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PubMed1990

Multiple system degeneration with glutamate dehydrogenase deficiency: pathology and biochemistry.

The neuropathological findings in a patient with antemortem diagnosis of olivopontocerebellar atrophy (OPCA) and reduced leucocytic glutamate dehydrogenase (GDH) activity included cerebellar cortical degeneration, most marked in the superior vermis, mild atrophy of the pons and the inferior olivary nucleus, marked reduction of anterior horn cells at all levels and gliosis in both lateral columns. GDH activities and their thermolability in "soluble" and "particulate" fractions in the cerebral cortex, cerebellar hemisphere and vermis were not significantly different from the values in two control brains. GDH mRNA in the patient's brain was not altered in size or amount.

Aged↗

PubMed1979

Serum glutamic oxalacetic transaminase, glutamic pyruvic transaminase, gamma-glutamyl transpeptidase and glutamic dehydrogenase levels in favism.

Serum GOT, GPT, gamma-GT and GLDH were determined in 15 G-6-PD-deficient subjects during a hemolytic crisis and hemoglobinuria due to ingestion of fresh fava beans. The same G-6-PD-deficient subjects were studied again 2 months after the crisis, when they were asymptomatic. 15 normal healthy children served as controls. A statistically significant increase, above normal, in serum GOT, GLDH and gamma-GT was observed in the favic subjects during the crisis. All the values reverted to normal in the asymptomatic period.

Alanine Transaminase↗

PubMed1990

The inhibition of glutamate dehydrogenase by some antipsychotic drugs.

The phenothiazines chlorpromazine and perphenazine and the butyrophenone haloperidol were shown to be reversible inhibitors of glutamate dehydrogenase (GDH). Inhibition by chlorpromazine was found to be partial, whereas haloperidol and perphenazine would, apparently, give full inhibition at saturating concentrations. Double-reciprocal plots of the difference between activities in the absence and presence of the inhibitor against the inhibitor concentration were linear with chlorpromazine and perphenazine but parabolic when haloperidol was used as the inhibitor. Comparisons between the responses of commercially available preparations of ox liver GDH, which have been shown to have suffered limited proteolysis during purification, and the ox brain enzyme prepared by a procedure which does not result in such proteolysis, revealed the latter preparation to be more sensitive to inhibition because of a higher apparent affinity for these drugs. The apparent dissociation constants for enzyme-drug interactions were, however considerably higher than the concentrations that have been reported to occur in vivo following chronic administration of chlorpromazine and haloperidol. This casts doubt on earlier claims that inhibition of GDH may be involved in the antipsychotic actions of these drugs, although it might be a factor in the side effects associated with the use of such compounds.

Animals↗

PubMed2003

Induction of glutamate dehydrogenase in the ovine fetal liver by dexamethasone infusion during late gestation.

Glucocorticoids near term are known to upregulate many important enzyme systems prior to birth. Glutamate dehydrogenase (GDH) is a mitochondrial enzyme that catalyzes both the reversible conversion of ammonium nitrogen into organic nitrogen (glutamate production) and the oxidative deamination of glutamate resulting in 2-oxoglutarate. The activity of this enzyme is considered to be of major importance in the development of catabolic conditions leading to gluconeogenesis prior to birth. Ovine hepatic GDH mRNA expression and activity were determined in near-term (130 days of gestation, term 147 +/- 4 days) control and acutely dexamethasone-treated (0.07 mg(-1) hr(-1) for 26 hr) fetuses. Dexamethasone infusion had no effect on placental or fetal liver weights. Dexamethasone infusion for 26 hr significantly increased hepatic GDH mRNA expression. This increased GDH mRNA expression was accompanied by an increase in hepatic mitochondrial GDH activity, from 30.0 +/- 7.4 to 58.2 +/- 8.1 U GDH/U CS (citrate synthase), and there was a significant correlation between GDH mRNA expression and GDH activity. The generated ovine GDH sequence displayed significant similarity with published human, rat, and murine GDH sequence. These data are consistent with the in vivo studies that have shown a redirection of glutamine carbon away from net hepatic glutamate release and into the citric acid cycle through the forward reaction catalyzed by GDH, i.e., glutamate to oxoglutarate.

Animals↗

PubMed2003

Histidine 454 plays an important role in polymerization of human glutamate dehydrogenase.

