; Silber, Ariel M.

    2011-01-01

    The inhibition of some glutamate metabolic pathways could lead to diminished parasite survival. In this study, the effects of L-methionine sulfoximine (MS), DL-methionine sulfone (MSO), and DL-methionine sulfoxide (MSE), three glutamate analogs, on several biological processes were evaluated. We found that these analogs inhibited the growth of epimastigotes cells and showed a synergistic effect with stress conditions such as temperature, nutritional starvation, and oxidative stress. The specific activity for the reductive amination of α-ketoglutaric acid, catalyzed by the NADP+-linked glutamate dehydrogenase, showed an increase in the NADP+ levels, when MS, MSE, and MSO were added. It suggests an eventual conversion of the compounds tested by the T. cruzi cells. The fact that trypomastigote bursting was not significantly inhibited when infected cells were treated with these compounds, remarks the existence of relevant metabolic differences among the different life-cycle stages. It must be considered when proposing a new therapeutic drug. PMID:21629861

  4. Metabolism of trans, trans-muconaldehyde, a cytotoxic metabolite of benzene, in mouse liver by alcohol dehydrogenase Adh1 and aldehyde reductase AKR1A4

    SciTech Connect

    Short, Duncan M.; Lyon, Robert; Watson, David G.; Barski, Oleg A.; McGarvie, Gail; Ellis, Elizabeth M. . E-mail: Elizabeth.ellis@strath.ac.uk

    2006-01-15

    The reductive metabolism of trans, trans-muconaldehyde, a cytotoxic metabolite of benzene, was studied in mouse liver. Using an HPLC-based stopped assay, the primary reduced metabolite was identified as 6-hydroxy-trans, trans-2,4-hexadienal (OH/CHO) and the secondary metabolite as 1,6-dihydroxy-trans, trans-2,4-hexadiene (OH/OH). The main enzymes responsible for the highest levels of reductase activity towards trans, trans-muconaldehyde were purified from mouse liver soluble fraction first by Q-sepharose chromatography followed by either blue or red dye affinity chromatography. In mouse liver, trans, trans-muconaldehyde is predominantly reduced by an NADH-dependent enzyme, which was identified as alcohol dehydrogenase (Adh1). Kinetic constants obtained for trans, trans-muconaldehyde with the native Adh1 enzyme showed a V {sub max} of 2141 {+-} 500 nmol/min/mg and a K {sub m} of 11 {+-} 4 {mu}M. This enzyme was inhibited by pyrazole with a K {sub I} of 3.1 {+-} 0.57 {mu}M. Other fractions were found to contain muconaldehyde reductase activity independent of Adh1, and one enzyme was identified as the NADPH-dependent aldehyde reductase AKR1A4. This showed a V {sub max} of 115 nmol/min/mg and a K {sub m} of 15 {+-} 2 {mu}M and was not inhibited by pyrazole.

  5. 2-Butanol and butanone production in Saccharomyces cerevisiae through combination of a B12 dependent dehydratase and a secondary alcohol dehydrogenase using a TEV-based expression system.

    PubMed

    Ghiaci, Payam; Norbeck, Joakim; Larsson, Christer

    2014-01-01

    2-Butanol and its chemical precursor butanone (methyl ethyl ketone--MEK) are chemicals with potential uses as biofuels and biocommodity chemicals. In order to produce 2-butanol, we have demonstrated the utility of using a TEV-protease based expression system to achieve equimolar expression of the individual subunits of the two protein complexes involved in the B12-dependent dehydratase step (from the pdu-operon of Lactobacillus reuteri), which catalyze the conversion of meso-2,3-butanediol to butanone. We have furthermore identified a NADH dependent secondary alcohol dehydrogenase (Sadh from Gordonia sp.) able to catalyze the subsequent conversion of butanone to 2-butanol. A final concentration of 4±0.2 mg/L 2-butanol and 2±0.1 mg/L of butanone was found. A key factor for the production of 2-butanol was the availability of NADH, which was achieved by growing cells lacking the GPD1 and GPD2 isogenes under anaerobic conditions. PMID:25054226

  6. 2-Butanol and Butanone Production in Saccharomyces cerevisiae through Combination of a B12 Dependent Dehydratase and a Secondary Alcohol Dehydrogenase Using a TEV-Based Expression System

    PubMed Central

    Ghiaci, Payam; Norbeck, Joakim; Larsson, Christer

    2014-01-01

    2-Butanol and its chemical precursor butanone (methyl ethyl ketone – MEK) are chemicals with potential uses as biofuels and biocommodity chemicals. In order to produce 2-butanol, we have demonstrated the utility of using a TEV-protease based expression system to achieve equimolar expression of the individual subunits of the two protein complexes involved in the B12-dependent dehydratase step (from the pdu-operon of Lactobacillus reuterii), which catalyze the conversion of meso-2,3-butanediol to butanone. We have furthermore identified a NADH dependent secondary alcohol dehydrogenase (Sadh from Gordonia sp.) able to catalyze the subsequent conversion of butanone to 2-butanol. A final concentration of 4±0.2 mg/L 2-butanol and 2±0.1 mg/L of butanone was found. A key factor for the production of 2-butanol was the availability of NADH, which was achieved by growing cells lacking the GPD1 and GPD2 isogenes under anaerobic conditions. PMID:25054226

  7. Synthesis of cinnamyl alcohol from cinnamaldehyde with Bacillus stearothermophilus alcohol dehydrogenase as the isolated enzyme and in recombinant E. coli cells.

    PubMed

    Pennacchio, Angela; Rossi, Mosè; Raia, Carlo A

    2013-07-01

    The synthesis of the aroma chemical cinnamyl alcohol (CMO) by means of enzymatic reduction of cinnamaldehyde (CMA) was investigated using NADH-dependent alcohol dehydrogenase from Bacillus stearothermophilus both as an isolated enzyme, and in recombinant Escherichia coli whole cells. The influence of parameters such as reaction time and cofactor, substrate, co-substrate 2-propanol and biocatalyst concentrations on the bioreduction reaction was investigated and an efficient and sustainable one-phase system developed. The reduction of CMA (0.5 g/L, 3.8 mmol/L) by the isolated enzyme occurred in 3 h at 50 °C with 97% conversion, and yielded high purity CMO (≥98%) with a yield of 88% and a productivity of 50 g/genzyme. The reduction of 12.5 g/L (94 mmol/L) CMA by whole cells in 6 h, at 37 °C and no requirement of external cofactor occurred with 97% conversion, 82% yield of 98% pure alcohol and a productivity of 34 mg/gwet cell weight. The results demonstrate the microbial system as a practical and efficient method for larger-scale synthesis of CMO. PMID:23686507

  8. Genetics Home Reference: lactate dehydrogenase deficiency

    MedlinePlus

    ... dehydrogenase-B pieces (subunits) of the lactate dehydrogenase enzyme. This enzyme is found throughout the body and is important ... cells. There are five different forms of this enzyme, each made up of four protein subunits. Various ...

  9. The glutamate homeostasis hypothesis of addiction.

    PubMed

    Kalivas, Peter W

    2009-08-01

    Addiction is associated with neuroplasticity in the corticostriatal brain circuitry that is important for guiding adaptive behaviour. The hierarchy of corticostriatal information processing that normally permits the prefrontal cortex to regulate reinforcement-seeking behaviours is impaired by chronic drug use. A failure of the prefrontal cortex to control drug-seeking behaviours can be linked to an enduring imbalance between synaptic and non-synaptic glutamate, termed glutamate homeostasis. The imbalance in glutamate homeostasis engenders changes in neuroplasticity that impair communication between the prefrontal cortex and the nucleus accumbens. Some of these pathological changes are amenable to new glutamate- and neuroplasticity-based pharmacotherapies for treating addiction. PMID:19571793

  10. Nonfunctional tricarboxylic acid cycle and the mechanism of glutamate biosynthesis in Acetobacter suboxydans.

    PubMed

    Greenfield, S; Claus, G W

    1972-12-01

    Acetobacter suboxydans does not contain an active tricarboxylic acid cycle, yet two pathways have been suggested for glutamate synthesis from acetate catalyzed by cell extracts: a partial tricarboxylic acid cycle following an initial condensation of oxalacetate and acetyl coenzyme A. and the citramalate-mesaconate pathway following an initial condensation of pyruvate and acetyl coenzyme A. To determine which pathway functions in growing cells, acetate-1-(14)C was added to a culture growing in minimal medium. After growth had ceased, cells were recovered and fractionated. Radioactive glutamate was isolated from the cellular protein fraction, and the position of the radioactive label was determined. Decarboxylation of the C5 carbon removed 100% of the radioactivity found in the purified glutamate fraction. These experiments establish that growing cells synthesize glutamate via a partial tricarboxylic acid cycle. Aspartate isolated from these hydrolysates was not radioactive, thus providing further evidence for the lack of a complete tricarboxylic acid cycle. When cell extracts were analyzed, activity of all tricarboxylic acid cycle enzymes, except succinate dehydrogenase, was demonstrated. PMID:4640504

  11. Synaptic Glutamate Spillover Due to Impaired Glutamate Uptake Mediates Heroin Relapse

    PubMed Central

    Scofield, Michael D.; Boger, Heather; Hensley, Megan; Kalivas, Peter W.

    2014-01-01

    Reducing the enduring vulnerability to relapse is a therapeutic goal in treating drug addiction. Studies with animal models of drug addiction show a marked increase in extrasynaptic glutamate in the core subcompartment of the nucleus accumbens (NAcore) during reinstated drug seeking. However, the synaptic mechanisms linking drug-induced changes in extrasynaptic glutamate to relapse are poorly understood. Here, we discovered impaired glutamate elimination in rats extinguished from heroin self-administration that leads to spillover of synaptically released glutamate into the nonsynaptic extracellular space in NAcore and investigated whether restoration of glutamate transport prevented reinstated heroin seeking. Through multiple functional assays of glutamate uptake and analyzing NMDA receptor-mediated currents, we show that heroin self-administration produced long-lasting downregulation of glutamate uptake and surface expression of the transporter GLT-1. This downregulation was associated with spillover of synaptic glutamate to extrasynaptic NMDA receptors within the NAcore. Ceftriaxone restored glutamate uptake and prevented synaptic glutamate spillover and cue-induced heroin seeking. Ceftriaxone-induced inhibition of reinstated heroin seeking was blocked by morpholino-antisense targeting GLT-1 synthesis. These data reveal that the synaptic glutamate spillover in the NAcore results from reduced glutamate transport and is a critical pathophysiological mechanism underling reinstated drug seeking in rats extinguished from heroin self-administration. PMID:24741055

  12. Regional brain glutamate transport in rats at normal and raised concentrations of circulating glutamate.

    PubMed

    Hawkins, R A; DeJoseph, M R; Hawkins, P A

    1995-08-01

    The permeability of the blood-brain barrier to glutamate was measured by quantitative autoradiography in brains of control rats (average plasma glutamate concentration of 95 microns) and rats infused with glutamate (average plasma glutamate concentration of 837 microns). Measurements of glutamate permeability were initiated by the injection of [14C]glutamate and stopped at 1 min to avoid the accumulation of [14C]glutamate metabolites. Glutamate entered the brain at a slow rate, with an average permeability-surface area product of 7 microliters.min-g-1, except in those areas known to have fenestrated capillaries. Glutamate accumulated in the choroid plexus of ventricles, but did not seem to enter the cerebrospinal fluid in detectable amounts regardless of the circulating concentration. Glutamate accumulated in circumventricular organs, such as the median eminence, where the radioactivity was localized without detectable spread. Infusion of glutamate to create high plasma concentrations did not result in greater spread of [14C]glutamate beyond the immediate vicinity of the circumventricular organs. PMID:7648616

