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PubMed1990

Failure of glutamate dehydrogenase system to predict oxygenation state of human skeletal muscle.

In a recent study, the total tissue contents of glutamate (Glu), ammonium (NH+4), and 2-oxoglutarate (2-OG) were used to estimate changes in the mitochondrial redox state ([NAD+]/[NADH]) of contracting skeletal muscle with intact circulation [Am. J. Physiol. 253 (Cell Physiol. 22): C263-C268, 1987]. These metabolites participate in the glutamate dehydrogenase (GDH) reaction, which, based on a number of assumptions, theoretically enables calculation of the mitochondrial redox state as follows (brackets indicate concentrations): [NAD+]/[NADH] = ([NH+4] [2-OG])/[( Glu]Kapp), where Kapp is the apparent equilibrium constant for GDH. The purpose of this study was to determine whether changes in the total tissue contents of Glu, NH+4, and 2-OG could be used to predict a reduction of the mitochondrial redox state in anoxic skeletal muscle. Anoxia was induced in the quadriceps femoris muscle by 10 min of circulatory occlusion (low metabolic rate) and isometric contraction to fatigue (high metabolic rate). The mean (+/- SE) value for the metabolite ratio ([NH+4][2-OG]/[Glu]) at rest was 6 +/- 3 mmol/kg dry wt (x 10(-4]. No significant change occurred after circulatory occlusion (4 +/- 2 x 10(-4); P greater than 0.05), whereas an almost 60-fold increase was observed after isometric contraction (P less than 0.05). Because the muscle was anoxic under both conditions, a significant decrease in the metabolite ratio should have occurred. These data demonstrate that changes in total tissue contents of Glu, NH+4, and 2-OG cannot be used to estimate changes in the redox and oxygenation state of mitochondria in intact human skeletal muscle.

Adenosine Triphosphate↗

PubMed2001

An essential histidine residue in GTP binding domain of bovine brain glutamate dehydrogenase isoproteins.

Greater than 90% of the original activity of the enzymes remained after modification of histidine residues of glutamate dehydrogenase (GDH) isoproteins from bovine brains with diethyl pyrocarbonate (DEPC). This suggests that the DEPC modified histidine residues are not critically involved in the catalysis of the GDH isoproteins. The influence of DEPC modified histidine residue(s) on binding of GTP to GDH isoproteins was investigated by protection studies. These studies showed that inhibition of GDH isoproteins by GTP was protected by preincubation of GDH isoproteins with DEPC. The amount of protection was dependent on the concentration of DEPC. The GTP inhibition was fully protected by preincubation of GDH isoproteins with DEPC at saturating concentrations. These results indicate that the histidine residues may play an important role in the GTP binding on GDH isoproteins. Spectrophotometric studies showed that three histidine residues per enzyme subunit were able to react with DEPC in the absence of GTP, whereas two histidine residues per enzyme subunit interacted with DEPC when the enzymes were preincubated with GTP. These results indicate that one of the histidine residues is involved in the GTP binding domain of GDH isoproteins. The quantitative affinity chromatographic studies showed that the influence of GTP on the binding of GDH isoproteins to DEPC-Sepharose was significantly distinct for the two GDH isoproteins. GDH I was more sensitively affected by GTP than GDH II in the binding affinity for DEPC-Sepharose. ADP, another well-known allosteric regulator, showed no significant changes in the interaction of DEPC with GDH isoproteins.

Adenosine Triphosphate↗

PubMed1997

Re-activation of Clostridium symbiosum glutamate dehydrogenase from subunits denatured by urea.

