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PubMed1976

An NADP-dependent Glutamate Dehydrogenase in Chloroplasts from the Marine Green Alga Caulerpa simpliciuscula.

NADP-dependent glutamate dehydrogenase was partially purified from extracts of the marine siphonous green alga Caulerpa simpliciuscula. The enzyme had an apparent Km NH(4) (+) of 0.4 to 0.7 mm and was highly specific for NADPH, alpha-ketoglutarate, and ammonium ions.The bulk of the NADP-glutamate dehydrogenase was isolated with the chloroplast fraction in cell-free preparations of this alga and was released from these "chloroplast fractions" as a soluble enzyme on gentle lysis of chloroplast membranes.

Journal Article↗

PubMed2005

Increase in NADH-glutamate dehydrogenase activity by mercury in excised bean leaf segments.

Application of Hg to excised bean leaf segments increased the glutamate dehydrogenase (NADH-GDH) activity substantially. However, specific activity of the enzyme decreased at lower concentration of Hg, and increased to lesser extent at higher concentration of Hg. Mercury supply increased the glutamate synthase (NADH-GOGAT) activity also. Mercury supply increased the NADH-GDH activity in the presence of NH4NO3, but to a lesser extent than in the absence of NH4NO3. The specific activity of the enzyme decreased considerably at lower concentration of Hg, but increased significantly at higher concentration of Hg. An increase in NADH-GOGAT activity was observed in the presence of NH4NO3, but specific activity of the enzyme decreased marginally. Increase in GDH activity due to Hg remained unaffected by the supply of sucrose, but was reduced by glutamine and glutathione and enhanced by Al. The glutamate dehydrogenase (+Hg enzyme) from mercury treated leaf segments had higher value of S0.5 for NADH than the enzyme (-Hg enzyme) from material not treated with mercury indicating that Hg binding to enzyme prevented NADH binding to the enzyme possibly at thiol groups. However, + Hg enzyme has more reactivity, as apparent Vmax value was higher for it. It has been suggested that Hg activates the NADH-GDH enzyme in the bean leaf segments by binding to thiol groups of protein and pronounced increase in activity by Hg suggests a possible role of enzyme under Hg-stress.

Aluminum↗

PubMed1995

Glutamate dehydrogenase in astrocytes of the rat dentate gyrus following lesion of the entorhinal cortex.

Applying quantitative microscopic histochemistry, the activity of the mitochondrial glutamate dehydrogenase which is localized in astrocytes was determined in the molecular layer of the dentate gyrus of the rat hippocampus. This hippocampal region contains the important terminations of the glutamatergic perforant path. For comparison, determinations of the mitochondrial succinate dehydrogenase were performed, which is localized preferentially in terminals and dendrites. Two age groups of animals were examined: young adults (three months old) and aged subjects (24 months old). Both age groups were divided into controls, and animals killed three, 21 and 90 days following unilateral electrolytic lesion of the entorhinal cortex. The post-lesional shrinkage of the terminal field of the perforant path, ipsilateral to the lesion side, was determined and considered in the evaluation of enzymatic data. Statistic analysis revealed that ipsilateral to the lesion side there was a significant decrease of glutamate and succinate dehydrogenase activities in the terminal field of the perforant path three, 21 and 90 days following lesion. It is reasonable to assume that the decrease of succinate dehydrogenase activity (50-60%) was caused by the loss of mitochondria localized in degenerating terminals, whereas the decrease of glutamate dehydrogenase activity (20-30%) was related to the decrease of glutamatergic transmission following lesion. In the terminal field of the perforant path contralateral to the lesion side both significant increases and decreases of enzyme activities were measured following lesion. From these results it is concluded that the hippocampus contralateral to the lesion side cannot be considered as an appropriate intraindividual control. The comparison between young and aged animals showed no differences in the demonstration of glutamate dehydrogenase and only restricted differences in the activity level of succinate dehydrogenase post-lesion. Therefore, it is reasonable to assume that the post-lesional reactivity of the enzymes studied was very similar in both age groups.

Age Factors↗

PubMed2003

Regulatory effects of 5'-deoxypyridoxal on glutamate dehydrogenase activity and insulin secretion in pancreatic islets.