  13. Glutamate: Tastant and Neuromodulator in Taste Buds.

    PubMed

    Vandenbeuch, Aurelie; Kinnamon, Sue C

    2016-07-01

    In taste buds, glutamate plays a double role as a gustatory stimulus and neuromodulator. The detection of glutamate as a tastant involves several G protein-coupled receptors, including the heterodimer taste receptor type 1, member 1 and 3 as well as metabotropic glutamate receptors (mGluR1 and mGluR4). Both receptor types participate in the detection of glutamate as shown with knockout animals and selective antagonists. At the basal part of taste buds, ionotropic glutamate receptors [N-methyl-d-aspartate (NMDA) and non-NMDA] are expressed and participate in the modulation of the taste signal before its transmission to the brain. Evidence suggests that glutamate has an efferent function on taste cells and modulates the release of other neurotransmitters such as serotonin and ATP. This short article reviews the recent developments in the field with regard to glutamate receptors involved in both functions as well as the influence of glutamate on the taste signal. PMID:27422519

  14. Glutamate Transporter-Mediated Glutamate Secretion in the Mammalian Pineal Gland

    PubMed Central

    Kim, Mean-Hwan; Uehara, Shunsuke; Muroyama, Akiko; Hille, Bertil; Moriyama, Yoshinori; Koh, Duk-Su

    2008-01-01

    Glutamate transporters are expressed throughout the central nervous system where their major role is to clear released glutamate from presynaptic terminals. Here we report a novel function of the transporter in rat pinealocytes. This electrogenic transporter conducted inward current in response to L-glutamate and L- or D-aspartate and depolarized the membrane in patch clamp experiments. Ca2+ imaging demonstrated that the transporter-mediated depolarization induced a significant Ca2+ influx through voltage-gated Ca2+ channels. The Ca2+ rise finally evoked glutamate exocytosis as detected by carbon-fiber amperometry and by high-performance liquid chromatography. In pineal slices with densely packed pinealocytes, glutamate released from the cells effectively activated glutamate transporters in neighboring cells. The Ca2+ signal generated by KCl depolarization or acetylcholine propagated through several cell layers by virtue of the regenerative ‘glutamate-induced glutamate release’. Therefore we suggest that glutamate transporters mediate synchronized elevation of L-glutamate and thereby efficiently down-regulate melatonin secretion via previously identified inhibitory metabotropic glutamate receptors in the pineal gland. PMID:18945893

  15. Glutamate Racemase Mutants of Bacillus anthracis

    PubMed Central

    Oh, So-Young; Richter, Stefan G.; Missiakas, Dominique M.

    2015-01-01

    ABSTRACT d-Glutamate is an essential component of bacterial peptidoglycan and a building block of the poly-γ-d-glutamic acid (PDGA) capsule of Bacillus anthracis, the causative agent of anthrax. Earlier work suggested that two glutamate racemases, encoded by racE1 and racE2, are each essential for growth of B. anthracis, supplying d-glutamic acid for the synthesis of peptidoglycan and PDGA capsule. Earlier work could not explain, however, why two enzymes that catalyze the same reaction may be needed for bacterial growth. Here, we report that deletion of racE1 or racE2 did not prevent growth of B. anthracis Sterne (pXO1+ pXO2−), the noncapsulating vaccine strain, or of B. anthracis Ames (pXO1+ pXO2+), a fully virulent, capsulating isolate. While mutants with deletions in racE1 and racE2 were not viable, racE2 deletion delayed vegetative growth of B. anthracis following spore germination and caused aberrant cell shapes, phenotypes that were partially restored by exogenous d-glutamate. Deletion of racE1 or racE2 from B. anthracis Ames did not affect the production or stereochemical composition of the PDGA capsule. A model is presented whereby B. anthracis, similar to Bacillus subtilis, utilizes two functionally redundant racemase enzymes to synthesize d-glutamic acid for peptidoglycan synthesis. IMPORTANCE Glutamate racemases, enzymes that convert l-glutamate to d-glutamate, are targeted for antibiotic development. Glutamate racemase inhibitors may be useful for the treatment of bacterial infections such as anthrax, where the causative agent, B. anthracis, requires d-glutamate for the synthesis of peptidoglycan and poly-γ-d-glutamic acid (PDGA) capsule. Here we show that B. anthracis possesses two glutamate racemase genes that can be deleted without abolishing either bacterial growth or PDGA synthesis. These data indicate that drug candidates must inhibit both glutamate racemases, RacE1 and RacE2, in order to block B. anthracis growth and achieve therapeutic

  16. Acetylated Chitosan Oligosaccharides Act as Antagonists against Glutamate-Induced PC12 Cell Death via Bcl-2/Bax Signal Pathway

    PubMed Central

    Hao, Cui; Gao, Lixia; Zhang, Yiran; Wang, Wei; Yu, Guangli; Guan, Huashi; Zhang, Lijuan; Li, Chunxia

    2015-01-01

    Chitosan oligosaccharides (COSs), depolymerized products of chitosan composed of β-(1→4) d-glucosamine units, have broad range of biological activities such as antitumour, antifungal, and antioxidant activities. In this study, peracetylated chitosan oligosaccharides (PACOs) and N-acetylated chitosan oligosaccharides (NACOs) were prepared from the COSs by chemcal modification. The structures of these monomers were identified using NMR and ESI-MS spectra. Their antagonist effects against glutamate-induced PC12 cell death were investigated. The results showed that pretreatment of PC12 cells with the PACOs markedly inhibited glutamate-induced cell death in a concentration-dependent manner. The PACOs were better glutamate antagonists compared to the COSs and the NACOs, suggesting the peracetylation is essential for the neuroprotective effects of chitosan oligosaccharides. In addition, the PACOs pretreatment significantly reduced lactate dehydrogenase release and reactive oxygen species production. It also attenuated the loss of mitochondrial membrane potential. Further studies indicated that the PACOs inhibited glutamate-induced cell death by preventing apoptosis through depressing the elevation of Bax/Bcl-2 ratio and caspase-3 activation. These results suggest that PACOs might be promising antagonists against glutamate-induced neural cell death. PMID:25775423

  17. Glutamate-gated Chloride Channels*

    PubMed Central

    Wolstenholme, Adrian J.

    2012-01-01

    Glutamate-gated chloride channels (GluCls) are found only in protostome invertebrate phyla but are closely related to mammalian glycine receptors. They have a number of roles in these animals, controlling locomotion and feeding and mediating sensory inputs into behavior. In nematodes and arthropods, they are targeted by the macrocyclic lactone family of anthelmintics and pesticides, making the GluCls of considerable medical and economic importance. Recently, the three-dimensional structure of a GluCl was solved, the first for any eukaryotic ligand-gated anion channel, revealing a macrocyclic lactone-binding site between the channel domains of adjacent subunits. This minireview will highlight some unique features of the GluCls and illustrate their contribution to our knowledge of the entire Cys loop ligand-gated ion channel superfamily. PMID:23038250

  18. Evaluation of the VIDAS glutamate dehydrogenase assay for the detection of Clostridium difficile.

    PubMed

    Shin, Bo-Moon; Lee, Eun Joo; Moon, Jung Wha; Lee, Seon Yeong

    2016-08-01

    We evaluated the performance of the VIDAS GDH assay for the detection of Clostridium difficile. In total, 350 fecal specimens collected from patients clinically suspected of having CDI were analyzed by C. difficile culture and enzyme-linked fluorescent immunoassay (VIDAS GDH); the results were compared with those of toxigenic C. difficile culture (TC), PCR (Xpert C. difficile assay), and toxin AB EIA (VIDAS CDAB). The numbers of culture-positive and culture-negative samples were 108 and 242, respectively. The concordance between the GDH assay and C. difficile culture was 90.3%. With PCR, 12 more samples were found to be positive in GDH-positive/C. difficile culture-negative specimens. Thus, the concordance between GDH assay and C. difficile culture/PCR was 93.7%. The sensitivity, specificity, positive predictive value, and negative predictive value of the VIDAS GDH assay were 97.2%, 87.2%, 77.2%, and 98.6%, respectively, based on the C. difficile culture, and 97.5%, 91.7%, 86.0%, and 98.6%, respectively, based on C. difficile culture/PCR. Positivity rates of the GDH assay were partially associated with those of semi-quantitative C. difficile cultures, which were maximized in grade 3 (>100 colony-forming unit [CFU]) compared with grade 1 (<10 CFU). We evaluated the two-step or three-step algorithm using GDH assay as a first step. No toxin EIA-positive case was found among GDH-negative samples, and 60.8% (48/79) were TC- and/or PCR-positive among the GDH-positive/toxin EIA-negative samples. Thus, approximately 25% of the 350 samples required a confirmatory test (TC or PCR) in the GDH-toxin EIA algorithm, whereas only 2.3% of the total samples in GDH-PCR algorithm was discrepant and required another confirmatory test like TC. PMID:27282799

  19. Purification and Characterization of the Bifunctional Enzyme Lysine-Ketoglutarate Reductase-Saccharopine Dehydrogenase from Maize.

    PubMed Central

    Goncalves-Butruille, M.; Szajner, P.; Torigoi, E.; Leite, A.; Arruda, P.

    1996-01-01

    The first enzyme of the lysine degradation pathway in maize (Zea mays L.), lysine-ketoglutarate reductase, condenses lysine and [alpha]-ketoglutarate into saccharopine using NADPH as a cofactor, whereas the second, saccharopine dehydrogenase, converts saccharopine to [alpha]-aminoadipic-[delta]-semialdehyde and glutamic acid using NAD+ or NADP+ as a cofactor. The reductase and dehydrogenase activities are optimal at pH 7.0 and 9.0, respectively. Both enzyme activities, co-purified on diethylaminoethyl-cellulose and gel filtration columns, were detected on nondenaturing polyacrylamide gels as single bands with identical electrophoretic mobilities and share tissue specificity for the endosperm. The highly purified preparation containing the reductase and dehydrogenase activities showed a single polypeptide band of 125 kD on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The native form of the enzyme is a dimer of 260 kD. Limited proteolysis with elastase indicated that lysine-ketoglutarate reductase and saccharopine dehydrogenase from maize endosperm are located in two functionally independent domains of a bifunctional polypeptide. PMID:12226216

  20. Replication of the Shrimp Virus WSSV Depends on Glutamate-Driven Anaplerosis

    PubMed Central

    Li, Chun-Yuan; Wang, Yi-Jan; Huang, Shiao-Wei; Cheng, Cheng-Shun; Wang, Han-Ching