Although previous chemical modification studies have suggested several residues to be involved in the maintenance of the quaternary structure of glutamate dehydrogenase (GDH), there are conflicting views for the polymerization process and no clear evidence has been reported yet. In the present study, cassette mutagenesis at seven putative positions (Lys333, Lys337, Lys344, Lys346, Ser445, Gly446, and His454) was performed using a synthetic human GDH gene to examine the polymerization process. Of the mutations at the seven different sites, only the mutagenesis at His454 results in depolymerization of the hexameric GDH into active trimers as determined by HPLC gel filtration analysis and native gradient polyacrylamide gel electrophoresis. The mutagenesis at His454 has no effects on expression or stability of the protein. The K(M) values for NADH and 2-oxoglutarate were 1.5-fold and 2.5-fold greater, respectively, for the mutant GDH than for wild-type GDH, indicating that substitution at position 454 had appreciable effects on the affinity of the enzyme for both NADH and 2-oxoglutarate. The V(max) values were similar for wild-type and mutant GDH. The k(cat)/K(M) value of the mutant GDH was reduced up to 2.8-fold. The decreased efficiency of the mutant, therefore, results from the increase in K(M) values for NADH and 2-oxoglutarate. The results with cassette mutagenesis and HPLC gel filtration analysis suggest that His454 is involved in the polymerization process of human GDH.

Biopolymers↗

PubMed2006

Unregulated insulin secretion by pancreatic beta cells in hyperinsulinism/hyperammonemia syndrome: role of glutamate dehydrogenase, ATP-sensitive potassium channel, and nonselective cation channel.

The hyperinsulinism/hyperammonemia (HI/HA) syndrome is caused by "gain of function" of glutamate dehydrogenase (GDH). Several missense mutations have been found; however, cell behaviors triggered by the excessive GDH activity have not been fully demonstrated. This study was aimed to clarify electrophysiological mechanisms underlying the dysregulated insulin secretion in pancreatic beta cells with GDH mutations. GDH kinetics and insulin secretion were measured in MIN6 cells overexpressing the G446D and L413V. Membrane potentials and channel activity were recorded under the perforated-patch configuration that preserved intracellular environments. In mutant MIN6 cells, sensitivity of GDH to guanosine triphosphate (GTP) was reduced and insulin secretion at low glucose concentrations was enhanced. The basal GDH activity was elevated in L413V bearing a mutation in the antenna-like structure. The L413V cells were depolarized without glucose, often accompanying by repetitive Ca2+ firings. The depolarization was maintained in the presence of adenosine triphosphate (ATP) and disappeared by depleting ATP, suggesting that the depolarization depended on intracellular ATP. In L413V cells, the ATP-sensitive potassium channel (K(ATP) channel) was suppressed and the nonselective cation channel (NSCC) was potentiated, while sensitivity of the channels to their specific blockers or agonists was not impaired. These data suggest that the L413V cells increase the intracellular ATP/adenosine diphosphate (ADP) ratio, which in turn causes sustained depolarization not only by closure of the K(ATP) channel, but also by opening of the NSCC. The resultant activation of the voltage-gated Ca2+ channel appears to induce hyperinsulinism. The present study provides evidence that multiple channels cooperate in unregulated insulin secretion in pancreatic beta cells of the HI/HA syndrome.

Adenosine Triphosphate↗

PubMed1973

The distribution and characteristics of nitrate reductase and glutamate dehydrogenase in the maize seedling.

In a study on 3-day maize (Zea mays) seedlings, grown on nitrate, requirements were established for the maximum extraction and optimum stabilization of nitrate reductase in vitro. With the primary root, 5 mm cysteine were required in the extraction medium, but for the scutellum, which has a high level of endogenous thiol, the use of additional thiol resulted in a reduced yield of a more labile enzyme. Activity of the root and scutella nitrate reductase was obtained with either NADH or NADPH, but that of the root enzyme with NADPH was only demonstrated in the absence of phosphate.Before leaf expansion, the nitrate reductase in the maize seedling was mainly in the scutellum. The enzyme present in the primary root was predominantly in the apical region (0-2 mm). In contrast, glutamate dehydrogenase was concentrated in the mature basal region of the root (30-60 mm). A high level of nitrate (approximately 100 mm) was required to saturate the induction of nitrate reductase in the root tip, mature root, and scutellum. The concentration of nitrate required to give half the maximum level of enzyme induced was the same for each region (29 mm).After leaf expansion, more than 90% of the nitrate reductase was in the shoot, mainly in the leaf blade, and a marked decrease occurred in the level of the enzyme in the scutellum. A large proportion of the glutamate dehydrogenase was still found in the root.