In a study of the re-activation of urea-denatured clostridial glutamate dehydrogenase (GDH) the maximum re-activation achieved without any added ligands was about 6%, but with NAD+ and 2-oxoglutarate in combination about 70%. NAD+ alone was also effective but 2-oxoglutarate was not, in striking contrast with the opposite pattern for protection of this enzyme against unfolding in urea [Aghajanian, Martin and Engel (1995) Biochem. J. 311,905-910]. The extent of re-activation was not increased by raising the incubation temperature to 37 degrees C and was independent of the time of enzyme denaturation. CD and fluorimetric studies showed that dilution of denatured enzyme into potassium phosphate buffer led to rapid (half-time <3-5 s)formation of 'structured' intermediates with secondary structure similar to that of native enzyme. These intermediate molecules were inactive, behaved as monomers on a size-exclusion column, and were unable to associate to give the native hexameric structure. Addition of NAD+ facilitated isomerization of these 'structured' monomers into a form(s) capable of re-activation. A side effect in the refolding process was non-specific aggregation, depending on final enzyme concentration. The hexamer fraction from re-activated samples, however, showed the same specific activity as native enzyme. The portion of the enzyme that is not lost through aggregation thus appears to regain the native structure fully. Detailed time-course studies showed that re-activation follows second-order kinetics, suggesting that formation of a dimer may be the rate-limiting step. The possible mechanism for the unfolding and refolding processes of clostridial GDH and effects of coenzyme and substrate on these are discussed in relation to the known crystal structure.

Clostridium↗

PubMed1977

The interaction of phospholipid membranes and detergents with glutamate dehydrogenase.

1. Both the anionic detergent sodium dodecylsulphate and the cationic detergent cetyltrimethylammonium bromide quenched the protein fluorescence of glutamate dehydrogenase. The anionic compound was more effective and brought about 50% quenching at a detergent concentration of 0.4 mM. The zwitterionic amphiphile, lysolecithin, did not quench the protein fluorescence and neither did the short-chain detergent n-hexylsulphonate, which under the range of concentrations examined (less than 1 mM) does not form micelles. 2. The zwitterionic phospholipid, phosphatidylcholine, did not quench the protein fluorescence but the anionic phospholipids, phosphatidylserine and cardiolipin, induced a reversible quenching of the enzyme fluorescence. These observations confirm the specificity of the phospholipid-enzyme interactions as deduced from the kinetic studies of the preceding paper. The degree of quenching brought about by the phospholipids decreased with increasing ionic strength and increasing pH and could be substantially reduced by basic proteins. An electrostatic contribution to the interaction is inferred from these results. 3. The binding of the anionic phospholipids to the enzyme is manifested in a further enhancement of the fluorescence of a 1-anilinonaphthalene-8-sulphonate-enzyme complex. The presence of substrates and allosteric effectors affect the interaction of the lipids with the enzyme as indicated by the magnitude of this increase in fluorescence. The enhancement of fluorescence of NADH when bound to the enzyme was not affected by the binding of the lipids. 4. The complex formed between the enzyme and phosphatidylserine/phosphatidylcholine can be solubilized in isooctane. The photolability of the aqueous protein when subjected to irradiation at 280 nm is suppressed in the isooctane-soluble complex. 5. Phosphatidylserine brings about a rapid (t 1/2 is about 150 ms at a lipid concentration of 0.75 mM) dissociation of the linear aggregates formed between the enzyme oligomers. 6. A model of the enzyme-lipid-membrane complex, consistent with these results, is proposed. It is suggested that the enzyme is an allotopic protein and that the dissociation of the enzyme in vitro may involve binding sites on the protein which are designed for interaction with the cardiolipin of the inner mitochondrial membrane, when the enzyme is in the mitochondrial matrix.

Ammonium Chloride↗

PubMed1998

The NADH-dependent glutamate dehydrogenase enzyme of Bacteroides fragilis Bf1 is induced by peptides in the growth medium.

Bacteroides fragilis Bf1 possesses two enzymes having glutamate dehydrogenase (GDH) activity. One is dual cofactor NAD(P)H-dependent, while the other has NADH-specific activity. The gene encoding the NADH-GDH (gdhB) was cloned by complementation of the glutamate auxotrophic mutant Escherichia coli MX3004 and the recombinant protein was characterized with respect to the GDH activities present in the parental organism grown under different nitrogen conditions. The NAD(P)H-dependent GDH of B. fragilis was confirmed to be most active under high ammonia conditions, but the NADH-specific GDH levels were increased by high peptide concentrations in the growth medium and not regulated by the levels of ammonia. Northern blotting analysis showed that gdhB regulation was at the transcription level, with a single transcript of approximately 1.6 kb being produced. GDH activity was demonstrated by zymography of the parental and recombinant enzymes. The recombinant GDH was NADH-specific and co-migrated with the equivalent enzyme band from B. fragilis cell extracts. The gdhB structural gene comprises 1335 bp and encodes a protein of 445 aa (49 kDa). Comparisons of the derived protein sequence with that of GDH from other bacteria indicated that significant sequence homology and conservation of functional domains exists with enzymes of Family I-type hexameric GDH proteins.