    2016-01-01

    Infection with the white spot syndrome virus (WSSV) induces a metabolic shift in shrimp that resembles the “Warburg effect” in mammalian cells. This effect is triggered via activation of the PI3K-Akt-mTOR pathway, and it is usually accompanied by the activation of other metabolic pathways that provide energy and direct the flow of carbon and nitrogen. Here we show that unlike the glutamine metabolism (glutaminolysis) seen in most cancer cells to double deaminate glutamine to produce glutamate and the TCA cycle intermediate α-ketoglutarate (α-KG), at the WSSV genome replication stage (12 hpi), although glutaminase (GLS) expression was upregulated, only glutamate was taken up by the hemocytes of WSSV-infected shrimp. At the same time, we observed an increase in the activity of the two enzymes that convert glutamate to α-KG, glutamate dehydrogenase (GDH) and aspartate aminotransferase (ASAT). α-ketoglutarate concentration was also increased. A series of inhibition experiments suggested that the up-regulation of GDH is regulated by mTORC2, and that the PI3K-mTORC1 pathway is not involved. Suppression of GDH and ASAT by dsRNA silencing showed that both of these enzymes are important for WSSV replication. In GDH-silenced shrimp, direct replenishment of α-KG rescued both ATP production and WSSV replication. From these results, we propose a model of glutamate-driven anaplerosis that fuels the TCA cycle via α-KG and ultimately supports WSSV replication. PMID:26751681

  1. Different pools of glutamate receptors mediate sensitivity to ambient glutamate in the cochlear nucleus

    PubMed Central

    Yang, Yang

    2015-01-01

    Ambient glutamate plays an important role in pathological conditions, such as stroke, but its role during normal activity is not clear. In addition, it is not clear how ambient glutamate acts on glutamate receptors with varying affinities or subcellular localizations. To address this, we studied “endbulb of Held” synapses, which are formed by auditory nerve fibers onto bushy cells (BCs) in the anteroventral cochlear nucleus. When ambient glutamate was increased by applying the glutamate reuptake inhibitor TFB-TBOA, BCs depolarized as a result of activation of N-methyl-d-aspartate receptors (NMDARs) and group I metabotropic glutamate receptors (mGluRs). Application of antagonists against NMDARs (in 0 Mg2+) or mGluRs caused hyperpolarization, indicating that these receptors were bound by a tonic source of glutamate. AMPA receptors did not show these effects, consistent with their lower glutamate affinity. We also evaluated the subcellular localization of the receptors activated by ambient glutamate. The mGluRs were not activated by synaptic stimulation and thus appear to be exclusively extrasynaptic. By contrast, NMDARs in both synaptic and extrasynaptic compartments were activated by ambient glutamate, as shown using the use-dependent antagonist MK-801. Levels of ambient glutamate appeared to be regulated in a spike-independent manner, and glia likely play a major role. These low levels of ambient glutamate likely have functional consequences, as even low concentrations of TBOA caused significant increases in BC spiking following synaptic stimulation. These results indicate that normal resting potential appears to be poised in the region of maximal sensitivity to small changes in ambient glutamate. PMID:25855696

  2. Different pools of glutamate receptors mediate sensitivity to ambient glutamate in the cochlear nucleus.

    PubMed

    Yang, Yang; Xu-Friedman, Matthew A

    2015-06-01

    Ambient glutamate plays an important role in pathological conditions, such as stroke, but its role during normal activity is not clear. In addition, it is not clear how ambient glutamate acts on glutamate receptors with varying affinities or subcellular localizations. To address this, we studied "endbulb of Held" synapses, which are formed by auditory nerve fibers onto bushy cells (BCs) in the anteroventral cochlear nucleus. When ambient glutamate was increased by applying the glutamate reuptake inhibitor TFB-TBOA, BCs depolarized as a result of activation of N-methyl-D-aspartate receptors (NMDARs) and group I metabotropic glutamate receptors (mGluRs). Application of antagonists against NMDARs (in 0 Mg(2+)) or mGluRs caused hyperpolarization, indicating that these receptors were bound by a tonic source of glutamate. AMPA receptors did not show these effects, consistent with their lower glutamate affinity. We also evaluated the subcellular localization of the receptors activated by ambient glutamate. The mGluRs were not activated by synaptic stimulation and thus appear to be exclusively extrasynaptic. By contrast, NMDARs in both synaptic and extrasynaptic compartments were activated by ambient glutamate, as shown using the use-dependent antagonist MK-801. Levels of ambient glutamate appeared to be regulated in a spike-independent manner, and glia likely play a major role. These low levels of ambient glutamate likely have functional consequences, as even low concentrations of TBOA caused significant increases in BC spiking following synaptic stimulation. These results indicate that normal resting potential appears to be poised in the region of maximal sensitivity to small changes in ambient glutamate. PMID:25855696

  3. Formaldehyde dehydrogenase preparations from Methylococcus capsulatus (Bath) comprise methanol dehydrogenase and methylene tetrahydromethanopterin dehydrogenase.

    PubMed

    Adeosun, Ekundayo K; Smith, Thomas J; Hoberg, Anne-Mette; Velarde, Giles; Ford, Robert; Dalton, Howard

    2004-03-01

    In methylotrophic bacteria, formaldehyde is an important but potentially toxic metabolic intermediate that can be assimilated into biomass or oxidized to yield energy. Previously reported was the purification of an NAD(P)(+)-dependent formaldehyde dehydrogenase (FDH) from the obligate methane-oxidizing methylotroph Methylococcus capsulatus (Bath), presumably important in formaldehyde oxidation, which required a heat-stable factor (known as the modifin) for FDH activity. Here, the major protein component of this FDH preparation was shown by biophysical techniques to comprise subunits of 64 and 8 kDa in an alpha(2)beta(2) arrangement. N-terminal sequencing of the subunits of FDH, together with enzymological characterization, showed that the alpha(2)beta(2) tetramer was a quinoprotein methanol dehydrogenase of the type found in other methylotrophs. The FDH preparations were shown to contain a highly active NAD(P)(+)-dependent methylene tetrahydromethanopterin dehydrogenase that was the probable source of the NAD(P)(+)-dependent formaldehyde oxidation activity. These results support previous findings that methylotrophs possess multiple pathways for formaldehyde dissimilation. PMID:14993320

  4. Vesicular Glutamate Transport Promotes Dopamine Storage and Glutamate Corelease In Vivo

    PubMed Central

    Hnasko, Thomas S.; Chuhma, Nao; Zhang, Hui; Goh, Germaine Y.; Sulzer, David; Palmiter, Richard D.; Rayport, Stephen; Edwards, Robert H.

    2010-01-01

    SUMMARY Dopamine neurons in the ventral tegmental area (VTA) play an important role in the motivational systems underlying drug addiction, and recent work has suggested that they also release the excitatory neurotransmitter glutamate. To assess a physiological role for glutamate corelease, we disrupted the expression of vesicular glutamate transporter 2 selectively in dopamine neurons. The conditional knockout abolishes glutamate release from midbrain dopamine neurons in culture and severely reduces their excitatory synaptic output in mesoaccumbens slices. Baseline motor behavior is not affected, but stimulation of locomotor activity by cocaine is impaired, apparently through a selective reduction of dopamine stores in the projection of VTA neurons to ventral striatum. Glutamate co-entry promotes monoamine storage by increasing the pH gradient that drives vesicular monoamine transport. Remarkably, low concentrations of glutamate acidify synaptic vesicles more slowly but to a greater extent than equimolar Cl−, indicating a distinct, presynaptic mechanism to regulate quantal size. PMID:20223200

  5. Metabolic fate and function of dietary glutamate in the gut

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Glutamate is a major constituent of dietary protein and is also consumed in many prepared foods as an additive in the form of monosodium glutamate. Evidence from human and animal studies indicates that glutamate is a major oxidative fuel for the gut and that dietary glutamate is extensively metabol...

  6. Emerging aspects of dietary glutamate metabolism in the developing gut

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Glutamate is a major constituent of dietary protein and is also consumed in many prepared foods as a flavour additive in the form of monosodium glutamate (MSG). Evidence from human and animal studies indicates that glutamate is the major oxidative fuel for the gut and that dietary glutamate is exten...

  7. Higher Ammonium Transamination Capacity Can Alleviate Glutamate Inhibition on Winter Wheat (Triticum aestivum L.) Root Growth under High Ammonium Stress

    PubMed Central

    Liu, Yang; Tian, Zhongwei; Muhammad, Abid; Zhang, Yixuan; Jiang, Dong; Cao, Weixing; Dai, Tingbo

    2016-01-01

    Most of the studies about NH4+ stress mechanism simply address the effects of free NH4+, failing to recognize the changed nitrogen assimilation products. The objective of this study was to elucidate the effects of glutamate on root growth under high ammonium (NH4+) conditions in winter wheat (Triticum aestivum L.). Hydroponic experiments were conducted using two wheat cultivars, AK58 (NH4+-sensitive) and Xumai25 (NH4+-tolerant) with either 5 mM NH4+ nitrogen (AN) as stress treatment or 5 mM nitrate (NO3-) nitrogen as control. To evaluate the effects of NH4+-assimilation products on plant growth, 1 μM L-methionine sulfoximine (MSO) (an inhibitor of glutamine synthetase (GS)) and 1 mM glutamates (a primary N assimilation product) were added to the solutions, respectively. The AN significantly reduced plant biomass, total root length, surface area and root volume in both cultivars, but less effect was observed in Xumai25. The inhibition effects were alleviated by the application of MSO but strengthened by the application of glutamate. The AN increased the activities of GS, glutamate dehydrogenase (GDH) in both cultivars, resulting in higher glutamate contents. However, its contents were decreased by the application of MSO. Compared to AK58, Xumai25 showed lower glutamate contents due to its higher activities of glutamic-oxaloacetic transaminase (GOT) and glutamic-pyruvic transaminase (GPT). With the indole-3-acetic acid (IAA) contents decreasing in roots, the ratio of shoot to root in IAA was increased, and further increased by the application of glutamate, and reduced by the application of MSO, but the ratio was lower in Xumai25. Meanwhile, the total soluble sugar contents and its root to shoot ratio also showed similar trends. These results indicate that the NH4+-tolerant cultivar has a greater transamination ability to prevent glutamate over-accumulation to maintain higher IAA transport ability, and consequently promoted soluble sugar transport to roots, further

  8. Higher Ammonium Transamination Capacity Can Alleviate Glutamate Inhibition on Winter Wheat (Triticum aestivum L.) Root Growth under High Ammonium Stress.