It has been known that glutamate, generated by glutamate dehydrogenase (GDH), acts as an intracellular messenger in insulin exocytosis in pancreatic beta cells. Here we demonstrate the correlation of GDH activity and insulin release in rat pancreatic islets perfused with 5'-deoxypyridoxal. Perfusion of islets with 5'-deoxypyridoxal, an effective inhibitor of GDH, reduced the islet GDH activity at concentration-dependent manner. Treatment of 5'-deoxypyridoxal up to 2 mM did not affect the cell viability. There was reduction in V(max) values on average about 60%, whereas no changes in K(m) values for substrates and coenzymes were observed. The concentration of GDH on the Western blot analysis and the level of GDH mRNA remained unchanged. The concentration of glutamate decreased by 52%, whereas the concentration of 2-oxoglutarate increased up to 2.3-fold in the presence of 5'-deoxypyridoxal. 5'-Deoxypyridoxal had no effects on inhibition by GTP and activation by ADP or L-leucine of islet GDH. In parallel with the inhibition of GDH activity, perfusion of islets with 5'-deoxypyridoxal reduced insulin release up to 2.5-fold. Although precise mechanism for correlation between GDH activity and insulin release remains to be studied further, our results suggest a possibility that the inhibitory effect of 5'-deoxypyridoxal on islet GDH activity may correlate with its effect on insulin release.

Adenosine Diphosphate↗

PubMed1980

Secondary structure predictions for the NAD-specific glutamate dehydrogenase of Neurospora crassa.

From the amino acid sequences of the three known fragments of the NAD-specific glutamate dehydrogenase of Neurospora crassa, the secondary structures have been predicted from the rules of Chou and Fasman (Chou, P.Y., and Fasman, G.D. (1979) Biophys. J. 26, 367-384). Comparison of these structures with those calculated for bovine glutamate dehydrogenase has shown that in the regions of homologous sequences containing identified functional regions, there is considerable homology of structure. From these predictions, it has been possible to identify a putative coenzyme-binding domain in the COOH-terminal part of the molecule similar to those of various NAD-specific dehydrogenases. Residues whose modification alters coenzyme binding are located in the putative coenzyme binding domain. The major sites of tryptic cleavage of the native enzyme, described in an accompanying paper (Haberland, M.E., Chen, C.-W., and Smith, E.L. (1980) J. Biol. Chem. 255, 7993-8000), are in regions of random coil structure.

Amino Acid Sequence↗

PubMed1975

The structural gene for NADP L-glutamate dehydrogenase in Aspergillus nidulans.

A total of 41 mutants lacking NADP L-glutamate dehydrogenase (NADP-GDH) activity have been studied. All the mutations were located at the gdhA locus within 0-1% recombination of gdhAI. Two mutants, gdhAI and gdhA2, out of five examined, produced cross-reacting material which neutralized NADP-GDH anti-serum. The mutant gdhA9 has altered Km values for all five substrates: ammonium, alpha-ketoglutarate, l-glutamate, NADPH and NADP. The mutant gdhA20 had temperature-sensitive growth, abnormal ammonium-regulation characteristics and thermolabile NADP-GDH activity. These results show that gdhA is the structural gene for NADP-GDH.

Aldehyde Oxidoreductases↗

PubMed1995

Steady-state in vivo glutamate dehydrogenase activity in rat brain measured by 15N NMR.

The in vivo activity of glutamate dehydrogenase (GDH) in the direction of reductive amination was measured in rat brain at steady-state concentrations of brain ammonia and glutamate after intravenous infusion of the substrate 15NH4+. The in vivo rate was determined from the steady-state fractional 15N enrichment of brain ammonia, measured by selective observation of 15NH4+ protons in brain extract by 1H-15N heteronuclear multiple-quantum coherence transfer NMR, and the rate of increase of brain [15N]glutamate and [2-15N]glutamine measured by 15N NMR. The in vivo GDH activity was 0.76-1.17 mumol/h/g, and 1.1-1.2 mumol/h/g at 1.0 +/- 0.17 mumol/g. Comparison of the observed in vivo GDH activity with the in vivo rates of glutamine synthesis and of phosphate-activated glutaminase suggests that, under mild hyperammonemia, GDH-catalyzed de novo synthesis can provide a minimum of 19% of the glutamate pool that is recycled from neurons to astrocytes through the glutamate-glutamine cycle.

Ammonia↗

PubMed1991

[Glutamate dehydrogenase and glutamine synthetase activity in the digestive tract in lambs in relation to age].