Amino Acid Sequence↗

PubMed2002

Structural and thermodynamic studies on a salt-bridge triad in the NADP-binding domain of glutamate dehydrogenase from Thermotoga maritima: cooperativity and electrostatic contribution to stability.

Cooperative interactions within ion-pair networks of hyperthermostable proteins are thought to be a major determinant for extreme protein stability. While the favorable thermodynamic contributions of optimized electrostatics in general as well as those of pairwise interactions have been documented, cooperativity between pairwise interactions has not yet been studied thermodynamically in proteins from hyperthermophiles. In this study we use the isolated cofactor binding domain of glutamate dehydrogenase from the hyperthermophilic bacterium Thermotoga maritima to analyze pairwise and cooperative interactions within the salt-bridge triad Arg190-Glu231-Lys193. The X-ray structure of the domain was solved at 1.43 A and reveals the salt-bridge network with surrounding solvent molecules in detail. All three participating charges in the network were mutated to alanine in all combinations. The X-ray structure of the variant lacking all three charges reveals that the removal of the side chains has no effect on the overall conformation of the protein. Using solvent denaturation and thermodynamic cycles, the interaction energies between each pair of residues in the network were determined in the presence and in the absence of the third residue. Both the Arg190-Glu231 ion pair and the Lys193-Glu231 salt bridge in the absence of the third residue, contribute favorably to the free energy for unfolding of the domain in urea. Using guanidinium chloride as denaturant reveals a strong cooperativity between the two ion-pair interactions, the presence of the second ion pair converts the first interaction from destabilizing into stabilizing by as much as 1.09 kcal/mol. The different energetics of the salt-bridge triad in urea and GdmCl are discussed with reference to the observed anion binding in the crystal structure at high ionic strength and their possible role in a highly charged, high-temperature environment such as the cytoplasm of hyperthermophiles.

Alanine↗

PubMed1994

Site and significance of chemically modifiable cysteine residues in glutamate dehydrogenase of Clostridium symbiosum and the use of protection studies to measure coenzyme binding.

Protein chemical studies of NAD(+)-dependent glutamate dehydrogenase (GDH; EC 1.4.1.2) from Clostridium symbiosum indicate only two cysteine residues/subunit, in good agreement with the gene sequence. Experiments with various thiol-modifying reagents reveal that in native clostridial GDH only one of these two cysteines is accessible for reaction. This residue does not react with iodoacetate, iodoacetamide, N-ethylmaleimide or N-phenylmaleimide, but reaction with either p-chloromercuribenzene sulphonate or 5,5'-dithiobis(2-nitrobenzoic acid) causes complete inactivation, preventable by NAD+ or NADH but not by glutamate or 2-oxoglutarate. Protection studies with combinations of substrates show that glutamate enhances protection by NADH, whereas 2-oxoglutarate diminishes it. These studies were also used to determine a dissociation constant (0.69 mM) for the enzyme-NAD+ complex. Similar data for NADH indicated mildly cooperative binding with a Hill coefficient of 1.32. The significance of these results is discussed in the light of the high-resolution crystallographic structure for clostridial GDH and in relation to information for GDH from other sources.

Amino Acid Sequence↗

PubMed1995

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

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

Journal Article↗

PubMed1999

The NAD-dependent glutamate dehydrogenase from the hyperthermophilic archaeon Pyrobaculum islandicum: cloning, sequencing, and expression of the enzyme gene(1).

The NAD-dependent glutamate dehydrogenase (GluDH) gene from the hyperthermophilic archaeon Pyrobaculum islandicum was cloned and expressed in Escherichia coli. Analysis of the nucleotide sequence revealed an open reading frame of 1266 bp encoding a protein of 421 amino acids with a molecular weight of 46,905. In the alignment of the amino acid sequence with those of mesophilic Clostridium symbiosum NAD-dependent GluDH and hyperthermophilic NADP-dependent enzymes from Thermococcus profundus and Pyrococcus furiosus, substitutions in the residues involved in dinucleotide binding were observed. On the other hand, the residues involved in glutamate binding were well conserved. This is the first description of the primary structure of NAD-dependent GluDH in hyperthermophilic archaea.