Journal Article↗

PubMed1985

Ox glutamate dehydrogenase. Comparison of the kinetic properties of native and proteolysed preparations.

Kinetic constants were determined for commercially available samples of ox liver glutamate dehydrogenase, which had previously been shown to have suffered limited proteolysis during preparation, with a range of substrates and effectors. These were compared with the values obtained with enzyme preparations purified in such a way as to prevent this proteolysis from occurring [McCarthy, Walker & Tipton (1980) Biochem. J. 191, 605-611]. The Km values and maximum velocities determined with different substrates revealed little difference between the two preparations although the proteolysed enzyme had lower Km values for NH4+ and glutamate when the activities were determined with NADPH and NADP+ respectively. This preparation was more sensitive to inhibition by Cl- ions but less sensitive to inhibition by high concentrations of the substrate NADH. The two preparations also differed in their sensitivities to allosteric effectors, with the proteolysed enzyme being the less sensitive to inhibition by GTP. At high concentrations of NADH, this preparation was also more sensitive to activation by ADP and ATP.

Adenosine Diphosphate↗

PubMed1994

Biochemical and genetical studies of NADP-specific glutamate dehydrogenase in the fission yeast Schizosaccharomyces pombe.

The initial velocity, pH and temperature optima, and Km values of Schizosaccharomyces pombe NADP-glutamate dehydrogenase (NADP-GDH:EC 1.4.1.4) have been determined. NADP-GDH was found to be specific for the substrates used in the reaction mixtures. NADP-GDH activity showed a sigmoidal response to changes in alpha-ketoglutarate concentrations, following Hill kinetics with a coefficient nH = 2. A two-fold and a three-fold increase in activity was found in extracts of cells grown on a medium containing cytosine or histidine as a sole nitrogen source, respectively, relative to the activity found in cells grown on other sole nitrogen sources including ammonium, adenine, arginine, aspartate, asparagine, glutamate, glutamine, leucine, lysine, proline, uridine and urea. Five NADP-GDH-defective mutants were isolated on the basis of no growth on ammonium plus allantoin as sole nitrogen sources. The mutants also failed to grow on allantoin alone but, in contrast, they were phenotypically indistinguishable from the wild-type growing on solid minimal medium with ammonium. Additionally, the mutants were found to grow as wild-type on minimal medium with alanine, arginine, asparagine, aspartate, glutamate, glutamine, leucine, ornithine and proline in the absence or presence of allantoin. In liquid minimal medium with ammonium as sole nitrogen source they had a slower growth than the wild-type. Normal growth was observed in cells grown on alanine, arginine, asparagine, aspartate, glutamate, glutamine, leucine, ornithine and proline. The mutants had undetectable levels of NADP-GDH activity, but retained wild-type levels of NAD-GDH, glutame synthase (GOGAT) and glutamine synthetase (GS).(ABSTRACT TRUNCATED AT 250 WORDS)

Cell Division↗

PubMed1984

Complete nucleotide sequence of the glutamate dehydrogenase gene from Escherichia coli K-12.

A 2.3-kb PstI- ClaI chromosomal DNA segment, carrying the complete coding region of the glutamate dehydrogenase (GDH) structural gene from Escherichia coli K-12, has been sequenced. The complete amino acid sequence (447 residues) of the GDH monomer has been deduced, and comparisons are made with reported amino acid sequences of GDH from other organisms.

Amino Acid Sequence↗

PubMed1983

Nucleotide sequence of the promoter and amino-terminal coding region of the glutamate dehydrogenase structural gene of Escherichia coli.

A 610-bp DNA fragment carrying the promoter and amino-terminal coding regions of the glutamate dehydrogenase (GDH) structural gene from Escherichia coli has been sequenced. The amino-terminal sequence of the enzyme was also determined to help localize the transcriptional and translational signals for this gene. Three possible promoters and a CRP binding site were identified by concensus criteria. The sequence of 102 amino acids at the amino terminus of the enzyme is compared with the amino acid sequence from other GDH enzymes.

Animals↗

PubMed1986

A study of derepression of NAD-specific glutamate dehydrogenase of Neurospora crassa.

