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PubMed1989

A spectrophotometric procedure for measuring oxoglutarate and determining aminotransferase activities using nicotinamide adenine dinucleotide phosphate-linked glutamate dehydrogenase from algae.

A new spectrophotometric procedure is described for determining glutamate-dependent activities of aspartate aminotransferase, alanine aminotransferase, and ornithine aminotransferase with NADPH-linked glutamate dehydrogenase (GDH) from nitrate-grown Stichococcus bacillaris. The algal NADPH-GDH is highly specific for oxoglutarate and can catalyze the reduction of this keto acid in the presence of high glutamate concentrations, and thus is suitable for the measurement of oxoglutarate produced in glutamate-dependent amino-transferase reactions. The alga produces large amounts of NADPH-GDH which can be adequately purified in a few simple steps. The purified enzyme can be stored at 4 degrees C for several weeks without any detectable loss of activity. The algal NADPH-GDH can also be used for the estimation of small amounts of oxoglutarate in aqueous extracts.

Animals↗
PubMed1996

NADP-specific glutamate dehydrogenase of Penicillium chrysogenum has a homohexamer structure.

The NADP-specific glutamate dehydrogenase of a high beta-lactam producing industrial strain of Penicillium chrysogenum was purified to homogeneity. The enzyme (M(r) = 339,000 +/- 34,000) was demonstrated to have a homohexamer quaternary structure with a subunit molecular mass of M(r) = 56,000 +/- 2000. The N-terminal sequence of the enzyme was also determined and was found to be highly homologous to other fungal NADP-specific glutamate dehydrogenase sequences.

Amino Acid Sequence↗
PubMed1981

Serum glutamate dehydrogenase as a marker of hepatocyte necrosis in alcoholic liver disease.

Serum glutamate dehydrogenase concentration was assessed as a marker of the degree of hepatocyte necrosis found at liver biopsy in 95 patients suspected of having alcoholic liver disease. Although the serum concentration was raised in 54 patients, no relation between it and the severity of hepatocyte necrosis could be established. Glutamate dehydrogenase was therefore not confirmed to be a useful indicator of hepatocyte necrosis in patients with chronic alcoholism.

Glutamate Dehydrogenase↗
PubMed1977

Regulatory effects of purine nucleotide analogs with liver glutamate dehydrogenase.

A total of 26 different purine nucleotides with specific modifications in the base moiety and/or in the polyphosphate chain as well as various combinations of nucleotides were tested as allosteric effectors of beef liver glutamate dehydrogenase (L-glutamate : NAD(P)+ oxidoreductase (deaminating), EC 1.4.1.3). The capacity of these nucleotide analogs to activate or to inhibit the glutamate dehydrogenase activity is expressed quantitatively and scaled between the extreme effects of ADP and GTP, respectively. The significance of distinct structural elements for the enzyme-effector interaction is discussed. While the inhibitory GTP site is less specific, accepting many natural and most modified nucleoside triphosphates as inhibitors, the activating ADP site shows a much higher specificity for nucleotides as activators.

Allosteric Regulation↗
PubMed1981

Reactivation of the phospho form of the NAD-dependent glutamate dehydrogenase by a yeast protein phosphatase.

A protein phosphatase was isolated from the yeast, Candida utilis, which could reactivate (dephosphorylate) the phosphorylated form of the NAD-dependent glutamate dehydrogenase. The protein could also dephosphorylate casein, histone and kemptide (a heptapeptide corresponding to the phosphorylation site of liver pyruvate kinase). Reactivation of the phosphorylated glutamate dehydrogenase was stimulated by the simultaneous addition of NAD and L-glutamate; 2-oxoglutarate, NH+4 and NADH had no effect. The reactivation of phosphorylated glutamate dehydrogenase could be inhibited by phosphate, pyrophosphate and fluoride.

Candida↗
PubMed1977

Glutamate dehydrogenase and glutamine synthetase activity in some organs of ruminants and monogastric animals.

A comparative study of glutamate dehydrogenase (GLDH 1.4.1.2) and glutamine synthetase (GS 6.3.1.2.) activity in liver, kidney and spleen homogenates from cattle, sheep, pigs and chickens showed that chicken liver contained on an average 3.5%, pig liver 8.3% and bovine liver 45.6% of the glutamate dehydrogenase activity present in sheep liver. Relatively low trace activity was found in the spleen and kidneys, except for the renal cortex of cattle (32% of activity in the liver). GS activity was the highest in chicken liver; in pigs it amounted to 33.40%, in cattle to 24.2% and in sheep to 19.7% of this activity. No marked interspecies differences were found in the values in the kidneys and spleen. It can be concluded from the results that the relatively high GLDH activity in the liver of ruminants compared with pigs and chicken is associated with the greater ability of ruminants to utilize ammonia. The higher GS activity and lower GLDH activity in chicken liver can be attributed to higher uric acid synthesis from ammonia via glutamine and purine bases and the lower ability of birds to utilize ammonia for protein synthesis. The presence of alanine dehydrogenase was not demonstrated in chicken liver, where the maximum oxidation of NADH after the addition to pyruvate and ammonia substrate was found.