    PubMed

    Wang, Feng; Gao, Jingwen; Liu, Yang; Tian, Zhongwei; Muhammad, Abid; Zhang, Yixuan; Jiang, Dong; Cao, Weixing; Dai, Tingbo

    2016-01-01

    Most of the studies about NH4+ stress mechanism simply address the effects of free NH4+, failing to recognize the changed nitrogen assimilation products. The objective of this study was to elucidate the effects of glutamate on root growth under high ammonium (NH4+) conditions in winter wheat (Triticum aestivum L.). Hydroponic experiments were conducted using two wheat cultivars, AK58 (NH4+-sensitive) and Xumai25 (NH4+-tolerant) with either 5 mM NH4+ nitrogen (AN) as stress treatment or 5 mM nitrate (NO3-) nitrogen as control. To evaluate the effects of NH4+-assimilation products on plant growth, 1 μM L-methionine sulfoximine (MSO) (an inhibitor of glutamine synthetase (GS)) and 1 mM glutamates (a primary N assimilation product) were added to the solutions, respectively. The AN significantly reduced plant biomass, total root length, surface area and root volume in both cultivars, but less effect was observed in Xumai25. The inhibition effects were alleviated by the application of MSO but strengthened by the application of glutamate. The AN increased the activities of GS, glutamate dehydrogenase (GDH) in both cultivars, resulting in higher glutamate contents. However, its contents were decreased by the application of MSO. Compared to AK58, Xumai25 showed lower glutamate contents due to its higher activities of glutamic-oxaloacetic transaminase (GOT) and glutamic-pyruvic transaminase (GPT). With the indole-3-acetic acid (IAA) contents decreasing in roots, the ratio of shoot to root in IAA was increased, and further increased by the application of glutamate, and reduced by the application of MSO, but the ratio was lower in Xumai25. Meanwhile, the total soluble sugar contents and its root to shoot ratio also showed similar trends. These results indicate that the NH4+-tolerant cultivar has a greater transamination ability to prevent glutamate over-accumulation to maintain higher IAA transport ability, and consequently promoted soluble sugar transport to roots, further

  9. Glutamate Receptor Stimulation Up-Regulates Glutamate Uptake in Human Müller Glia Cells.

    PubMed

    López-Colomé, Ana María; López, Edith; Mendez-Flores, Orquidia G; Ortega, Arturo

    2016-07-01

    Glutamate, the main excitatory amino acid in the vertebrate retina, is a well know activator of numerous signal transduction pathways, and has been critically involved in long-term synaptic changes acting through ionotropic and metabotropic glutamate receptors. However, recent findings underlining the importance of intensity and duration of glutamate stimuli for specific neuronal responses, including excitotoxicity, suggest a crucial role for Na(+)-dependent glutamate transporters, responsible for the removal of this neurotransmitter from the synaptic cleft, in the regulation of glutamate-induced signaling. Transporter proteins are expressed in neurons and glia cells, albeit most of glutamate uptake occurs in the glial compartment. Within the retina, Müller glia cells are in close proximity to glutamatergic synapses and participate in the recycling of glutamate through the glutamate/glutamine shuttle. In this context, we decided to investigate a plausible role of glutamate as a regulatory signal for its own transport in human retinal glia cells. To this end, we determined [(3)H]-D-aspartate uptake in cultures of spontaneously immortalized human Müller cells (MIO-M1) exposed to distinct glutamatergic ligands. A time and dose-dependent increase in the transporter activity was detected. This effect was dependent on the activation of the N-methyl D-aspartate subtype of glutamate receptors, due to a dual effect: an increase in affinity and an augmented expression of the transporter at the plasma membrane, as established via biotinylation experiments. Furthermore, a NMDA-dependent association of glutamate transporters with the cystoskeletal proteins ezrin and glial fibrillary acidic protein was also found. These results add a novel mediator of the glutamate transporter modulation and further strengthen the notion of the critical involvement of glia cells in synaptic function. PMID:27017513

  10. Cellobiose dehydrogenase in cellulose degradation

    SciTech Connect

    Eriksson, L.; Igarashi, Kiyohiko; Samejima, Masahiro

    1996-10-01

    Cellobiose dehydrogenase is produced by a variety of fungi. Although it was already discovered during the 70`s, it`s role in cellulose and lignin degradation is yet ambiguous. The enzyme contains both heme and FAD as prosthetic groups, and seems to have a domain specifically designed to bind the enzyme to cellulose. It`s affinity to amorphous cellulose is higher than to crystalline cellulose. We will report on the binding behavior of the enzyme, its usefulness in elucidation of cellulose structures and also, possibilities for applications such as its use in measuring individual and synergistic mechanisms for cellulose degradation by endo- and exo-glucanases.

  11. Changes in metabolic proteins in ex vivo rat retina during glutamate-induced neural progenitor cell induction.

    PubMed

    Tokuda, Kazuhiro; Kuramitsu, Yasuhiro; Baron, Byron; Kitagawa, Takao; Tokuda, Nobuko; Kobayashi, Masaaki; Kimura, Kazuhiro; Sonoda, Koh-Hei; Nakamura, Kazuyuki

    2016-08-01

    Understanding how energy metabolism and related proteins influence neural progenitor cells in adult tissues is critical for developing new strategies in clinical tissue regeneration therapy. We have recently reported that a subtoxic concentration of glutamate-induced neural progenitor cells in the mature ex vivo rat retina. We herein explore changes in the metabolic pathways during the process. We firstly observed an increase in lactate and lactate dehydrogenase concentration in the glutamate-treated retina. We then investigated the levels of glycolytic enzymes and confirmed significant upregulation of pyruvate kinase M type (PKM), especially PKM2, enolase, phosphoglycerate mutase 1 (PGAM1), and inosine-5'-monophosphate dehydrogenase (IMPDH1) in the glutamate-treated retina compared to the untreated retina. An analysis of the subcellular localization of PKM2 revealed nuclear translocation in the treated retina, which has been reported to regulate cell cycle proliferation and glycolytic enzymes. Our findings indicate that the mature rat retina undergoes an increase in aerobic glycolysis. PKM2, both in the cytoplasm and in the nucleus, may thus play an important role during neural progenitor cell induction, as it does in other proliferating cells. PMID:27421851

  12. Glutamate Receptor Dynamics in Dendritic Microdomains

    PubMed Central

    Newpher, Thomas M.; Ehlers, Michael D.

    2008-01-01

    Among diverse factors regulating excitatory synaptic transmission, the abundance of postsynaptic glutamate receptors figures prominently in molecular memory and learning-related synaptic plasticity. To allow for both long-term maintenance of synaptic transmission and acute changes in synaptic strength, the relative rates of glutamate receptor insertion and removal must be tightly regulated. Interactions with scaffolding proteins control the targeting and signaling properties of glutamate receptors within the postsynaptic membrane. In addition, extrasynaptic receptor populations control the equilibrium of receptor exchange at synapses and activate distinct signaling pathways involved in plasticity. Here, we review recent findings that have shaped our current understanding of receptor mobility between synaptic and extrasynaptic compartments at glutamatergic synapses, focusing on AMPA and NMDA receptors. We also examine the cooperative relationship between intracellular trafficking and surface diffusion of glutamate receptors that underlies the expression of learning-related synaptic plasticity. PMID:18498731

  13. 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

  14. [Glutamate transporter dysfunction and major mental illnesses].

    PubMed

    Tanaka, Kohichi

    2016-01-01

    Glutamate is the main excitatory neurotransmitter in the central nervous system and plays an important role in most aspects of normal brain function. In spite of its importance as a neurotransmitter, excess glutamate is toxic to neurons. Clearance of extracellular glutamate is critical for maintenance of low extracellular glutamate concentration, and occurs in large part through the activity of GLT1 (EAAT2) and GLAST (EAAT1), which are primarily expressed by astrocytes. Rare variants and down-regulation of GLT1 and GLAST, in psychiatric disorders have been reported. In this review, we demonstrate that various kinds of GLT1 and/or GLAST knockout mice replicate many aspects of the behavioral abnormalities seen in major mental illnesses including schizophrenia, depression, obsessive -compulsive disorders, autism, epilepsy and addiction. PMID:26793898

  15. DNA nanopore translocation in glutamate solutions

    NASA Astrophysics Data System (ADS)

    Plesa, C.; van Loo, N.; Dekker, C.

    2015-08-01

    Nanopore experiments have traditionally been carried out with chloride-based solutions. Here we introduce silver/silver-glutamate-based electrochemistry as an alternative, and study the viscosity, conductivity, and nanopore translocation characteristics of potassium-, sodium-, and lithium-glutamate solutions. We show that it has a linear response at typical voltages and can be used to detect DNA translocations through a nanopore. The glutamate anion also acts as a redox-capable thickening agent, with high-viscosity solutions capable of slowing down the DNA translocation process by up to 11 times, with a corresponding 7 time reduction in signal. These results demonstrate that glutamate can replace chloride as the primary anion in nanopore resistive pulse sensing.

  16. Δ1-Pyrroline-5-Carboxylate/Glutamate Biogenesis Is Required for Fungal Virulence and Sporulation

    PubMed Central

    Yao, Ziting; Zou, Chengwu; Zhou, Hui; Wang, Jinzi; Lu, Lidan; Li, Yang; Chen, Baoshan

    2013-01-01

    Proline dehydrogenase (Prodh) and Δ1-pyrroline-5-carboxylate dehydrogenase (P5Cdh) are two key enzymes in the cellular biogenesis of glutamate. Recombinant Prodh and P5Cdh proteins of the chestnut blight fungus Cryphonectria parasitica were investigated and showed activity in in vitro assays. Additionally, the C. parasitica Prodh and P5Cdh genes were able to complement the Saccharomyces cerevisiae put1 and put2 null mutants, respectively, to allow these proline auxotrophic yeast mutants to grow on media with proline as the sole source of nitrogen. Deletion of the Prodh gene in C. parasitica resulted in hypovirulence and a lower level of sporulation, whereas deletion of P5Cdh resulted in hypovirulence though no effect on sporulation; both Δprodh and Δp5cdh mutants were unable to grow on minimal medium with proline as the sole nitrogen source. In a wild-type strain, the intracellular level of proline and the activity of Prodh and P5Cdh increased after supplementation of exogenous proline, though the intracellular Δ1-pyrroline-5-carboxylate (P5C) content remained unchanged. Prodh and P5Cdh were both transcriptionally down-regulated in cells infected with hypovirus. The disruption of other genes with products involved in the conversion of arginine to ornithine, ornithine and glutamate to P5C, and P5C to proline in the cytosol did not appear to affect virulence; however, asexual sporulation was reduced in the Δpro1 and Δpro2 mutants. Taken together, our results showed that Prodh, P5Cdh and related mitochondrial functions are essential for virulence and that proline/glutamate pathway components may represent down-stream targets of hypovirus regulation in C. parasitica. PMID:24039956

  17. [Glutamate neurotransmission, stress and hormone secretion].

    PubMed

    Jezová, D; Juránková, E; Vigas, M

    1995-11-01

    Glutamate neurotransmission has been investigated in relation to several physiological processes (learning, memory) as well as to neurodegenerative and other disorders. Little attention has been paid to its involvement in neuroendocrine response during stress. Penetration of excitatory amino acids from blood to the brain is limited by the blood-brain barrier. As a consequence, several toxic effects but also bioavailability for therapeutic purposes are reduced. A free access to circulating glutamate is possible only in brain structures lacking the blood-brain barrier or under conditions of its increased permeability. Excitatory amino acids were shown to stimulate the pituitary hormone release, though the mechanism of their action is still not fully understood. Stress exposure in experimental animals induced specific changes in mRNA levels coding the glutamate receptor subunits in the hippocampus and hypothalamus. The results obtained with the use of glutamate receptor antagonists indicate that a number of specific receptor subtypes contribute to the stimulation of ACTH release during stress. The authors provided also data on the role of NMDA receptors in the control of catecholamine release, particularly in stress-induced secretion of epinephrine. These results were the first piece of evidence on the involvement of endogenous excitatory amino acids in neuroendocrine activation during stress. Neurotoxic effects of glutamate in animals are well described, especially after its administration in the neonatal period. In men, glutamate toxicity and its use as a food additive are a continuous subject of discussions. The authors found an increase in plasma cortisol and norepinephrine, but not epinephrine and prolactin, in response to the administration of a high dose of glutamate. It cannot be excluded that these effects might be induced even by lower doses in situations with increased vulnerability to glutamate action (age, individual variability). (Tab. 1, Fig. 6, Ref. 44

  18. Glutamic Acid Decarboxylation in Chlorella12

    PubMed Central

    Lane, T. R.; Stiller, Mary

    1970-01-01

    The decarboxylation of endogenous free glutamic acid by Chlorella pyrenoidosa, Marburg strain, was induced by a variety of metabolic poisons, by anaerobic conditions, and by freezing and thawing the cells. The rate of decarboxylation was proportional to the concentration of inhibitor present. Possible mechanisms which relate the effects of the various conditions on glutamate decarboxylation and oxygen consumption by Chlorella are discussed. Images PMID:5429350

