PubMed Health⌕ Search

SEARCH · PubMed Health

Results for “GLUTAMATE DEHYDROGENASE”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 883 records · Page 49Linked to original sources
PubMed1994

Identification of the reactive cysteine in clostridial glutamate dehydrogenase by site-directed mutagenesis and proof that this residue is not strictly essential.

Cys320 of clostridial glutamate dehydrogenase, a residue close to the coenzyme binding site, has been replaced by serine. The mutant enzyme was successfully overproduced and purified by using the normal protocol for the wild-type enzyme and also behaved indistinguishably from wild-type enzyme on native and SDS-PAGE. The specific activity was significantly enhanced in assays at both pH 7 (+90%) and pH 8 (+38%). Detailed initial-rate kinetics revealed that at pH 7 this increase was mainly attributable to a higher maximum rate, since the Km values for both substrates were marginally increased. In the mutant enzyme the inactivating reaction with DTNB that characterizes the wild-type enzyme is completely eliminated. This proves that inactivation of the wild-type enzyme is due to modification of Cys320, that nevertheless Cys320 is not strictly essential for catalytic activity and that the remaining cysteine residue at position 144 is inaccessible to DTNB. Provision of an engineered subunit with a correct native structure but with its DTNB titre decreased from 1 to 0 mol/mol now offers a valuable tool for counting subunits in hybrid oligomers.

Base Sequence↗
PubMed2006

Green tea polyphenols modulate insulin secretion by inhibiting glutamate dehydrogenase.

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.

Adenosine Diphosphate↗
PubMed1996

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

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

Acetanilides↗
PubMed1975

The amino acid sequence of Neurospora NADP-specific glutamate dehydrogenase. Peptides from digestion with a staphylococcal proteinase.

The extracellular proteinase of Staphylococcus aureus strain V8 was used to digest the NADP-specific glutamate dehydrogenase of Neurospora crassa. Of 35 non-overlapping peptides expected from the glutamate content of the polypeptide chain, 29 were isolated and substantially sequenced. The sequences obtained were valuable in providing overlaps for the alignment of about two-thirds of the sequences found in tryptic peptides [Wootton, J. C., Taylor, J, G., Jackson, A. A., Chambers, G. K. & Fincham, J. R. S. (1975) Biochem. J. 149, 739-748]. The blocked N-terminal peptide of the protein was isolated. This peptide was sequenced by mass spectrometry, and found to have N-terminal N-acetylserine by Howard R. Morris and Anne Dell, whose results are presented as an Appendix to the main paper. The staphylococcal proteinase showed very high specificity for glutamyl bonds in the NH4HCO3 buffer used. Partial splits of two aspartyl bonds, both Asp-Ile, were probably attributable to the proteinase. No cleavage of glutaminyl or S-carboxymethylcysteinyl bonds was found. Additional experimental detail has been deposited as Supplementary Publication SUP 50053 (5 pages) with the British Library (Lending Division), Boston Spa, Wetherby, W. Yorkshire LS23 7BQ, U.K, from whom copies may be obtained under the terms given in Biochem. J. (1975) 1458 5.

Amino Acid Sequence↗
PubMed1989

Kinetic advantages of hetero-enzyme complexes with glutamate dehydrogenase and the alpha-ketoglutarate dehydrogenase complex.

We have found previously (Fahien, L.A., Kmiotek, E.H., MacDonald, M. J., Fibich, B., and Mandic, M. (1988) J. Biol. Chem. 263, 10687-10697) that glutamate-malate oxidation can be enhanced by cooperative binding of mitochondrial aspartate aminotransferase and malate dehydrogenase to the alpha-ketoglutarate dehydrogenase complex. The present results demonstrate that glutamate dehydrogenase, which forms binary complexes with these enzymes, adds to this ternary complex and thereby increases binding of the other enzymes. Kinetic evidence for direct transfer of alpha-ketoglutarate and NADH, within these complexes, has been obtained by measuring steady-state rates of E2 when most of the substrate or coenzyme is bound to the aminotransferase or glutamate dehydrogenase (E1). Rates significantly greater than those which can be accounted for by the concentration of free ligand, calculated from the measured values of the E1-ligand dissociation constants, require that the E1-ligand complex serve as a substrate for E2 (Srivastava, D. K., and Bernhard, S. A. (1986) Curr. Tops. Cell Regul. 28, 1-68). By this criterion, NADH is transferred directly from glutamate dehydrogenase to malate dehydrogenase and alpha-ketoglutarate is channeled from the aminotransferase to both glutamate dehydrogenase and the alpha-ketoglutarate dehydrogenase complex. Similar evidence indicates that GTP bound to an allosteric site on glutamate dehydrogenase functions as a substrate for succinic thiokinase. The potential physiological advantages to channeling of activators and inhibitors as well as substrates within multienzyme complexes organized around the alpha-ketoglutarate dehydrogenase complex are discussed.