Developmental dynamics was investigated in the activity of glutamate dehydrogenase (GDH, E.C. 1.4.1.2.-4) and glutamine synthetase (GS, E.C. 6.3.1.2) in different parts of the digestive tract of lambs, in dependence on the age from 10 to 90 days; the goal of these investigations was to elucidate in greater detail the role of the above enzymes in nitrogen metabolism. The activity of GDH, and of the coenzymes NADH and NADPH, was followed in the digesta because simple organisms (bacteria, fungi, plants) have two glutamate dehydrogenases: they differ from each other by coenzyme specificity, unlike GDH from animal sources which can utilize both NADH coenzyme and NADPH coenzyme (Fahien et al., 1965; Frieden, 1964). The following activities of GDH and GS were found out in trials with lambs at the age of 10, 20, 30, 40 and 90 days, as to the different parts of digestive tract: in the tissues of rumen, omasum, reticulum, spleen, duodenum, jejunum, ileum, int. caecum and colon the activity of GDH (NADH) varied from 0.031 to 0.305 nkat/mg dry matter, in the digesta from 0 to 2.92 nkat/mg dry matter. An investigation of GDH (NADH, NADPH) dynamics in the digesta of lambs showed the relatively high activity of GDH (NADH) in the digesta of colon at the age of 10 days and that of GDH (NADPH) in the digesta of int. caecum. The activity of GDH (NADH) was also found to be high in the digesta of int. caecum at the age of 20 days. In that period the activity of GDH (NADH, NADPH) in the digesta of rumen, omasum and reticulum was zero.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1989

Effects of sodium nitroprusside on blood glucose concentration, B-cell morphology and islet glutamate dehydrogenase activity in mice.

Mice injected with sodium nitroprusside (NaNP) exhibited a marked, transient hyperglycemia and enhanced activity of glutamate dehydrogenase in the pancreatic islets. Ultrastructurally, the islet B-cells of NaNP-treated mice showed expanded granular endoplasmic reticulum, prominent Golgi complex, increased amount of secretory granules, mitochondrial enlargement and vacuolation, and mitochondrion-secretory granule complexes. Stereological analyses disclosed increased volume of endoplasmic reticulum, Golgi complex and mitochondria, and increased number of secretory granules in the B-cells 1 h after the injection of NaNP. Isolated mouse islets exposed to NaNP showed stimulated activity of glutamate dehydrogenase both in the presence of 2 and 18 mM glucose, whereas the release of insulin was stimulated at 2 mM glucose, but inhibited at 18 mM glucose. The observations demonstrate that NaNP induces transiently altered structure and function in mouse islet B-cells.

Animals↗

PubMed1994

Flow injection determination of glutamate in human serum and rat brain samples with immobilized glutamate oxidase and glutamate dehydrogenase reactors.

Two methods are proposed for the determination of regional concentrations of glutamate in the rat brain as well as in human serum. Glutamate oxidase was immobilized on non-porous glass beads and glutamate dehydrogenase was immobilized on glass derivatives. These supports were employed for the construction of Single Bead String Reactors and Packed Bed Reactors, respectively, which in turn were linked to Flow Injection Analysis systems with either photometric or fluorometric detection. Analytical working curves are linear in the range 1-200 mumol/l for packed bed reactors and 10-500 mmol/l for single bead string reactors. The samples were pretreated depending on their origin and the applied measuring system. Optimal dilution factors were established for the two techniques. Optimal dilution ratios were established and the influence of several added substances was investigated. Recovery and method comparison studies including high performance liquid chromatography verified the accuracy of the proposed methods. Results from within-day and between-day measurements gave relative standard deviations of 4.7 and 5.9% for serum samples and 2.5 and 4.0% for brain samples, respectively.

Amino Acid Oxidoreductases↗

PubMed1986

Reaction of the 2',3'-dialdehyde derivative of NADPH at a nucleotide site of bovine liver glutamate dehydrogenase.