  19. Neuroprotective effects of atorvastatin against glutamate-induced excitotoxicity in primary cortical neurones.

    PubMed

    Bösel, Julian; Gandor, Florin; Harms, Christoph; Synowitz, Michael; Harms, Ulrike; Djoufack, Pierre Chryso; Megow, Dirk; Dirnagl, Ulrich; Hörtnagl, Heide; Fink, Klaus B; Endres, Matthias

    2005-03-01

    Statins [3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors] exert cholesterol-independent pleiotropic effects that include anti-thrombotic, anti-inflammatory, and anti-oxidative properties. Here, we examined direct protective effects of atorvastatin on neurones in different cell damage models in vitro. Primary cortical neurones were pre-treated with atorvastatin and then exposed to (i) glutamate, (ii) oxygen-glucose deprivation or (iii) several apoptosis-inducing compounds. Atorvastatin significantly protected from glutamate-induced excitotoxicity as evidenced by propidium iodide staining, nuclear morphology, release of lactate dehydrogenase, and mitochondrial tetrazolium metabolism, but not from oxygen-glucose deprivation or apoptotic cell death. This anti-excitototoxic effect was evident with 2-4 days pre-treatment but not with daily administration or shorter-term pre-treatment. The protective properties occurred independently of 3-hydroxy-3-methylglutaryl-CoA reductase inhibition because co-treatment with mevalonate or other isoprenoids did not reverse or attenuate neuroprotection. Atorvastatin attenuated the glutamate-induced increase of intracellular calcium, which was associated with a modulation of NMDA receptor function. Taken together, atorvastatin exerts specific anti-excitotoxic effects independent of 3-hydroxy-3-methylglutaryl-CoA reductase inhibition, which has potential therapeutic implications. PMID:15748157

  20. SOD2 Mediates Amifostine-Induced Protection against Glutamate in PC12 Cells

    PubMed Central

    Jia, Ji; Zhang, Lei; Shi, Xiaolei; Wu, Mingchun; Zhou, Xiang; Liu, Xiaonan; Huo, Tingting

    2016-01-01

    Background. Cytoprotectant amifostine attenuates radiation-induced oxidative injury by increasing intracellular manganese superoxide dismutase (SOD2) in peripheral tissue. However, whether amifostine could protect neuronal cells against oxidative injury has not been reported. The purpose of this study is to explore the protection of amifostine in PC12 cells. Methods. PC12 cells exposed to glutamate were used to mimic neuronal oxidative injury. SOD assay kit was taken to evaluate intracellular Cu/Zn SOD (SOD1) and SOD2 activities; western blot analysis and immunofluorescence staining were performed to investigate SOD2 protein expression; MTT, lactate dehydrogenase (LDH), release and cell morphology were used to evaluate cell injury degree, and apoptotic rate and cleaved caspase-3 expression were taken to assess apoptosis; mitochondrial superoxide production, intracellular reactive oxygen species (ROS), and glutathione (GSH) and catalase (CAT) levels were evaluated by reagent kits. Results. Amifostine increased SOD2 activity and expression, decreased cell injury and apoptosis, reduced mitochondrial superoxide production and intracellular ROS generation, and restored intracellular GSH and CAT levels in PC12 cells exposed to glutamate. SOD2-siRNA, however, significantly reversed the amifostine-induced cytoprotective and antioxidative actions. Conclusion. SOD2 mediates amifostine-induced protection in PC12 cells exposed to glutamate. PMID:26770652

  1. Yeast nitrogen catabolite repression is sustained by signals distinct from glutamine and glutamate reservoirs.

    PubMed

    Fayyad-Kazan, Mohammad; Feller, A; Bodo, E; Boeckstaens, M; Marini, A M; Dubois, E; Georis, I

    2016-01-01

    Nitrogen catabolite repression (NCR) is a wide transcriptional regulation program enabling baker's yeast to downregulate genes involved in the utilization of poor nitrogen sources when preferred ones are available. Nowadays, glutamine and glutamate, the major nitrogen donors for biosyntheses, are assumed to be key metabolic signals regulating NCR. NCR is controlled by the conserved TORC1 complex, which integrates nitrogen signals among others to regulate cell growth. However, accumulating evidence indicate that the TORC1-mediated control of NCR is only partial, arguing for the existence of supplementary regulatory processes to be discovered. In this work, we developed a genetic screen to search for new players involved in NCR signaling. Our data reveal that the NADP-glutamate dehydrogenase activity of Gdh1 negatively regulates NCR-sensitive gene transcription. By determining the total, cytoplasmic and vacuolar pools of amino acids, we show that there is no positive correlation between glutamine/glutamate reservoirs and the extent of NCR. While our data indicate that glutamine could serve as initial trigger of NCR, they show that it is not a sufficient signal to sustain repression and point to the existence of yet unknown signals. Providing additional evidence uncoupling TORC1 activity and NCR, our work revisits the dogmas underlying NCR regulation. PMID:26419331

  2. Ionotropic Glutamate Receptors & CNS Disorders

    PubMed Central

    Bowie, Derek

    2008-01-01

    Disorders of the central nervous system (CNS) are complex disease states that represent a major challenge for modern medicine. Although etiology is often unknown, it is established that multiple factors such as defects in genetics and/or epigenetics, the environment as well as imbalance in neurotransmitter receptor systems are all at play in determining an individual’s susceptibility to disease. Gene therapy is currently not available and therefore, most conditions are treated with pharmacological agents that modify neurotransmitter receptor signaling. Here, I provide a review of ionotropic glutamate receptors (iGluRs) and the roles they fulfill in numerous CNS disorders. Specifically, I argue that our understanding of iGluRs has reached a critical turning point to permit, for the first time, a comprehensive re-evaluation of their role in the cause of disease. I illustrate this by highlighting how defects in AMPA receptor trafficking are important to Fragile X mental retardation and ectopic expression of kainate (KA) receptor synapses contributes to the pathology of temporal lobe epilepsy. Finally, I discuss how parallel advances in studies of other neurotransmitter systems may allow pharmacologists to work towards a cure for many CNS disorders rather than developing drugs to treat their symptoms. PMID:18537642

  3. Therapeutic Potential of Metabotropic Glutamate Receptor Modulators

    PubMed Central

    Hovelsø, N; Sotty, F; Montezinho, L.P; Pinheiro, P.S; Herrik, K.F; Mørk, A

    2012-01-01

    Glutamate is the main excitatory neurotransmitter in the central nervous system (CNS) and is a major player in complex brain functions. Glutamatergic transmission is primarily mediated by ionotropic glutamate receptors, which include NMDA, AMPA and kainate receptors. However, glutamate exerts modulatory actions through a family of metabotropic G-protein-coupled glutamate receptors (mGluRs). Dysfunctions of glutamatergic neurotransmission have been implicated in the etiology of several diseases. Therefore, pharmacological modulation of ionotropic glutamate receptors has been widely investigated as a potential therapeutic strategy for the treatment of several disorders associated with glutamatergic dysfunction. However, blockade of ionotropic glutamate receptors might be accompanied by severe side effects due to their vital role in many important physiological functions. A different strategy aimed at pharmacologically interfering with mGluR function has recently gained interest. Many subtype selective agonists and antagonists have been identified and widely used in preclinical studies as an attempt to elucidate the role of specific mGluRs subtypes in glutamatergic transmission. These studies have allowed linkage between specific subtypes and various physiological functions and more importantly to pathological states. This article reviews the currently available knowledge regarding the therapeutic potential of targeting mGluRs in the treatment of several CNS disorders, including schizophrenia, addiction, major depressive disorder and anxiety, Fragile X Syndrome, Parkinson’s disease, Alzheimer’s disease and pain. PMID:22942876

  4. Betaine aldehyde dehydrogenase in sorghum.

    PubMed Central

    Wood, A J; Saneoka, H; Rhodes, D; Joly, R J; Goldsbrough, P B

    1996-01-01

    The ability to synthesize and accumulate glycine betaine is wide-spread among angiosperms and is thought to contribute to salt and drought tolerance. In plants glycine betaine is synthesized by the two-step oxidation of choline via the intermediate betaine aldehyde, catalyzed by choline monooxygenase and betaine aldehyde dehydrogenase (BADH). Two sorghum (Sorghum bicolor) cDNA clones, BADH1 and BADH15, putatively encoding betaine aldehyde dehydrogenase were isolated and characterized. BADH1 is a truncated cDNA of 1391 bp. BADH15 is a full-length cDNA clone, 1812 bp in length, predicted to encode a protein of 53.6 kD. The predicted amino acid sequences of BADH1 and BADH15 share significant homology with other plant BADHs. The effects of water deficit on BADH mRNA expression, leaf water relations, and glycine betaine accumulation were investigated in leaves of preflowering sorghum plants. BADH1 and BADH15 mRNA were both induced by water deficit and their expression coincided with the observed glycine betaine accumulation. During the course of 17 d, the leaf water potential in stressed sorghum plants reached -2.3 MPa. In response to water deficit, glycine betaine levels increased 26-fold and proline levels increased 108-fold. In severely stressed plants, proline accounted for > 60% of the total free amino acid pool. Accumulation of these compatible solutes significantly contributed to osmotic potential and allowed a maximal osmotic adjustment of 0.405 MPa. PMID:8934627

  5. STRUCTURE AND KINETICS OF MONOFUNCTIONAL PROLINE DEHYDROGENASE FROM THERMUS THERMOPHILUS

    PubMed Central

    White, Tommi A.; Krishnan, Navasona; Becker, Donald F.; Tanner, John J.