Transfer of Neurospora crassa mycelium from a 1% (w/v) sucrose medium to carbon-free or 1% (w/v) glutamate medium results in the onset of derepression of the catabolic NAD-specific glutamate dehydrogenase (NAD-GDH), within 30 min of the shift. Immunoprecipitation of in vivo pulse-labelled NAD-GDH demonstrated that this enzyme was synthesized de novo, correlating with increasing enzyme activity in shifted cells. Derepression was shown to be under transcriptional control by using the RNA synthesis inhibitor, picolinic acid, and by immunoprecipitation of the in vitro translation products of poly(A)-containing mRNA from repressed and derepressed cells. A brief (5 min) shift to derepression medium followed by a return to 1% (w/v) sucrose medium was sufficient to trigger synthesis of abundant NAD-GDH transcripts and low levels of the active enzyme. A secondary level of translational control is proposed to account for the discrepancy between the detectable levels of NAD-GDH transcripts and protein, following transient derepression.

Autoradiography↗

PubMed1966

Conformational changes and the regulation of glutamate-dehydrogenase activity.

1. The effect of NADH and the non-competitive inhibitor GTP on the optical-rotatory-dispersion properties of glutamate dehydrogenase has been studied. 2. Analysis of the data in terms of the a(0) and b(0) parameters of the Moffitt-Yang equation indicates that a conformational change is induced either by NADH or by GTP in the presence of small amounts of NADH. 3. Sedimentation measurements under comparable conditions showed that the enzyme reversibly dissociates into sub-units but that this dissociation is only secondary to the conformational changes. 4. Fluorescence measurements showed that the binding constant of NADH and the number of binding sites on the enzyme increased in the presence of GTP. 5. This is confirmed by studies of fluorescence polarization, which in addition showed that the movement of NADH on the enzyme surface is more restricted in the presence of GTP. 6. The relation of these results to possible regulatory mechanisms is discussed.

Chemical Phenomena↗

PubMed1997

Alteration of the quaternary structure of glutamate dehydrogenase from Clostridium symbiosum by a single mutation distant from the subunit interfaces.

X-ray crystallographic studies have previously shown that glutamate dehydrogenase from Clostridium symbiosum is a homohexamer. Mutation of the active-site aspartate-165 to histidine causes an alteration in the structural properties of the enzyme. The mutant enzyme, D165H exists predominantly as a single species of lower molecular mass than the wild-type enzyme as indicated by gel filtration and sedimentation velocity analysis. The latter technique gives an S20,w value for D165H of (6.07 +/- 0.01)S which compares with (11.08 +/- 0.01)S for the wild-type, indicative of alteration of the homohexameric quaternary structure of the native enzyme to a dimeric form, a result confirmed by sedimentation equilibrium experiments. Further support for this is provided by chemical modification by Ellman's reagent of cysteine-144 in the mutant, a residue which is buried at the dimer-dimer interface in the wild-type enzyme and is normally inaccessible to modification. The results suggest a possible structural route for communication between the active sites and subunit interfaces which may be important for relaying signals between subunits in allosteric regulation of the enzyme.

Chromatography, Gel↗

PubMed1992

The effect of AMP on the NAD-dependent glutamate dehydrogenase during activation and morphogenesis in the cellular slime moulds.

In extracts from vegetative Dictyostelium discoideum V12 the basal NAD-dependent glutamate dehydrogenase (NAD-GDH) activity was low, but it increased on standing at 4 degrees C. When 0.1 mM-AMP was included in the assay mix, enzyme activity was stimulated nearly 30-fold. As the extract was allowed to age, the enzyme rapidly lost its ability to be stimulated by AMP. The response of NAD-GDH to AMP was also dependent on the stage of morphogenesis. The ratios of NAD-GDH activity assayed with and without AMP (+AMP/-AMP ratios) in freshly prepared extracts from cells at 0, 4, 8 and 12 h of development were similar, but declined later in morphogenesis. The +AMP/-AMP ratio decreased sharply during activation at 4 degrees C in extracts from cells at 0, 4, 16 and 20 h of development. By contrast, extracts from cells starved for 8 and 12 h remained more responsive to AMP throughout activation. Analysis of Western blots showed that vegetative NAD-GDH did not undergo any detectable proteolytic cleavage during 96 h of activation at 4 degrees C. Also, no change in molecular mass appeared to take place within the cells until culmination (20-24 h), when some breakdown products appeared. Activation of NAD-GDH also occurred in D. discoideum strains NC4 and AX3, and in D. mucoroides. In addition, the enzyme from these four strains was stimulated by AMP and the +AMP/-AMP ratio declined with similar kinetics during activation. The enzyme from Polysphondylium violaceum was not activated on standing, but it was stimulated by AMP. The effect of activation of NAD-GDH is discussed in relation to a postulated catabolic role for this enzyme.