Animals↗
PubMed2001

Allosteric behaviour of 1:5 hybrids of mutant subunits of Clostridium symbiosum glutamate dehydrogenase differing in their amino acid specificity.

Hybrid hexamers were made by refolding mixtures of two mutant forms of clostridial glutamate dehydrogenase. Mutant Cys320Ser (C320S) has a similar activity to the wild-type enzyme, but is unreactive with Ellman's reagent, 5,5'-dithiobis(2-nitrobenzoate) (DTNB). The triple mutant Lys89Leu/Ala163Gly/Ser380Ala (K89L/A163G/S380A), active with norleucine but not glutamate, is inactivated by DTNB, since the amino acid residue at position 320 is a cysteine residue. The chosen ratio favoured 1:5 hybrids of the triple mutant and C320S. The renatured mixture was treated with DTNB and separated on an NAD(+)-agarose column to which only C320S subunits bind tightly. Fractions were monitored for glutamate and norleucine activity and for releasable thionitrobenzoate to establish subunit stoichiometry. A fraction highly enriched in the 1:5 hybrid was identified. Homohexamers (C320S with 40 mM glutamate and 1 mM NAD(+) at pH 8.8, or K89L/A163G/S380A with 70 mM norleucine and 1 mM NAD(+) at pH 8.5) showed allosteric activation; succinate activated C320S approx. 50-fold (EC(50)=70 mM, h=2.4), and glutarate gave approx. 30-fold activation (EC(50)=35 mM, h=2.3). For the triple mutant, corresponding values were 80 mM and 2.2 for succinate, and 75 mM and 1.7 for glutarate, but maximal activation was only about 2-fold. In the 1:5 hybrid, with only one norleucine-active subunit per hexamer, responses to glutarate and succinate were still co-operative, and activation was more extensive than in the triple mutant homohexamer. A single norleucine-active subunit can thus respond co-operatively to a substrate analogue at the other five inactive sites. On the other hand, similar hyperbolic dependence on the norleucine concentration for the hybrid and the triple mutant homohexamer reflected the inability of C320S subunits to bind norleucine. With glutamate at pH 8.8, an h value of 3.6 was obtained for the 1:5 hybrid, in contrast with an h value of 5.2 for the C320S homohexamer. The "foreign" subunit evidently impedes inter-subunit communication to some extent.

Chromatography, Affinity↗
PubMed1977

[Comparative study of the multiple molecular forms of NAD-dependent glutamate dehydrogenase in the liver and femoral muscle at different stages of swine embryogenesis[].

The multiplicity of NAD-dependent glutamate dehydrogenase (GDH) was studied in the liver and femoral muscle of 30, 60 and 108 days old pig embryos by the method of discelectrophoresis in polyacrilamide gel. The GDH was shown to be presented in the tissues under study by 3--4 multiple molecular forms. Specific ratios of the multiple GDH forms were established in the tissues under study at different stages of embryogenesis.

Animals↗
PubMed1987

Localization of two L-glutamate dehydrogenases in the coral Acropora latistella.

The coral Acropora latistella has been shown to contain two distinct L-glutamate dehydrogenases. An NADPH-specific GDH, closely resembling the enzyme from other coelenterates, is located in the cytosol; a distinct NADH-specific GDH is localized within the mitochondria. This is in contrast to earlier reports, where the NADPH-specific enzyme was thought to be the only GDH present and was assumed to be mitochondrial. Preliminary kinetic and stability data are presented for the two GDHs.

Animals↗
PubMed1984

The effects of magnesium ions on the interactions of ox brain and liver glutamate dehydrogenase with ATP and GTP.