    2009-01-01

    Proline dehydrogenase (PRODH) and Δ1-pyrroline-5-carboxylate dehydrogenase (P5CDH) catalyze the two-step oxidation of proline to glutamate. They are distinct monofunctional enzymes in all eukaryotes and some bacteria, but are fused into bifunctional enzymes known as Proline utilization A (PutA) in other bacteria. Here we report the first structure and biochemical data for a monofunctional PRODH. The 2.0 Å resolution structure of Thermus thermophilus PRODH reveals a distorted (βα)8 barrel catalytic core domain and a hydrophobic α-helical domain located above the carboxyl terminal ends of the strands of the barrel. Although the catalytic core is similar to that of the PutA PRODH domain, the FAD conformation of T. thermophilus PRODH is remarkably different and likely reflects unique requirements for membrane association and communication with P5CDH. Also, the FAD of T. thermophilus PRODH is highly solvent exposed compared to PutA due to a 4-Å shift of helix 8. Structure-based sequence analysis of the PutA/PRODH family led us to identify 9 conserved motifs involved in cofactor and substrate recognition. Biochemical studies show that the midpoint potential of the FAD is −75 mV and the kinetic parameters for proline are Km=27 mM and kcat=13 s−1. 3,4-dehydro-L-proline was found to be an efficient substrate and L-tetrahydro-2-furoic acid is a competitive inhibitor (KI=1.0 mM). Finally, we demonstrate that T. thermophilus PRODH reacts with O2 producing superoxide. This is significant because superoxide production underlies the role of human PRODH in p53-mediated apoptosis, implying commonalities between eukaryotic and bacterial monofunctional PRODHs. PMID:17344208

  6. The Degradation of 14C-Glutamic Acid by L-Glutamic Acid Decarboxylase.

    ERIC Educational Resources Information Center

    Dougherty, Charles M; Dayan, Jean

    1982-01-01

    Describes procedures and semi-micro reaction apparatus (carbon dioxide trap) to demonstrate how a particular enzyme (L-Glutamic acid decarboxylase) may be used to determine the site or sites of labeling in its substrate (carbon-14 labeled glutamic acid). Includes calculations, solutions, and reagents used. (Author/SK)

  7. Glutamate receptor ligands as anxiolytics.

    PubMed

    Chojnacka-Wójcik, E; Kłodzinska, A; Pilc, A

    2001-08-01

    The glutamatergic system has received considerable attention over recent years as a potential target for anxiolytic drugs. In spite of the pronounced anxiolytic-like effects of competitive and non-competitive antagonists of NMDA receptors in animal models of anxiety, these substances can not be regarded as potential anxiolytic drugs, mainly due to their side-effect profiles (eg, ataxia, myorelaxation, impairment of learning and memory processes and psychotomimetic effects). Antagonists and partial agonists of the glycine, receptor inhibit function of the NMDA receptor complex and evoke in animals an anxiolytic-like response. Although data concerning anti-anxiety-like effects of glycine, receptor antagonists are not very promising, studies are underway to develop new, brain-penetrating agents devoid of side effects. Further developments are necessary to more fully elucidate the possible involvement of AMPA/kainate receptors in anxiety. The recent discovery of metabotropic glutamate receptors, which modulate the function of the glutamatergic system, offers new hope for discovery of a new generation of anxiolytics. MPEP, a highly selective, brain penetrable, noncompetitive mGlu5 receptor antagonist, evokes anxiolytic-like effects in several animal models of anxiety, remaining remarkably free of side effects. LY-354740, a selective brain-penetrable group II mGlu receptor agonist, evokes marked anxiolytic-like effects in animal models of anxiety. LY-354740 causes mild sedation in mice, does not disturb motor coordination and has no potential to cause dependence. Therefore mGlu receptor ligands may become the anxiolytics of the future, free from the side effects characteristic of benzodiazepines. PMID:11892923

  8. Single motoneuron succinate dehydrogenase activity.

    PubMed

    Chalmers, G R; Edgerton, V R

    1989-07-01

    We have developed a quantitative histochemical assay for measurement of succinate dehydrogenase (SDH) activity in single motoneurons. A computer image processing system was used to quantify the histochemical enzyme reaction product and to follow the time course of the reaction. The optimal concentration for each of the ingredients of the incubation medium for the SDH reaction was determined and the importance of using histochemical "blanks" in the determination of enzymatic activity was demonstrated. The enzymatic activity was linear with respect to reaction time and tissue thickness. The procedure described meets the criteria generally considered essential for establishment of a quantitative histochemical assay. The assay was then used to examine the SDH activity of cat and rat motoneurons. It was found that motoneurons with a small soma size had a wide range of SDH activity, whereas those with a large soma size were restricted to low SDH activity. PMID:2732457

  9. Glucose-6-Phosphate Dehydrogenase Deficiency.

    PubMed

    Luzzatto, Lucio; Nannelli, Caterina; Notaro, Rosario

    2016-04-01

    G6PD is a housekeeping gene expressed in all cells. Glucose-6-phosphate dehydrogenase (G6PD) is part of the pentose phosphate pathway, and its main physiologic role is to provide NADPH. G6PD deficiency, one of the commonest inherited enzyme abnormalities in humans, arises through one of many possible mutations, most of which reduce the stability of the enzyme and its level as red cells age. G6PD-deficient persons are mostly asymptomatic, but they can develop severe jaundice during the neonatal period and acute hemolytic anemia when they ingest fava beans or when they are exposed to certain infections or drugs. G6PD deficiency is a global health issue. PMID:27040960

  10. Data of multiple regressions analysis between selected biomarkers related to glutamate excitotoxicity and oxidative stress in Saudi autistic patients.

    PubMed

    El-Ansary, Afaf

    2016-06-01

    This work demonstrates data of multiple regression analysis between nine biomarkers related to glutamate excitotoxicity and impaired detoxification as two mechanisms recently recorded as autism phenotypes. The presented data was obtained by measuring a panel of markers in 20 autistic patients aged 3-15 years and 20 age and gender matching healthy controls. Levels of GSH, glutathione status (GSH/GSSG), glutathione reductase (GR), glutathione-s-transferase (GST), thioredoxin (Trx), thioredoxin reductase (TrxR) and peroxidoxins (Prxs I and III), glutamate, glutamine, glutamate/glutamine ratio glutamate dehydrogenase (GDH) in plasma and mercury (Hg) in red blood cells were determined in both groups. In Multiple regression analysis, R (2) values which describe the proportion or percentage of variance in the dependent variable attributed to the variance in the independent variables together were calculated. Moreover, β coefficients values which show the direction either positive or negative and the contribution of the independent variable relative to the other independent variables in explaining the variation of the dependent variable were determined. A panel of inter-related markers was recorded. This paper contains data related to and supporting research articles currently published entitled "Mechanism of nitrogen metabolism-related parameters and enzyme activities in the pathophysiology of autism" [1], "Novel metabolic biomarkers related to sulfur-dependent detoxification pathways in autistic patients of Saudi Arabia [2], and "A key role for an impaired detoxification mechanism in the etiology and severity of autism spectrum disorders" [3]. PMID:26933667

  11. Modular pathway engineering of Corynebacterium glutamicum for production of the glutamate-derived compounds ornithine, proline, putrescine, citrulline, and arginine.

    PubMed

    Jensen, Jaide V K; Eberhardt, Dorit; Wendisch, Volker F

    2015-11-20

    The glutamate-derived bioproducts ornithine, citrulline, proline, putrescine, and arginine have applications in the food and feed, cosmetic, pharmaceutical, and chemical industries. Corynebacterium glutamicum is not only an excellent producer of glutamate but also of glutamate-derived products. Here, engineering targets beneficial for ornithine production were identified and the advantage of rationally constructing a platform strain for the production of the amino acids citrulline, proline, and arginine, and the diamine putrescine was demonstrated. Feedback alleviation of N-acetylglutamate kinase, tuning of the promoter of glutamate dehydrogenase gene gdh, lowering expression of phosphoglucoisomerase gene pgi, along with the introduction of a second copy of the arginine biosynthesis operon argCJB(A49V,M54V)D into the chromosome resulted in a C. glutamicum strain producing ornithine with a yield of 0.52 g ornithine per g glucose, an increase of 71% as compared to the parental ΔargFRG strain. Strains capable of producing 0.41 g citrulline per g glucose, 0.29 g proline per g glucose, 0.30 g arginine per g glucose, and 0.17 g putrescine per g glucose were derived from the ornithine-producing platform strain by plasmid-based overexpression of appropriate pathway modules with one to three genes. PMID:26393954

  12. Glycolytic flux controls d-serine synthesis through glyceraldehyde-3-phosphate dehydrogenase in astrocytes

    PubMed Central

    Suzuki, Masataka; Sasabe, Jumpei; Miyoshi, Yurika; Kuwasako, Kanako; Muto, Yutaka; Hamase, Kenji; Matsuoka, Masaaki; Imanishi, Nobuaki; Aiso, Sadakazu

    2015-01-01

    d-Serine is an essential coagonist with glutamate for stimulation of N-methyl-d-aspartate (NMDA) glutamate receptors. Although astrocytic metabolic processes are known to regulate synaptic glutamate levels, mechanisms that control d-serine levels are not well defined. Here we show that d-serine production in astrocytes is modulated by the interaction between the d-serine synthetic enzyme serine racemase (SRR) and a glycolytic enzyme, glyceraldehyde 3-phosphate dehydrogenase (GAPDH). In primary cultured astrocytes, glycolysis activity was negatively correlated with d-serine level. We show that SRR interacts directly with GAPDH, and that activation of glycolysis augments this interaction. Biochemical assays using mutant forms of GAPDH with either reduced activity or reduced affinity to SRR revealed that GAPDH suppresses SRR activity by direct binding to GAPDH and through NADH, a product of GAPDH. NADH allosterically inhibits the activity of SRR by promoting the disassociation of ATP from SRR. Thus, astrocytic production of d-serine is modulated by glycolytic activity via interactions between GAPDH and SRR. We found that SRR is expressed in astrocytes in the subiculum of the human hippocampus, where neurons are known to be particularly vulnerable to loss of energy. Collectively, our findings suggest that astrocytic energy metabolism controls d-serine production, thereby influencing glutamatergic neurotransmission in the hippocampus. PMID:25870284

  13. Flavor Preferences Conditioned by Dietary Glutamate.

    PubMed

    Ackroff, Karen; Sclafani, Anthony

    2016-07-01

    Our understanding of the molecular basis of umami taste and its appetitive qualities has been greatly aided by studies in laboratory rodents. This review describes methods for testing responses to the prototypical umami substance monosodium glutamate (MSG) in rodents. Two techniques, forced exposure to MSG and 2-bottle choice tests with ascending concentrations, were used to evaluate the responses to the taste of umami itself, and 2 other methods used oral or postoral MSG to modify the responses to other flavors. Intake and preference for MSG are enhanced in mice by experience with MSG and with other nutrients with positive postoral effects. In addition, flavor preferences are enhanced in mice and rats by gastric or intestinal MSG infusions via an associative learning process. Even mice with an impaired or absent ability to taste MSG can learn to prefer a flavor added to an MSG solution, supporting the notion that glutamate acts postorally. The more complex flavor of dashi seasoning, which includes umami substances (inosinate, glutamate), is attractive to rodents, but dashi does not condition flavor preferences. Details of the postoral glutamate detection process and the nature of the signal involved in learned preferences are still uncertain but probably involve gastric or intestinal sensors or both and vagal transmission. Some findings suggest that postoral glutamate effects may enhance food preferences in humans, but this requires further study. PMID:27422522

  14. Glutamate Metabolism in Major Depressive Disorder

    PubMed Central

    Abdallah, Chadi G.; Jiang, Lihong; De Feyter, Henk M.; Fasula, Madonna; Krystal, John H.; Rothman, Douglas L.; Mason, Graeme F.; Sanacora, Gerard

    2015-01-01

    Objective Emerging evidence suggests abnormalities in amino acid neurotransmitter function and impaired energy metabolism contribute to the underlying pathophysiology of Major Depressive Disorder (MDD). To test whether impairments in energetics and glutamate neurotransmitter cycling are present in MDD we used in vivo 13C magnetic resonance spectroscopy (13C MRS) to measure these fluxes in individuals diagnosed with MDD relative to non-depressed subjects. Method 1H MRS and 13C MRS data were collected on 23 medication-free MDD and 17 healthy subjects. 1H MRS provided total glutamate and GABA concentrations, and 13C MRS, coupled with intravenous infusion of [1-13C]-glucose, provided measures of the neuronal tricarboxylic acid cycle (VTCAN) for mitochondrial energy production, GABA synthesis, and glutamate/glutamine cycling, from voxels placed in the occipital cortex. Results Our main finding was that mitochondrial energy production of glutamatergic neurons was reduced by 26% in MDD subjects (t = 2.57, p = 0.01). Paradoxically we found no difference in the rate of glutamate/glutamine cycle (Vcycle). We also found a significant correlation between glutamate concentrations and Vcycle considering the total sample. Conclusions We interpret the reduction in mitochondrial energy production as being due to either mitochondrial dysfunction or a reduction in proper neuronal input or synaptic strength. Future MRS studies could help distinguish these possibilities. PMID:25073688

  15. Glucose-6-Phosphate Dehydrogenase and Its Deficiency in Mutants of Corynebacterium glutamicum

    PubMed Central

    Ihnen, Ernel D.; Demain, Arnold L.