Animals↗
PubMed1982

Regulation of bovine glutamate dehydrogenase. The effects of pH and ADP.

The activity of bovine liver glutamate dehydrogenase is affected in several ways depending on substrate concentrations and pH. At ph 6.5 and below, both oxidative deamination and reductive amination reactions are inhibited by ADP. At pH 7.0 and above both activatory and inhibitory effects can be observed depending on substrate concentrations. The effects are explicable in terms of a model with ADP binding at both a regulatory site and competing with coenzyme at the active site. The activatory effects of ADP result from destabilization of various abortive complexes by ADP binding to its regulatory site. The concerted effects of pH and ADP lead to a potentiation of either activation effects or inhibition effects depending on conditions. A consideration of in vivo concentrations of the various substrates involved and intramitochondrial pH and adenine nucleotide levels suggests that in vivo the reductive amination reaction is favored. It is suggested that glutamate dehydrogenase may be intimately involved with regulation of the urea cycle by responding to changes in the mitochondrial ammonia levels.

Adenosine Diphosphate↗
PubMed1989

Affinity labeling of a glutamyl peptide in the coenzyme binding site of NADP+-specific glutamate dehydrogenase of Salmonella typhimurium by 2-[(4-bromo-2,3-dioxobutyl)thio]-1,N6-ethenoadenosine 2',5'-bisphosphate.

NADP+-specific glutamate dehydrogenase from Salmonella typhimurium, cloned and expressed in Escherichia coli, has been purified to homogeneity. The nucleotide sequence of S. typhimurium gdhA was determined and the amino acid sequence derived. The nucleotide analogue 2-[(4-bromo-2,3-dioxobutyl)thio]-1,N6-ethenoadenosine 2',5'-bisphosphate (2-BDB-T epsilon A-2',5'-DP) reacts irreversibly with the enzyme to yield a partially inactive enzyme. After about 60% loss of activity, no further inactivation is observed. The rate of inactivation exhibits a nonlinear dependence on 2-BDB-T epsilon A-2',5'-DP concentration with kmax = 0.160 min-1 and KI = 300 microM. Reaction of 200 microM 2-BDB-T epsilon A-2',5'-DP with glutamate dehydrogenase for 120 min results in the incorporation of 0.94 mol of reagent/mol of enzyme subunit. The coenzymes, NADPH and NADP+, completely protect the enzyme against inactivation by the reagent and decrease the reagent incorporation from 0.94 to 0.5 mol of reagent/mol enzyme subunit, while the substrate alpha-ketoglutarate offers only partial protection. These results indicate that 2-BDB-T epsilon A-2',5'-DP functions as an affinity label of the coenzyme binding site and that specific reaction occurs at only about 0.5 sites/enzyme subunit or 3 sites/hexamer. Glutamate dehydrogenase modified with 200 microM 2-BDB-T epsilon A-2',5'-DP in the absence and presence of coenzyme was reduced with NaB3H4, carboxymethylated, and digested with trypsin. Labeled peptides were purified by high performance liquid chromatography and characterized by gas phase sequencing. Two peptides modified by the reagent were isolated and identified as follows: Phe-Cys(CM)-Gln-Ala-Leu-Met-Thr-Glu-Leu-Tyr-Arg and Leu-Cys(CM)-Glu-Ile-Lys. These two peptides were located within the derived amino acid sequence as residues 146-156 and 282-286. In the presence of NADPH, which completely prevents inactivation, only peptide 146-156 was labeled. This result indicates that modification of the pentapeptide causes loss of activity. Glutamate 284 in this peptide is the probable reaction target and is located within the coenzyme binding site.

Adenosine Diphosphate↗
PubMed1998

Enzymological characteristics of the hyperthermostable NAD-dependent glutamate dehydrogenase from the archaeon Pyrobaculum islandicum and effects of denaturants and organic solvents.