The 2',3'-dialdehyde derivative of NADPH (oNADPH) acts as a coenzyme for the reaction catalyzed by bovine liver glutamate dehydrogenase. Incubation of 250 microM oNADPH with enzyme for 300 min at 30 degrees C and pH 8.0 yields covalent incorporation of 1.0 mol of oNADPH/mol of enzyme subunit. The modified enzyme has a functional catalytic site and is activated by ADP, but is no longer inhibited by high NADH concentrations and exhibits decreased sensitivity to GTP inhibition. Using the change in inhibition by 600 microM NADH or 1 microM GTP to monitor the reaction leads to rate constants of 44.0 and 41.5 min-1 M-1, respectively, suggesting that loss of inhibition by the two regulatory compounds results from reaction by oNADPH at a single location. The oNADPH incorporation is proportional to the decreased inhibition by 600 microM NADH or 1 microM GTP, extrapolating to less than 1 mol of oNADPH/mol of subunit when the maximum change in NADH or GTP inhibition has occurred. Modified enzyme is still 93% inhibited at saturating levels of GTP, although its K1 is increased 20-fold to 4.6 microM. The kinetic effects caused by oNADPH are not prevented by alpha-ketoglutarate, ADP, 5 mM NADH, or 200 microM GTP alone, but are prevented by 5 mM NADH with 200 microM GTP. Incorporation of oNADPH into enzyme at 255 min is 0.94 mol/mol of peptide chain in the absence of ligands but only 0.53 mol/mol of peptide chain in the presence of the protectants 5 mM NADH plus 200 microM GTP. These results indicate that oNADPH modifies specifically about 0.4-0.5 sites/enzyme subunit or about 3 sites/enzyme hexamer and that reaction occurs at a GTP-dependent inhibitory NADH site of glutamate dehydrogenase.

Animals↗

PubMed1983

The radiation inactivation of glutamate dehydrogenase.

Pulse radiolysis and 60Co gamma radiolysis were used to study the effects of ionizing radiations on the activity of glutamate dehydrogenase. Hydroxyl radicals are considerably more effective than hydrated electrons in causing loss of enzymatic activity. Evidence is also presented that the free radical anions (SCN)-.2, (Br)-.2, and (I)-.2 react with the enzyme and cause a loss of enzymatic activity. The results implicate the possible involvement of cysteine, tyrosine, and tryptophan residues in the activity of glutamate dehydrogenase.

Binding Sites↗

PubMed1977

Studies of glutamate dehydrogenase. Analysis of quaternary structure and contact areas between the polypeptide chains.

Cross-linking of the unimer of glutamate dehydrogenase from beef liver (consisting of six polypeptide chains each having a molecular weight of 56000) with dimethyladipimidate and subsequent analysis by sodium dodecylsulfate electrophoresis shows predominantly the trimeric species (molecular weight 168000). Treatment with dimethylimidates of other chain length yields significantly less trimeric species indicating that the amino groups being cross-linked are within a distance of about 0.85 nm. Comparison of the molar amount of incorporated [14C]dimethyladipimidate with the number of modified amino groups (determined with trinitrobenzenesulfonic acid) shows that although 8-9 of the 34 amino groups have reacted, only 2-3 of them are involved in cross-links. Reaction with dimethylimidates inactivates the enzyme. The loss of the activity is partly concomitant to cross-linking to the trimeric species and not simply due to the modification of essential lysine residues. This is supported by the fact that, although more lysine residues react with mono-functional methylimidates, the loss of activity is reduced. Purified chymotryptic and tryptic peptides of the radioactive-labeled trimeric species were subjected to sequence analysis. Six peptides containing 75% of the total label were identified: one involves the amino-terminal residue alanine-1 and the others involve lysine-105, lysine-154, lysine-269, lysine-358 and lysine-399. Quantitative analysis of the specific radioactivity of each peptide/mol lysine leads to the conclusion that only lysine-105, lysine-154, lysine-269 and lysine-358 participate in cross-links, lysine-269 and lysine-358, respectively, being at isologous and lysine-105 cross-linked with lysine-154 at heterologous contact domains of the enzyme. A model for the planar arrangement of the trimeric species in the quaternary structure of glutamate dehydrogenase is discussed. It includes both isologous and heterologous contact areas between the polypeptide chains.

Amino Acid Sequence↗

PubMed1977

Glutamate dehydrogenase and aspartate aminotransferase in Trypanosoma cruzi.

1. The cultured, epimastigote-form of Trypanosoma cruzi contains NADP-linked glutamate dehydrogenase (EC 1.4.1.4), with a molecular weight of about 280,000, similar to the enzyme from Plasmodium chabaudi and different from the enzymes from higher animal sources. 2. T. cruzi also contains aspartate aminotransferase (EC 2.6.1.1), with properties similar to those of the enzyme from mammals. 3. The concerted action of the transaminase and glutamate dehydrogenase might be responsible for the production of NH3 which characterizes the protein catabolism in T. cruzi.

Animals↗

PubMed1980

Glutamate dehydrogenase from peas: isolation, quaternary structure, and influence of cations on activity.