The effects of ATP and GTP on the activities of ox liver and brain glutamate dehydrogenase were determined in the absence and presence of added Mg2+ ions. Although GTP was an inhibitor of the enzyme reaction assayed in the direction of NAD+ reduction, the magnesium complex of this nucleotide had no effect on the activity. Similarly the magnesium complex of ATP was without effect on the activity of the enzyme although the free nucleotide was an activator. These results suggest that it is important to take account of magnesium complex formation when considering the regulatory actions of these nucleotides.

Adenosine Triphosphate↗
PubMed1998

Effect of pyridoxine and insulin administration on brain glutamate dehydrogenase activity and blood glucose control in streptozotocin-induced diabetic rats.

Blood glucose level and kinetic parameters of glutamate dehydrogenase (GDH) were measured in the cerebellum, brain stem and cerebral cortex of control, insulin treated, pyridoxine treated, pyridoxine and insulin treated and untreated streptozotocin-diabetic rats. The combined administration of insulin and pyridoxine was found to be better in controlling the hyperglycaemia. Insulin with pyridoxine treatment brought back the increased maximal velocity of GDH during diabetes to control state. Also, there was an increase in Michaelis-Menten constant. These results suggest that pyridoxine and insulin together serve a better control for diabetes.

Animals↗
PubMed2001

A Japanese case of congenital hyperinsulinism with hyperammonemia due to a mutation in glutamate dehydrogenase (GLUD1) gene.

We describe a Japanese case of neonatal hyperinsulinism due to a de novo mutation (Gly446Asp) in glutamate dehydrogenase gene (GLUD1). A boy suffered from hypoglycemic coma with relative hyperinsulinemia on day 1 after birth, and received subtotal pancreatectomy. Examination of the resected pancreas revealed a diffuse increase in endocrine cells, consistent with 'nesidioblastosis'. He is now 15 years old and has exhibited mild but persistent hyperammonemia, which is a very unique feature of the disorder caused by GLUD1 activating mutations. He has also been suffering from seizures and mental retardation. Thus, GLUD1 mutations can be a cause of congenital hyperinsulinism in Japanese.

Allosteric Regulation↗
PubMed1993

The gdhA1 point mutation in Escherichia coli K12 CLR207 alters a key lysine residue of glutamate dehydrogenase.

gdhA1 is a spontaneous mutant of Escherichia coli that causes complete loss of activity of the NADP-specific glutamate dehydrogenase (GDH) encoded by the gdhA gene. The gdhA1 mutational site has been identified by recombinational mapping, polymerase chain reaction (PCR) amplification and DNA sequencing, as an A to G transition at nucleotide 274 of the gdhA coding sequence, resulting in an amino acid change of lysine 92 to glutamic acid. The mutant enzyme forms hybrid hexamers with a wild-type GDH, providing a useful system for analysis of conformational integrity of mutational variants.

Base Sequence↗
PubMed1985

The Klebsiella aerogenes glutamate dehydrogenase (gdhA) gene: cloning, high-level expression and hybrid enzyme formation in Escherichia coli.

The NADP-dependent glutamate dehydrogenase gene of Klebsiella aerogenes was cloned in E. coli in the expression plasmid pRK9. The cloned gene shows a high level of expression in E. coli in the hybrid plasmid pKG3 and such expression is independent of the vector promoter, as shown by experiments in which the promoter was deleted. Active hybrid GDH hexamers were shown in cell-free extracts of an E. coli strain carrying cloned gdhA genes of both E. coli and K. aerogenes. The nucleotide sequence of the N-terminal coding region of the K. aerogenes gdhA gene was determined and found to be strongly homologous with that of E. coli.

Base Sequence↗
PubMed2000

Electrochemical study of the effect of ADP and AMP on the kinetics of glutamate dehydrogenase.

A chronoamperometric method based on the 'diffusion' layer concept of the convective system was used to assay the glutamate dehydrogenase (GLDH) activity. Once the reaction was initiated by adding the enzyme GLDH into a well-stirred nicotinamide adenine dinucleotide (NADH, coenzyme) solution, the steady-state oxidation limiting current of NADH would decrease linearly in a short time. The major advantage of this method is that it directly indicates the continuous in-situ change of the coenzyme concentration, thus, the real initial reaction rate of enzyme-catalyzed reaction, V0, can be determined. Using this method, the effect of adenosine-5'-monophosphate (AMP) and adenosine-5'-diphosphate (ADP) on the GLDH activity has been monitored. The results showed that ADP and AMP could increase the activity of GLDH. This activation mechanism was proposed by the voltammetric study.