NAD-dependent glutamate dehydrogenase (L-glutamate:NAD oxidoreductase, deaminating; EC 1.4.1.2) was purified to homogeneity from a crude extract of the continental hyperthermophilic archaeon Pyrobaculum islandicum by two successive Red Sepharose CL-4B affinity chromatographies. The enzyme is the most thermostable NAD-dependent dehydrogenase found to date; the activity was not lost after incubation at 100 degrees C for 2 h. The enzyme activity increased linearly with temperature, and the maximum was observed at ca. 90 degrees C. The enzyme has a molecular mass of about 220 kDa and consists of six subunits with identical molecular masses of 36 kDa. The enzyme required NAD as a coenzyme for L-glutamate deamination and was different from the NADP-dependent glutamate dehydrogenase from other hyperthermophiles. The Km values for NAD, L-glutamate, NADH, 2-oxoglutarate, and ammonia were 0.025, 0.17, 0.0050, 0.066, and 9.7 mM, respectively. The enzyme activity was significantly increased by the addition of denaturants such as guanidine hydrochloride and some water-miscible organic solvents such as acetonitrile and tetrahydrofuran. When fluorescence of the enzyme was measured in the presence of guanidine hydrochloride, a significant emission spectrum change and a shift in the maximum were observed but not in the presence of urea. These results indicate that this hyperthermophilic enzyme may have great potential in applications to biosensor and bioreactor processes.

Amino Acid Sequence↗
PubMed1979

Occurrence of thermolabile and regulatory NAD-linked glutamate dehydrogenase in Pseudomonas fluorescens.

NAD-linked glutamate dehydrogeanse [EC 1.4.1.2] was detected together with NADP-linked glutamate dehydrogenase [EC 1.4.1.4] and aspartase [EC 4.3.1.1] in Pseudomonas fluorescens cells. The three enzymes were distinctly separated by DEAE-Sephadex column chromatography. The NAD-linked enzyme was extremely thermolabile and was rapidly inactivated even at temperatures as low as 35--40 degrees C. The combined addition of NAD+ and glutamate, however, effectively stabilized the enzyme. The glutamate saturation profile of the NAD-linked enzyme exhibited cooperativity with a Hill coefficient (n) of 1.4. ATP inhibited the enzyme in an allosteric manner, increasing the n value to 2.2. These results suggest a novel type of metabolic regulation shared by the three enzymes in the biosynthesis and catabolism of amino acids.

Glutamate Dehydrogenase↗
PubMedDate not supplied

[The effect of neutral polymers on glutamate dehydrogenase activity of mouse liver mitochondria upon administration of endotoxin].

It is shown that neutral polymers administered intraperitoneally to intact animals considerably affect glutamate dehydrogenase activity in the liver cell mitochondria as well as in the supernatant. Of the tested polymers, only polyvinyl methylacetamide and dextran inhibit a decrease in the level of mitochondrial enzyme activity which develops with administration of endotoxin. Polyvinyl pyrrolidone with molecular weight of 100 kDa, polyvinyl methylacetamide, dextran and polyvinyl caprolactam prevent an increase of glutamate dehydrogenase activity in the supernatant in case of endotoxin administration to animals. It is possibly a result of the effect of the mitochondrial structure stabilization by the above polymers. A physiological effect of polyvinyl pyrrolidone revealed as an effect on the activity level of mitochondrial glutamate dehydrogenase and in the supernatant after endotoxin administration to animals, depends on the molecular weight of the polymer.

Animals↗
PubMed1984

Catalytic activity of bovine glutamate dehydrogenase requires a hexamer structure.

Previous workers have shown that the hexamers of glutamate dehydrogenase are dissociated first into trimers and subsequently into monomers by increasing guanidinium chloride concentrations. In renaturation experiments it is shown that trimers of glutamate dehydrogenase can be reassociated to give the hexamer form of the enzyme, with full regain of activity. Monomeric subunits produced at high guanidinium chloride concentrations cannot be renatured. The trimer form of the enzyme is shown to have no catalytic activity, although the hexamer form in guanidinium chloride has full activity.

Animals↗
PubMed1989

Purification and properties of NADP-dependent glutamate dehydrogenase from Streptomyces fradiae.