    1969-01-01

    Corynebacterium glutamicum is a member of a group of taxonomically related glutamate-excreting bacteria which utilize glucose both by the Embden-Meyerhof and the pentose phosphate pathways, the latter sequence accounting for 10 to 38% of the glucose metabolized. Some of the properties of glucose-6-phosphate dehydrogenase in crude extracts of C. glutamicum were studied. The enzyme was rapidly inactivated by dilution in tris (hydroxymethyl)aminomethane-hydrochloride buffer. This inactivation was prevented by the presence of 0.45 m NaCl. Mg++ was required for enzyme activity, but Mn++, Ca++, Sr++, and Ba++ were equally effective. Growth of the organism under differing conditions did not markedly affect the specific activity of the enzyme. A generally applicable method for detecting colonies deficient in glucose-6-phosphate dehydrogenase was developed. Mutants so obtained were found to be auxotrophic for tryptophan. Upon reversion of the tryptophan requirement, the revertants still retained the property of glucose-6-phosphate dehydrogenase deficiency. Neither the mutants nor the revertants could grow as rapidly as the parent culture in glucose, in gluconate, or in a complex medium. PMID:5788701

  16. Genetic Examination of Initial Amino Acid Oxidation and Glutamate Catabolism in the Hyperthermophilic Archaeon Thermococcus kodakarensis

    PubMed Central

    Yokooji, Yuusuke; Sato, Takaaki; Fujiwara, Shinsuke; Imanaka, Tadayuki

    2013-01-01

    Amino acid catabolism in Thermococcales is presumed to proceed via three steps: oxidative deamination of amino acids by glutamate dehydrogenase (GDH) or aminotransferases, oxidative decarboxylation by 2-oxoacid:ferredoxin oxidoreductases (KOR), and hydrolysis of acyl-coenzyme A (CoA) by ADP-forming acyl-CoA synthetases (ACS). Here, we performed a genetic examination of enzymes involved in Glu catabolism in Thermococcus kodakarensis. Examination of amino acid dehydrogenase activities in cell extracts of T. kodakarensis KUW1 (ΔpyrF ΔtrpE) revealed high NADP-dependent GDH activity, along with lower levels of NAD-dependent activity. NADP-dependent activities toward Gln/Ala/Val/Cys and an NAD-dependent threonine dehydrogenase activity were also detected. In KGDH1, a gene disruption strain of T. kodakarensis GDH (Tk-GDH), only threonine dehydrogenase activity was detected, indicating that all other activities were dependent on Tk-GDH. KGDH1 could not grow in a medium in which growth was dependent on amino acid catabolism, implying that Tk-GDH is the only enzyme that can discharge the electrons (to NADP+/NAD+) released from amino acids in their oxidation to 2-oxoacids. In a medium containing excess pyruvate, KGDH1 displayed normal growth, but higher degrees of amino acid catabolism were observed compared to those for KUW1, suggesting that Tk-GDH functions to suppress amino acid oxidation and plays an anabolic role under this condition. We further constructed disruption strains of 2-oxoglutarate:ferredoxin oxidoreductase and succinyl-CoA synthetase. The two strains displayed growth defects in both media compared to KUW1. Succinate generation was not observed in these strains, indicating that the two enzymes are solely responsible for Glu catabolism among the multiple KOR and ACS enzymes in T. kodakarensis. PMID:23435976

  17. Single channel kinetics of a glutamate receptor.

    PubMed Central

    Kerry, C J; Kits, K S; Ramsey, R L; Sansom, M S; Usherwood, P N

    1987-01-01

    The glutamate receptor-channel of locust muscle membrane was studied using the patch-clamp technique. Muscles were pretreated with concanavalin A to block receptor-channel desensitization, thus facilitating analysis of receptor-channel gating kinetics. Single channel kinetics were analyzed to aid in identification of the molecular basis of channel gating. Channel dwell-time distributions and dwell-time autocorrelation functions were calculated from single channel data recorded in the precence of 10-4M glutamate. Analysis of the dwell time distributions in terms of mixtures of exponential functions revealed there to be at least three open states of the receptor-channel and at least four closed states. Autocorrelation function analysis showed there to be at least three pathways linking the open states with the closed. This results in a minimal scheme for gating of the glutamate receptor-channel, which is suggestive of allosteric models of receptor-channel gating. PMID:2436676

  18. Single Channel Kinetics of a Glutamate Receptor

    PubMed Central

    Kerry, Cathryn J.; Kits, Karel S.; Ramsey, Robert L.; Sansom, Mark S. P.; Usherwood, Peter N. R.

    1986-01-01

    The glutamate receptor-channel of locust muscle membrane was studied using the patch-clamp technique. Muscles were pretreated with concanavalin A to block receptor-channel desensitization, thus facilitating analysis of receptor-channel gating kinetics. Single channel kinetics were analyzed to aid in identification of the molecular basis of channel gating. Channel dwell-time distributions and dwell-time autocorrelation functions were calculated from single channel data recorded in the presence of 10-4 M glutamate. Analysis of the dwell time distributions in terms of mixtures of exponential functions revealed there to be at least three open states of the receptor-channel and at least four closed states. Autocorrelation function analysis showed there to be at least three pathways linking the open states with the closed. This results in a minimal scheme for gating of the glutamate receptor-channel, which is suggestive of allosteric models of receptor-channel gating. PMID:19431683

  19. The safety evaluation of monosodium glutamate.

    PubMed

    Walker, R; Lupien, J R

    2000-04-01

    L-Glutamic acid and its ammonium, calcium, monosodium and potassium salts were evaluated by the Joint FAO/WHO Expert Committee on Food Additives (JECFA) in 1988. The Committee noted that intestinal and hepatic metabolism results in elevation of levels in systemic circulation only after extremely high doses given by gavage (>30mg/kg body weight). Ingestion of monosodium glutamate (MSG) was not associated with elevated levels in maternal milk, and glutamate did not readily pass the placental barrier. Human infants metabolized glutamate similarly to adults. Conventional toxicity studies using dietary administration of MSG in several species did not reveal any specific toxic or carcinogenic effects nor were there any adverse outcomes in reproduction and teratology studies. Attention was paid to central nervous system lesions produced in several species after parenteral administration of MSG or as a consequence of very high doses by gavage. Comparative studies indicated that the neonatal mouse was most sensitive to neuronal injury; older animals and other species (including primates) were less so. Blood levels of glutamate associated with lesions of the hypothalamus in the neonatal mouse were not approached in humans even after bolus doses of 10 g MSG in drinking water. Because human studies failed to confirm an involvement of MSG in "Chinese Restaurant Syndrome" or other idiosyncratic intolerance, the JECFA allocated an "acceptable daily intake (ADI) not specified" to glutamic acid and its salts. No additional risk to infants was indicated. The Scientific Committee for Food (SCF) of the European Commission reached a similar evaluation in 1991. The conclusions of a subsequent review by the Federation of American Societies for Experimental Biology (FASEB) and the Federal Drug Administration (FDA) did not discount the existence of a sensitive subpopulation but otherwise concurred with the safety evaluation of JECFA and the SCF. PMID:10736380

  20. GLT-1: The elusive presynaptic glutamate transporter.

    PubMed

    Rimmele, Theresa S; Rosenberg, Paul A

    2016-09-01

    Historically, glutamate uptake in the CNS was mainly attributed to glial cells for three reasons: 1) none of the glutamate transporters were found to be located in presynaptic terminals of excitatory synapses; 2) the putative glial transporters, GLT-1 and GLAST are expressed at high levels in astrocytes; 3) studies of the constitutive GLT-1 knockout as well as pharmacological studies demonstrated that >90% of glutamate uptake into forebrain synaptosomes is mediated by the operation of GLT-1. Here we summarize the history leading up to the recognition of GLT-1a as a presynaptic glutamate transporter. A major issue now is understanding the physiological and pathophysiological significance of the expression of GLT-1 in presynaptic terminals. To elucidate the cell-type specific functions of GLT-1, a conditional knockout was generated with which to inactivate the GLT-1 gene in different cell types using Cre/lox technology. Astrocytic knockout led to an 80% reduction of GLT-1 expression, resulting in intractable seizures and early mortality as seen also in the constitutive knockout. Neuronal knockout was associated with no obvious phenotype. Surprisingly, synaptosomal uptake capacity (Vmax) was found to be significantly reduced, by 40%, in the neuronal knockout, indicating that the contribution of neuronal GLT-1 to synaptosomal uptake is disproportionate to its protein expression (5-10%). Conversely, the contribution of astrocytic GLT-1 to synaptosomal uptake was much lower than expected. In contrast, the loss of uptake into liposomes prepared from brain protein from astrocyte and neuronal knockouts was proportionate with the loss of GLT-1 protein, suggesting that a large portion of GLT-1 in astrocytic membranes in synaptosomal preparations is not functional, possibly because of a failure to reseal. These results suggest the need to reinterpret many previous studies using synaptosomal uptake to investigate glutamate transport itself as well as changes in glutamate

  1. Mood disorders: regulation by metabotropic glutamate receptors.

    PubMed

    Pilc, Andrzej; Chaki, Shigeyuki; Nowak, Gabriel; Witkin, Jeffrey M

    2008-03-01

    Medicinal therapies for mood disorders neither fully serve the efficacy needs of patients nor are they free of side-effect issues. Although monoamine-based therapies are the primary current treatment approaches, both preclinical and clinical findings have implicated the excitatory neurotransmitter glutamate in the pathogenesis of major depressive disorders. The present commentary focuses on the metabotropic glutamate receptors and their relationship to mood disorders. Metabotropic glutamate (mGlu) receptors regulate glutamate transmission by altering the release of neurotransmitter and/or modulating the post-synaptic responses to glutamate. Convergent biochemical, pharmacological, behavioral, and clinical data will be reviewed that establish glutamatergic neurotransmission via mGlu receptors as a biologically relevant process in the regulation of mood and that these receptors may serve as novel targets for the discovery of small molecule modulators with unique antidepressant properties. Specifically, compounds that antagonize mGlu2, mGlu3, and/or mGlu5 receptors (e.g. LY341495, MGS0039, MPEP, MTEP) exhibit biochemical effects indicative of antidepressant effects as well as in vivo activity in animal models predictive of antidepressant efficacy. Both preclinical and clinical data have previously been presented to define NMDA and AMPA receptors as important targets for the modulation of major depression. In the present review, we present a model suggesting how the interplay of glutamate at the mGlu and at the ionotropic AMPA and NMDA receptors might account for the antidepressant-like effects of glutamatergic- and monoaminergic-based drugs affecting mood in patients. The current data lead to the hypothesis that mGlu-based compounds and conventional antidepressants impact a network of interactive effects that converge upon a down regulation of NMDA receptor function and an enhancement in AMPA receptor signaling. PMID:18164691

  2. Circuit Mapping by UV Uncaging of Glutamate

    PubMed Central

    Shepherd, Gordon M. G.