Amino Acid Sequence↗

PubMed1979

[Comparison of glutamate dehydrogenase and glutamine synthetase activities in the roots and aerial organs of an obligate halophyte: Suaeda maritima var. macrocarpa and a glycophyte: Phaseolus vulgaris, grown in presence of different concentration of NaCl].

The glutamate dehydrogenase and glutamine synthetase activities of an obligate halophyte, Suaeda maritima var. macrocarpa and a glycophyte. Phaseolus vulgaris are compared in function of salinity (increasing concentrations of NaCl) of the culturing solution. In culture, addition of NaCl stimulates glutamine synthetase activity and lowers glutamine dehydrogenase activity in the aerial organs and in the roots of Suaeda as opposed to what is observed in the glycophyte. Hence the obligatory halophily of Suaeda is related to an increase of the glutamine synthetase activity in a sal-trich medium corresponding to the stimulation of nitrate reductase and proteogenesis.

Glutamate Dehydrogenase↗

PubMed1991

Physiological and genetic analysis of the carbon regulation of the NAD-dependent glutamate dehydrogenase of Saccharomyces cerevisiae.

We found that cells of Saccharomyces cerevisiae have an elevated level of the NAD-dependent glutamate dehydrogenase (NAD-GDH; encoded by the GDH2 gene) when grown with a nonfermentable carbon source or with limiting amounts of glucose, even in the presence of the repressing nitrogen source glutamine. This regulation was found to be transcriptional, and an upstream activation site (GDH2 UASc) sufficient for activation of transcription during respiratory growth conditions was identified. This UAS was found to be separable from a neighboring element which is necessary for the nitrogen source regulation of the gene, and strains deficient for the GLN3 gene product, required for expression of NAD-GDH during growth with the activating nitrogen source glutamate, were unaffected for the expression of NAD-GDH during growth with activating carbon sources. Two classes of mutations which prevented the normal activation of NAD-GDH in response to growth with nonfermentable carbon sources, but which did not affect the nitrogen-regulated expression of NAD-GDH, were found and characterized. Carbon regulation of GDH2 was found to be normal in hxk2, hap3, and hap4 strains and to be only slightly altered in a ssn6 strain; thus, in comparison with the regulation of previously identified glucose-repressed genes, a new pathway appears to be involved in the regulation of GDH2.

Blotting, Northern↗

PubMed1971

Radiation-target molecular weights of urease and of L-glutamate dehydrogenase, and their relevance to the size of the functional subunits.

Activities of the oligomeric enzymes urease and l-glutamate dehydrogenase were measured after exposure as dry preparations to various doses of electron radiation. Inactivation curves were exponential. ;Target sizes' deduced from these were small compared with the molecular weights of the whole enzyme molecules, but accorded well with independent estimates of the sizes of functional subunits. It was concluded that, when these were in associated from, there could be no transfer of absorbed energy between subunits.

Air↗

PubMedDate not supplied

[Purification and properties of the NADP-specific glutamate dehydrogenase of Candida tropicalis feed yeasts].

From the cell-free extract of fodder yeast Candida tropicalis NADP-specific glutamate dehydrogenase was isolated and partially purified (75-fold) by means of fractional precipitation by ammonium sulphate and ion-exchange chromatography on DEAE-cellulose. The preparation was investigated with the aid of polyacrylamide gel electrophoresis. Kinetic characteristics of the enzyme in the cell-free extract and partially purified preparation were derived.

Animal Feed↗

PubMed1983

Beef liver glutamate dehydrogenase: a study of the oxidation of various alternative amino acid substrates retaining the correct spacing of the two carboxylate groups.

1. Several glutamate analogues substituted at the beta- or gamma-carbon atoms have been tested as substrates for glutamate dehydrogenase. 2. The two gamma-methyl derivatives and DL-beta-methylglutamate give the same pH optimum (8.7) as L-glutamate, but show inhibition by ADP and activation by GTP as pH 8, unlike glutamate and like the monocarboxylic substrate L-norvaline, which gives a pH optimum of 10. 3. L-gamma-methyleneglutamate, the poorest substrate tested (0.28% of rate with glutamate) gives a high pH optimum (10), like norvaline, but shows marked activation by both ADP (13-fold) and GTP (27-fold). 4. Despite the correct dicarboxylate spacing, all the analogues were much poorer substrates than L-norvaline.

