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At least 73 records · Page 4Linked to original sources
PubMed1977

Biogenesis of the mitochondrial matrix enzyme, glutamate dehydrogenase, in rat liver cells. II. Significance of binding of glutamate dehydrogenase to microsomal membrane.

1. Glutamate dehydrogenase and malate dehydrogenase solubilized from liver microsomes were able to rebind to microsomal vesicles while the corresponding dehydrogenases extracted from mitochondria showed no affinity for microsomes. 2. Competition was noticed between microsomal glutamate dehydrogenase and microsomal malate dehydrogenase in the binding to microsomal membranes. Mitochondrial malate dehydrogenase or bovine serum albumin did not inhibit the binding of microsomal glutamate dehydrogenase to microsomes. 3. Binding of microsomal glutamate dehydrogenase to microsomal membranes decreased when microsomes was preincubated with trypsin. 4. Rough microsomal glutamate dehydrogenase was more efficiently bound to rough microsomes than smooth microsomes. Conversely, smooth microsomal glutamate dehydrogenase had higher affinity for smooth microsomes than for rough microsomes. 5. A difference was noticed among the glutamate dehydrogenase isolated from rough and smooth microsomes, and from mitochondria, which suggested the possibility of minor post-translational modification of enzyme molecules in the transport from the site of synthesis to mitochondria.

Animals↗
PubMed1985

Effect of chronic ethanol or acetaldehyde on hepatic alcohol and aldehyde dehydrogenases, aminotransferases and glutamate dehydrogenase.

Ethanol or acetaldehyde orally administered (15% and 2% respectively in drinking water) to male Wistar rats for three months induced alterations in the main liver enzymes responsible for ethanol metabolism, aspartate and alanine aminotransferases and NAD glutamate dehydrogenase. Ethanol produced a significant decrease in the activity of soluble alcohol dehydrogenase, while acetaldehyde induced alterations both in soluble and mitochondrial aldehyde dehydrogenases: soluble activity was significantly higher than in the control and ethanol-treated groups, and mitochondrial activity was significantly diminished. Both soluble aspartate and alanine aminotransferases showed pronounced increases by the chronic effect of acetaldehyde, while mitochondrial activities were practically unchanged by the effect of ethanol or acetaldehyde. Mitochondrial NAD glutamate dehydrogenase showed a rise in its activity both by the effect of chronic ethanol and acetaldehyde consumption. The level of metabolites assayed in liver extracts showed marked differences between ethanol and acetaldehyde treatment which indicates that ethanol produced a remarkable increase in glutamate, aspartate and free ammonia together with marked decrease in pyruvate and 2-oxoglutarate concentrations. Acetaldehyde consumption induced a significant decrease in 2-oxoglutarate and pyruvate concentrations. These observations suggest that ethanol has an important effect on the urea cycle enzymes, while the effect of acetaldehyde contributes to the impairment of the citric acid cycle.

Acetaldehyde↗
PubMed2001

Regulation of human glutamate dehydrogenases: implications for glutamate, ammonia and energy metabolism in brain.

Glutamate dehydrogenase (GDH) catalyzes the oxidative deamination of glutamate to alpha-ketoglutarate using NAD or NADP as cofactors. In mammalian brain, GDH is located predominantly in astrocytes, where it is probably involved in the metabolism of transmitter glutamate. The exact mechanisms that regulate glutamate fluxes through this pathway, however, have not been fully understood. In the human, GDH exists in heat-resistant and heat-labile isoforms, encoded by the GLUD1 (housekeeping) and GLUD2 (nerve tissue-specific) genes, respectively. These forms differ in their catalytic and allosteric properties. Kinetic studies showed that the K(m) value for glutamate for the nerve tissue GDH is within the range of glutamate levels in astrocytes (2.43 mM), whereas for the housekeeping enzyme, this value is significantly higher (7.64 mM; P < 0.01). The allosteric activators ADP (0.1-1.0 mM) and L-leucine (1.0-10.0 mM) induce a concentration-dependent enzyme stimulation that is proportionally greater for the nerve tissue-specific GDH (up to 1,600%) than for the housekeeping enzyme (up to 150%). When used together at lower concentrations, ADP (10-50 mM) and L-leucine (75-200 microM) act synergistically in stimulating GDH activity. GTP exerts a powerful inhibitory effect (IC(50) = 0.20 mM) on the housekeeping GDH; in contrast, the nerve tissue isoenzyme is resistant to GTP inhibition. Thus, although the housekeeping GDH is regulated primarily by GTP, the nerve tissue GDH activity depends largely on available ADP or L-leucine levels. Conditions associated with enhanced hydrolysis of ATP to ADP (e.g., intense glutamatergic transmission) are likely to activate nerve tissue-specific GDH leading to an increased glutamate flux through this pathway.