    2014-01-01

    In laser photostimulation, small clusters of neurons in brain slices are induced to fire action potentials by focal glutamate uncaging, and synaptic connectivity between photoexcited presynaptic neurons and individual postsynaptic neurons is assessed by intracellular recording of synaptic events. With a scanner, this process can be repeated sequentially across a patterned array of stimulus locations, generating maps of neurons’ local sources of synaptic inputs. Laser scanning photostimulation (LSPS) based on patterned glutamate uncaging offers an efficient, quantitative, optical-electrophysiological way to map synaptic circuits in brain slices. PMID:22949715

  3. Molecular physiology of vesicular glutamate transporters in the digestive system

    PubMed Central

    Li, Tao; Ghishan, Fayez K.; Bai, Liqun

    2005-01-01

    Glutamate is the major excitatory neurotransmitter in the mammalian central nervous system (CNS). Packaging and storage of glutamate into glutamatergic neuronal vesicles require ATP-dependent vesicular glutamate uptake systems, which utilize the electrochemical proton gradient as a driving force. Three vesicular glutamate transporters (VGLUT1-3) have been recently identified from neuronal tissue where they play a key role to maintain the vesicular glutamate level. Recently, it has been demonstrated that glutamate signaling is also functional in peripheral neuronal and non-neuronal tissues, and occurs in sites of pituitary, adrenal, pineal glands, bone, GI tract, pancreas, skin, and testis. The glutamate receptors and VGLUTs in digestive system have been found in both neuronal and endocrinal cells. The glutamate signaling in the digestive system may have significant relevance to diabetes and GI tract motility disorders. This review will focus on the most recent update of molecular physiology of digestive VGLUTs. PMID:15793854

  4. Comparison of biochemical parameters of benzamide riboside, a new inhibitor of IMP dehydrogenase, with tiazofurin and selenazofurin.

    PubMed

    Gharehbaghi, K; Sreenath, A; Hao, Z; Paull, K D; Szekeres, T; Cooney, D A; Krohn, K; Jayaram, H N

    1994-10-01

    The biochemical and cytotoxic activities of the IMP dehydrogenase (IMPDH) inhibitors benzamide riboside, tiazofurin, and selenazofurin were compared. These three C-nucleosides exert their cytotoxicity by forming an analogue of NAD, wherein nicotinamide is replaced by the C-nucleoside base. The antiproliferative activities of these three agents were compared in a panel of 60 human cancer cell lines. To examine the relationship of benzamide riboside and selenazofurin to tiazofurin, COMPARE computer analysis was performed, and correlation coefficients of 0.761 and 0.815 were obtained for benzamide riboside and selenazofurin, respectively. The biochemical activities of these agents were examined in human myelogenous leukemia K562 cells. Incubation of K562 cells for 4 hr with 10 microM each of benzamide riboside, selenazofurin and tiazofurin resulted in a 49, 71, and 26% decrease in IMPDH activity with a concurrent increase in intracellular IMP pools. As a consequence of IMPDH inhibition, GTP and dGTP concentrations were curtailed. These studies demonstrated that selenazofurin was the most potent of the three agents. To compare the cellular synthesis of NAD analogues of these agents, K562 cells were incubated with 10 microM each of benzamide riboside, tiazofurin and selenazofurin after prelabeling the cells with [2,8-3H]adenosine. The results demonstrated that benzamide riboside produced 2- and 3-fold more of NAD analogue (BAD) than tiazofurin and selenazofurin did. To elucidate the effects of the three compounds on other NAD-utilizing enzymes, the inhibitory activities of purified benzamide adenine dinucleotide (BAD), thiazole-4-carboxamide adenine dinucleotide (TAD) and selenazole-4-carboxamide adenine dinucleotide (SAD) were studied in commercially available purified preparations of lactate dehydrogenase, glutamate dehydrogenase and malate dehydrogenase. TAD and SAD did not inhibit these three dehydrogenases. Although BAD did not influence lactate and glutamate

  5. Genetics Home Reference: succinic semialdehyde dehydrogenase deficiency

    MedlinePlus

    ... a chemical that transmits signals in the brain (neurotransmitter) called gamma-amino butyric acid (GABA). The primary ... Diseases National Organization for Rare Disorders (NORD) Pediatric Neurotransmitter Disease Association GeneReviews (1 link) Succinic Semialdehyde Dehydrogenase ...

  6. Up-regulation of 2-oxoglutarate dehydrogenase as a stress response.

    PubMed

    Graf, Anastasia; Trofimova, Lidia; Loshinskaja, Alexandra; Mkrtchyan, Garik; Strokina, Anastasiia; Lovat, Maxim; Tylicky, Adam; Strumilo, Slawomir; Bettendorff, Lucien; Bunik, Victoria I

    2013-01-01

    2-Oxoglutarate dehydrogenase multienzyme complex (OGDHC) operates at a metabolic cross-road, mediating Ca(2+)- and ADP-dependent signals in mitochondria. Here, we test our hypothesis that OGDHC plays a major role in the neurotransmitter metabolism and associated stress response. This possibility was assessed using succinyl phosphonate (SP), a highly specific and efficient in vivo inhibitor of OGDHC. Animals exposed to toxicants (SP, ethanol or MnCl(2)), trauma or acute hypoxia showed intrinsic up-regulation of OGDHC in brain and heart. The known mechanism of the SP action as OGDHC inhibitor pointed to the up-regulation triggered by the enzyme impairment. The animal behavior and skeletal muscle or heart performance were tested to correlate physiology with the OGDHC regulation and associated changes in the glutamate and cellular energy status. The SP-treated animals exhibited interdependent changes in the brain OGDHC activity, glutamate level and cardiac autonomic balance, suggesting the neurotransmitter role of glutamate to be involved in the changed heart performance. Energy insufficiency after OGDHC inhibition was detectable neither in animals up to 25 mg/kg SP, nor in cell culture during 24 h incubation with 0.1 mM SP. However, in animals subjected to acute ethanol intoxication SP did evoke energy deficit, decreasing muscular strength and locomotion and increasing the narcotic sleep duration. This correlated with the SP-induced decrease in NAD(P)H levels of the ethanol-exposed neurons. Thus, we show the existence of natural mechanisms to up-regulate mammalian OGDHC in response to stress, with both the glutamate neurotransmission and energy production potentially involved in the OGDHC impact on physiological performance. This article is part of a Directed Issue entitled: Bioenergetic dysfunction, adaptation and therapy. PMID:22814169

  7. Phosphorylation site on yeast pyruvate dehydrogenase complex

    SciTech Connect

    Uhlinger, D.J.

    1986-01-01

    The pyruvate dehydrogenase complex was purified to homogeneity from baker's yeast (Saccharomyces cerevisiae). Yeast cells were disrupted in a Manton-Gaulin laboratory homogenizer. The pyruvate dehydrogenase complex was purified by fractionation with polyethylene glycol, isoelectric precipitation, ultracentrifugation and chromatography on hydroxylapatite. Final purification of the yeast pyruvate dehydrogenase complex was achieved by cation-exchange high pressure liquid chromatography (HPLC). No endogenous pyruvate dehydrogenase kinase activity was detected during the purification. However, the yeast pyruvate dehydrogenase complex was phosphorylated and inactivated with purified pyruvate dehydrogenase kinase from bovine kidney. Tryptic digestion of the /sup 32/P-labeled complex yielded a single phosphopeptide which was purified to homogeniety. The tryptic digest was subjected to chromatography on a C-18 reverse phase HPLC column with a linear gradient of acetonitrile. Radioactive fractions were pooled, concentrated, and subjected to anion-exchange HPLC. The column was developed with a linear gradient of ammonium acetate. Final purification of the phosphopeptide was achieved by chromatography on a C-18 reverse phase HPLC column developed with a linear gradient of acetonitrile. The amino acid sequence of the homogeneous peptide was determined by manual modified Edman degradation.

  8. Immobilization of Ni-Pd/core-shell nanoparticles through thermal polymerization of acrylamide on glassy carbon electrode for highly stable and sensitive glutamate detection.

    PubMed

    Yu, Huicheng; Ma, Zhenzhen; Wu, Zhaoyang

    2015-10-01

    The preparation of a persistently stable and sensitive biosensor is highly important for practical applications. To improve the stability and sensitivity of glutamate sensors, an electrode modified with glutamate dehydrogenase (GDH)/Ni-Pd/core-shell nanoparticles was developed using the thermal polymerization of acrylamide (AM) to immobilize the synthesized Ni-Pd/core-shell nanoparticles onto a glassy carbon electrode (GCE). The modified electrode was characterized by scanning electron microscopy (SEM), Fourier transform infrared (FTIR) spectroscopy, cyclic voltammetry (CV), and electrochemical impedance spectroscopy (EIS). Electrochemical data showed that the prepared biosensor had remarkably enhanced electrocatalytic activity toward glutamate. Moreover, superior reproducibility and excellent stability were observed (relative average deviation was 2.96% after continuous use of the same sensor for 60 times, and current responses remained at 94.85% of the initial value after 60 d). The sensor also demonstrated highly sensitive amperometric detection of glutamate with a low limit of detection (0.052 μM, S/N = 3), high sensitivity (4.768 μA μM(-1) cm(-2)), and a wide, useful linear range (0.1-500 μM). No interference from potential interfering species such as l-cysteine, ascorbic acid, and l-aspartate were noted. The determination of glutamate levels in actual samples achieved good recovery percentages. PMID:26481997

  9. Lycium chinensis Mill attenuates glutamate induced oxidative toxicity in PC12 cells by increasing antioxidant defense enzymes and down regulating ROS and Ca(2+) generation.

    PubMed

    Olatunji, Opeyemi J; Chen, Hongxia; Zhou, Yifeng

    2016-03-11

    Lycium chinensis Mill is a famous traditional Chinese medicine which displays several medicinal activities including antioxidant and neuroprotective activities. However, the mechanism of action towards the neuroprotective action has not been fully elucidated. This work was aimed at investigating the neuroprotective effects of L. chinensis Mill against glutamate-induced oxidative neurotoxicity in PC12 cells. Oxidative cell death was induced with 5mM glutamate in PC12 cells. Cell viability, LDH release, intracellular Ca(2+) concentration, reactive oxygen species (ROS) accumulation, GSH-Px, CAT and SOD antioxidant enzyme levels were measured. Our results indicated that pretreatment of PC12 cells with L. chinensis Mill extracts markedly attenuated the loss of cell viability, the release of lactate dehydrogenase (LDH), Ca(2+) overload, ROS generation, and cell apoptosis induced by glutamate toxicity. Furthermore, L. chinensis Mill extracts also significantly increased the levels of innate antioxidant enzymes GSH-Px, SOD and CAT in glutamate-induced PC12 cells. Conclusively, our results provided substantial evidence that L. chinensis Mill protected PC12 cells against glutamate-induced cell death by attenuating ROS generation, Ca(2+) influx, and increased the antioxidant defense capacity of PC12 cells against oxidative stress damages, suggesting the possible potential of extracts from the plant as sources of bioactive molecules in the treatment of neurodegenerative disorders. PMID:26536075

  10. GLUTAMATE NEUROTOXICITY IN RAT AUDITORY SYSTEM: COCHLEAR NUCLEAR COMPLEX

    EPA Science Inventory

    In other systems such as the hypothalamus and hippocampus, it has been shown that cells postsynaptic with respect to glutamatergic inputs degenerate when exposed to large doses of glutamate ("glutamate neurotoxicity"). e have shown that large doses of glutamate administered intra...