Adenosine Diphosphate↗
PubMed1970

Kinetic studies of glutamate dehydrogenase. The reductive amination of 2-oxoglutarate.

1. Kinetic studies of the reductive amination of 2-oxoglutarate catalysed by glutamate dehydrogenase with NADH and NADPH as coenzyme were made at pH7.0 and pH 8.0. The concentrations of both substrates and coenzymes were simultaneously varied over wide ranges. Lineweaver-Burk plots with respect to each substrate and coenzyme were linear, except that with high concentrations of 2-oxoglutarate or coenzyme inhibition occurred. There was no evidence of the negative homotropic interactions between the enzyme subunits that were revealed in previous kinetic studies of the reverse reaction. 2. The initial-rate results are shown to be inconsistent with any of the six possible compulsory-order mechanisms for this three-substrate reaction, and it is concluded that a random-order mechanism is the most likely one. On the basis of this mechanism, the dissociation constants of all the binary, ternary and quaternary complexes of the enzyme and substrates are calculated from initial-rate parameters. 3. The results are discussed in relation to those of earlier workers who concluded that the mechanism is of the compulsory-order type.

Glutamate Dehydrogenase↗
PubMed1971

Covalent attachment of diethylstilbestrol to glutamate dehydrogenase: implications for allosteric regulation.

An affinity labeling reagent for the estrogenic-binding site of bovine liver L-glutamate dehydrogenase (EC 1.4.1.3) was prepared by conversion of diethylstilbestrol to its alkylating analogue, bromoacetyldiethylstilbestrol. Under standard assay conditions, the analogue acted as a reversible allosteric ligand with regulatory activity much like that of diethylstilbestrol. However, incubation of the enzyme with the alkylating agent in the presence of DPNH resulted in a permanent decrease in glutamate (X form) and an increase in alanine (Y form) activities, and in covalent attachment of diethylstilbestrol in the ratio of 1 mol per subunit (of particle weight 52,000). The brominated analogue behaved as an affinity label that mimicked the allosteric effects of diethylstilbestrol. Diethylstilbestrol protection of the enzyme against alkylation by bromoacetylated sterol suggested competition for the same binding site, while ADP protection indicated a shift of protein equilibrium into the X form. The diethylstilbestrol-enzyme compound was desensitized (relative to the native enzyme) to allosteric reagents such as ADP and GTP. The results were consistent with conformational freezing of the modified protein molecule into the Y form.

Adenine Nucleotides↗
PubMed2001

Molecular characterisation of glutamate dehydrogenase gene defects in Japanese patients with congenital hyperinsulinism/hyperammonaemia.

Congenital hyperinsulinism and hyperammonaemia (CHH) is caused by dysregulation of glutamate dehydrogenase (GDH). We characterised the GDH gene in two Japanese patients with CHH. Patient 1 showed late-onset and mild hypoglycaemic episodes and mild hyperammonaemia, compared with patient 2. In GDH activity of lymphoblasts, patient 1 showed twofold higher basal GDH activity than control subjects and mild insensitivity for GTP inhibition. Patient 2 showed severe insensitivity for GTP inhibition, and similar allosteric stimulation by ADP in the controls. Genetic studies identified heterozygous and de novo L413V and G446D mutations in patients 1 and 2, respectively. COS cell expression study confirmed that both mutations were disease-causing gene. The insensitivity for GTP inhibition in L413V and G446D was emphasised in COS cell expression system as a result of the dosage effect of mutant GDH gene. L413V showed less impairment of GDH than G446D based on biochemical and genetic results, which was consistent with the clinical phenotype. Based on the structure of bovine GDH, G446D was located in GTP binding site of pivot helix and its surroundings, while L413V was located in alpha-helix of antenna-like structure. These different locations of mutations gave different effects on GDH enzyme. The antenna-like structure plays an important role in GDH activity.

Animals↗