The most active multiple molecular form of glutamate dehydrogenase from pea seeds has been enriched 30000-fold with a recovery of 60--70%. The preparation is homogenous as judged from gel electrophoresis of native and dodecylsulfate-denatured enzyme and from analytical ultracentrifugation. Specific activities were 530 U/mg in the reductive amination and 90 U/mg in the oxidative deamination reaction. A sedimentation coefficient of s20, w = 10.49 S was determined. The specific volume and the molecular weight of the native enzyme were found to be v2 = 0.759 cm3/g and Mr = 260000, respectively, by equilibrium sedimentation in H2O and 90% 2H2O buffers. Dodecylsulfate electrophoresis of the denatured enzyme yielded a molecular weight of Mr = 44000 for the polypeptide chain. From our data we propose glutamate dehydrogenase from peas to be a hexamer. The hexameric structure is confirmed by the appearance of six electrophoretic bands after cross-linking with diimidates. Enzyme which was treated with Dowex A1 chelating resin was found to be almost completely inactive in the absence of divalent metal ions. From several metals tested, calcium was most efficient in reactivating the enzyme; half-maximal activity was attained at about 5 microM calcium. In contrast to potassium ions, sodium ions were found to interfere with this regulatory mechanism by activating the enzyme at high concentrations.

Animals↗

PubMed1991

The gene for a halophilic glutamate dehydrogenase: sequence, transcription analysis and phylogenetic implications.

We have isolated and sequenced the gene for a putative NADP-dependent glutamate dehydrogenase from the extremely halophilic archaebacterium Halobacterium salinarium. This gene is transcribed as a unique RNA molecule of about 1700 nucleotides. The 5' end of the transcript contains characteristic consensus transcription initiation and promoter sequences observed in halophilic archaebacteria. The encoded polypeptide, with a predicted length of 435 amino acids, shows significant overall homology and conservation of functional domains when compared with different eubacterial and eukaryotic glutamate dehydrogenases. Surprisingly, the archaebacterial protein shares a larger number of identical amino acid residues with homologous polypeptides from higher eukaryotes than with those from unicellular eukaryotes and eubacteria.

Amino Acid Sequence↗

PubMed1992

NAD-dependent glutamate dehydrogenase from Pseudomonas aeruginosa is a membrane-bound enzyme.

Measurements of the deaminating activity of NAD-dependent glutamate dehydrogenase (NAD-GDH) in Pseudomonas aeruginosa strain 8602 (PAC 1) showed an initially constant rate that gave way to a 3.5-fold increased rate on prolonged incubation. Only the faster rate was observed when assay mixtures were preflushed with nitrogen or were treated with the detergent Triton X-100. Comparison of the intracellular distribution of NAD-GDH with marker enzymes showed it to be associated with the cytoplasmic membrane. The results suggest that NAD-GDH may be linked to oxygen through an electron-transport system.

Bacterial Proteins↗

PubMed1999

Insights into the mechanism of domain closure and substrate specificity of glutamate dehydrogenase from Clostridium symbiosum.

Comparisons of the structures of glutamate dehydrogenase (GluDH) and leucine dehydrogenase (LeuDH) have suggested that two substitutions, deep within the amino acid binding pockets of these homologous enzymes, from hydrophilic residues to hydrophobic ones are critical components of their differential substrate specificity. When one of these residues, K89, which hydrogen-bonds to the gamma-carboxyl group of the substrate l-glutamate in GluDH, was altered by site-directed mutagenesis to a leucine residue, the mutant enzyme showed increased substrate activity for methionine and norleucine but negligible activity with either glutamate or leucine. In order to understand the molecular basis of this shift in specificity we have determined the crystal structure of the K89L mutant of GluDH from Clostridium symbiosum. Analysis of the structure suggests that further subtle differences in the binding pocket prevent the mutant from using a branched hydrophobic substrate but permit the straight-chain amino acids to be used as substrates. The three-dimensional crystal structure of the GluDH from C. symbiosum has been previously determined in two distinct forms in the presence and absence of its substrate glutamate. A comparison of these two structures has revealed that the enzyme can adopt different conformations by flexing about the cleft between its two domains, providing a motion which is critical for orienting the partners involved in the hydride transfer reaction. It has previously been proposed that this conformational change is triggered by substrate binding. However, analysis of the K89L mutant shows that it adopts an almost identical conformation with that of the wild-type enzyme in the presence of substrate. Comparison of the mutant structure with both the wild-type open and closed forms has enabled us to separate conformational changes associated with substrate binding and domain motion and suggests that the domain closure may well be a property of the wild-type enzyme even in the absence of substrate.

Amino Acid Oxidoreductases↗