Streptomyces fradiae has two chromatographically distinct forms of glutamate dehydrogenase (GDH): one GDH utilizes NAD as coenzyme, the other uses NADP. The intracellular level of both GDHs is strongly regulated by the nitrogen source in the growth medium. NADP-dependent GDH was purified to homogeneity from crude extracts of S. fradiae. The Mr of the native enzyme was determined to be 200,000 by size-exclusion high-performance liquid chromatography whereas after sodium dodecyl sulphate-polyacrylamide gel electrophoresis one major band of Mr 49,000 was found, suggesting that the enzyme is a tetramer. The enzyme was highly specific for the substrates 2-oxoglutarate and L-glutamate, and required NADP, which could not be replaced by NAD, as a cofactor. The pH optimum was 9.2 for oxidative deamination of glutamate and 8.4 for reductive amination of 2-oxoglutarate. The Michaelis constants (Km) were 28.6 mM for L-glutamate and 0.12 mM for NADP. Km values for reductive amination were 1.54 mM for 2-oxoglutarate, 0.07 mM for NADPH and 30.8 mM for NH+4. The enzyme activity was significantly reduced by adenine nucleotides, particularly ATP.

Adenosine Diphosphate↗
PubMed1996

Glucocorticoid upregulation of glutamate dehydrogenase gene expression in vitro in astrocytes.

Glutamate, the major excitatory neurotransmitter, is preferentially catabolized in astrocytes by glutamate dehydrogenase (GDH). Treatment of an astrocytic cell line with hydrocortisone (10(-5) M) resulted in increased expression of GDH mRNA. Transfection of the cells with truncated parts of the GDH promoter showed that genomic responsive elements activated by hydrocortisone are localized in the -557/+1 region of the promoter. This control of GDH expression by glucocorticoids may be involved in their protective effect against glutamate excitotoxicity.

Animals↗
PubMed1995

Refolding of glutamate dehydrogenase from Bacillus acidocaldarius after guanidinium chloride-induced unfolding.

The hexameric NAD-dependent glutamate dehydrogenase from the thermophilic eubacterium Bacillus acidocaldarius is the first glutamate dehydrogenase which spontaneously refolds in vitro. After 24 h unfolding in 6 M guanidinium chloride at 20 degrees C, refolding was achieved by dilution of the denaturant. The yield of reconstitution obtained in the presence of 1,4 dithio-DL-threitol in the unfolding/refolding mixture was 75%. The unfolding/refolding equilibria have been studied by fluorescence, circular dichroism and catalytic activity, which was 50% inhibited at 0.08 M guanidinium chloride, a value 30-fold lower than the transition midpoint detected by physical changes. Refolding was attempted in the presence of various additives, at different temperatures and varying enzyme and residual guanidinium chloride concentration. The refolded enzyme, after removal of inactive aggregated species, completely resembles the native enzyme in term of its physicochemical and kinetic properties as well as thermophilicity.

Bacillus↗
PubMed1982

Glutamate dehydrogenase catalyzes the reduction of a Schiff base (delta 1-pyrroline-2-carboxylic acid) by NADPH.

alpha-Iminoglutarate has long been postulated as an obligatory intermediate in the glutamate dehydrogenase catalyzed reaction, but direct proof of its participation is lacking. We report here the glutamate dehydrogenase catalyzed reduction of delta 1-pyrroline-2-carboxylic acid (a cyclic-alpha-imino acid) to proline (an alpha-amino acid). The catalysis occurs at the normal catalytic site of the enzyme. The imine and the enzyme-NADPH complex are the active oxidant and reductant, respectively. The latter is about 500 times more reactive than NADPH itself. These findings provide direct evidence that the glutamate dehydrogenase catalyzed reaction does indeed proceed by way of an enzyme-bound form of an alpha-iminocarboxylic acid.

Animals↗
PubMed2003

Heat effect on the structure and activity of the recombinant glutamate dehydrogenase from a hyperthermophilic archaeon Pyrococcus horikoshii.

Glutamate dehydrogenase from Pyrococcus horikoshii (Pho-GDH) was cloned and overexpressed in Escherichia coli. The cloned enzyme with His-tag was purified to homogeneity by affinity chromatography and shown to be a hexamer enzyme of 290+/-8 kDa (subunit mass 48 kDa). Its optimal pH and temperature were 7.6 and 90 degrees C, respectively. The purified enzyme has outstanding thermostability (the half-life for thermal inactivation at 100 degrees C was 4 h). The enzyme shows strict specificity for 2-oxoglutarate and L-glutamate and requires NAD(P)H and NADP as cofactors but it does not reveal activity on NAD as cofactor. K(m) values of the recombinant enzyme are comparable for both substrates: 0.2 mM for L-glutamate and 0.53 mM for 2-oxoglutarate. The enzyme was activated by heating at 80 degrees C for 1 h, which was accompanied by the formation of its active conformation. Circular dichroism and fluorescence spectra show that the active conformation is heat-inducible and time-dependent.

Amino Acid Sequence↗