Adenosine Monophosphate↗

PubMed1999

Hyperinsulinism-hyperammonemia syndrome caused by mutant glutamate dehydrogenase accompanied by novel enzyme kinetics.

Hyperinsulinism-hyperammonemia syndrome (HHS) is a recently identified genetic disorder characterized by hyperinsulinemic hypoglycemia with concomitant hyperammonemia. In patients with HHS, activating mutations in the glutamate dehydrogenase (GDH) gene have been identified. GDH is a key enzyme linking glutamate metabolism with the Krebs cycle and catalyzes the conversion of glutamate to alpha-ketoglutarate. The activity of GDH is controlled by allosteric inhibition by GTP and, so far, all the mutations of HHS patients have been located within the GTP-binding site. Characteristically, GDH from these individuals have therefore normal basal activity in conjunction with a loss of GTP inhibition. In this study, however, we have identified a novel variant GDH in a patient with a more severe form of HHS. The mutation is located outside the GTP-binding site and the patient's GDH shows consistently higher activity, even in the absence of allosteric effectors. These results further support the hypothesis that the activating mutation of GDH is the cause of HHS. The mechanism leading to the activation of GDH, however, is not always related to the loss of GTP inhibition as was originally suggested.

Adenosine Diphosphate↗

PubMed2001

Identification of the GTP binding site of human glutamate dehydrogenase by cassette mutagenesis and photoaffinity labeling.

It has been reported that the hyperinsulinism-hyperammonemia syndrome is caused by mutations in glutamate dehydrogenase (GDH) gene that affects enzyme sensitivity to GTP-induced inhibition. To identify the GTP binding site(s) within human GDH, mutant GDHs at Tyr-266 or Lys-450 position were constructed by cassette mutagenesis. More than 90% of the initial activities were remained at the concentration of GTP up to 300 microm for the Lys-450 mutant GDHs regardless of their size, hydrophobicity, and ionization of the side chains, whereas the wild type GDH and the Tyr-266 mutant GDHs were completely inhibited by 30 microm GTP. The binding of GTP to the wild type GDH or the mutant GDHs was further examined by photoaffinity labeling with 8-[gamma-(32)P]azidoguanosine 5'-triphosphate (8-N(3)-GTP). Saturation of photoinsertion with 8-N(3)-GTP occurred apparent K(d) values near 20 microm for the wild type GDH or the Tyr-266 mutant GDH, and the photoinsertion of 8-N(3)-[gamma-(32)P]GTP was significantly decreased in the presence of 300 microm GTP. Unlike the wild type GDH or the Tyr-266 mutant GDH, less than 10% of photoinsertion was detected in the Lys-450 mutant GDH, and the photoinsertion was not affected by the presence of 300 microm GTP. The results with cassette mutagenesis and photoaffinity labeling demonstrate selectivity of the photoprobe for the GTP binding site and suggest that Lys-450, but not Tyr-266, is required for efficient binding of GTP to GDH. Interestingly, studies of the steady-state velocity showed that both the wild type GDH and the Tyr-266 mutant GDHs were inhibited by ATP at concentrations between 10 and 100 microm, whereas less than 10% of the initial activities of the Lys-450 mutant GDHs were diminished by ATP. These results indicate that Lys-450, but not Tyr-266, may be also responsible for the ATP inhibition; therefore, ATP bound to the GTP site.

Amino Acid Sequence↗

PubMed1981

Nitrogen control in Pseudomonas aeruginosa: a role for glutamine in the regulations of the synthesis of nadp-dependent glutamate dehydrogenase, urease and histidase.

In Pseudomonas aeruginosa the formation of urease, histidase and some other enzymes involved in nitrogen assimilation is repressed by ammonia in the growth medium. The key metabolite in this process appears to be glutamine or a product derived from it, since ammonia and glutamate did not repress urease and histidase synthesis in a mutant lacking glutamine synthetase activity when growth was limited for glutamine. The synthesis of these enzymes was repressed in cells growing in the presence of excess glutamine. High levels of glutamine were also required for the derepression of NADP-dependent glutamate dehydrogenase formation in the glutamine synthetase-negative mutant.

Ammonia↗