Amino Acids↗

PubMedDate not supplied

[Activity of glutamate dehydrogenase, gamma-glutamyltranspeptidase and creatine kinase in saliva in gingivitis].

In the human saliva (oral fluid), the activity of glutamate dehydrogenase tends to decrease in gingivitis and diminishes in periodontitis. That of gamma-glutamyl transpeptidase and creatine kinase increases in gingivitis. In periodontitis, their activity is higher than that in gingivitis. The above changes in enzymatic activities reflect metabolic changes in the gingiva and periodontium in inflammation. The saliva from the salivary ducts shows no activity of the above enzymes. They enter the mixed saliva (oral fluid) together with epithelial and other oral tissue cells, with leukocytes and microbes which manifold in gingivitis.

Case-Control Studies↗

PubMed1978

[Heterogeneity and regulation of glutamate dehydrogenase activity in mammalian brain and liver].

The present report concerns the study of the catalytic properties and the coenzyme affinity of glutamate dehydrogenase (GDH) and its isoenzymes in various preparations of the brain and liver as well as the different regulatory mechanisms controlling the ratio of the rates of biogenesis and breakdown of glutamate (Glu). The investigations carried out showed that GDH activity of various preparations of brain and liver (crystalline enzymes, cellular extracts and mitochondria) are markedly different from each other by their catalytic and regulatory properties as well as by their coenzyme activity. The data obtained make us conclude that nicotinamide-hypoxanthine-nucleotide (deaminoNAD) is a more effective coenzyme in the oxidative deamination of Glu, than other piridine nucleotides (NAD, NADP, deamino-NADP). It is supposed that in the formation of ammonia and amino acids in brain and especially liver, together with other known mechanisms an important role may be ascribed to the process of transdeamination. In this aspect, as a co-factor of oxidative deamination of Glu deamino-NAD (D-NAD) is thought to be of significant importance.

Animals↗

PubMed1990

[Cerebral form of glutamate dehydrogenase activity in autonomic paroxysms (attacks of panic)].

A study was made of the activity of the cerebral form of glutamate dehydrogenase (GDH) in 59 patients suffering from vegetative paroxysms (VP), 8 patients with multiple sclerosis and 15 normal test subjects. As compared to the normal test subjects, the patients with VP manifested higher activity and lability of the enzyme. The patients with VP demonstrating the "labile" and "stable" enzyme were subjected to a clinicopsychological analysis which revealed a graver disease course and more pronounced anxiety in the group with the "stable" enzyme. The role of GDH in the pathogenesis of VP is under discussion.

Adult↗

PubMed2006

Effect of olanzapine treatment on platelet glutamine synthetase-like protein and glutamate dehydrogenase immunoreactivity in schizophrenia.

According to contemporary views, the glutamatergic system is implicated in the pathogenesis of schizophrenia, and atypical neuroleptics exert their effects (at least partially) through the glutamatergic system. Immunoreactive glutamate-metabolising enzymes, such as glutamine synthetase-like protein (GSLP) and two glutamate dehydrogenase isoenzymes (GDH), have been discovered in human platelets. The amount of GSLP in the platelets of 40 chronic patients with schizophrenia was found to be significantly higher than in 33 controls (consistent with our previous finding of increased amounts of GSLP in the prefrontal cortex of chronic schizophrenia patients). Moreover, survival analysis of the group of patients treated with olanzapine for 28 weeks showed that the larger amount of GSLP measured in platelets before treatment, the shorter the treatment time needed to achieve a positive clinical response (defined a priori as > or = 20% reduction in PANSS total score from the initial level before the treatment). Hence, GSLP level may serve as a predictor of the treatment duration to achieve a positive outcome with olanzapine. Both GSLP and GDH were found significantly changed in the course of treatment; hence, treatment with olanzapine influences the amounts of glutamate-metabolising enzymes in the platelets of chronic schizophrenia patients.

Adult↗