Adenosine Diphosphate↗
PubMed2001

Contribution of glutamate dehydrogenase to mitochondrial glutamate metabolism studied by (13)C and (31)P nuclear magnetic resonance.

The relative contribution of glutamate dehydrogenase (GDH) and the aminotransferase activity to mitochondrial glutamate metabolism was investigated in dilute suspensions of purified mitochondria from potato (Solanum tuberosum) tubers. Measurements of glutamate-dependent oxygen consumption by mitochondria in different metabolic states were complemented by novel in situ NMR assays of specific enzymes that metabolize glutamate. First, a new assay for aminotransferase activity, based on the exchange of deuterium between deuterated water and glutamate, provided a method for establishing the effectiveness of the aminotransferase inhibitor amino-oxyacetate in situ, and thus allowed the contribution of the aminotransferase activity to glutamate oxidation to be assessed unambiguously. Secondly, the activity of GDH in the mitochondria was monitored in a coupled assay in which glutamine synthetase was used to trap the ammonium released by the oxidative deamination of glutamate. Thirdly, the reversibility of the GDH reaction was investigated by monitoring the isotopic exchange between glutamate and [(15)N]ammonium. These novel approaches show that the oxidative deamination of glutamate can make a significant contribution to mitochondrial glutamate metabolism and that GDH can support the aminotransferases in funneling carbon from glutamate into the TCA cycle.

Aminooxyacetic Acid↗
PubMed2002

Unregulated elevation of glutamate dehydrogenase activity induces glutamine-stimulated insulin secretion: identification and characterization of a GLUD1 gene mutation and insulin secretion studies with MIN6 cells overexpressing the mutant glutamate dehydrogenase.

Glutamate dehydrogenase (GDH) is important in normal glucose homeostasis. Mutations of GDH result in hyperinsulinism/hyperammonemia syndrome. Using PCR/single-strand conformation polymorphism analysis of the gene encoding GDH in 12 Japanese patients with persistent hyperinsulinemic hypoglycemia of infancy (PHHI), we found a mutation (Y266C) in one PHHI patient. This mutation was not found in any of the control or type 2 diabetic subjects. The activity of the mutant GDH (GDH266C), expressed in COS-7 cells, was constitutively elevated, and allosteric regulations by ADP and GTP were severely impaired. The effect of the unregulated increase in GDH activity on insulin secretion was examined by overexpressing GDH266C in an insulinoma cell line, MIN6. Although glutamine alone did not stimulate insulin secretion from control MIN6-lacZ, it remarkably stimulated insulin secretion from MIN6-GDH266C. This finding suggests that constitutively activated GDH enhances oxidation of glutamate, which is intracellularly converted from glutamine to alpha-ketoglutarate, a tricarboxylic acid cycle substrate, which thereby stimulates insulin secretion. Interestingly, insulin secretion is also exaggerated significantly at low glucose concentrations (2 and 5 mmol/l) but not at higher glucose concentrations (8--25 mmol/l). Our results directly illustrate the importance of GDH in the regulation of insulin secretion from pancreatic beta-cells.

Adenosine Diphosphate↗
PubMed1977

Biogenesis of the mitochondrial matrix enzyme, glutamate dehydrogenase, in rat liver cells. I. Subcellular localization, biosynthesis, and intracellular translocation of glutamate dehydrogenase.

1. The presence of glutamate dehydrogenase in the microsomal fraction of rat liver was confirmed. The identities of mitochondrial and microsomal glutamate dehydrogenases were proved by immunochemical methods and by SDS polyacrylamide gel electrophoresis of purified enzymes. 2. Synthesis of glutamate dehydrogenase by the membrane-bound ribosomes of rough endoplasmic reticulum was determined. Newly synthesized enzyme molecules were discharged on the cytoplasmic surface of endoplasmic reticulum membranes. 3. A precursor-product relationship was found between microsomal and mitochondrial glutamate dehydrogenases. About six hours were needed for the transport of glutamate dehydrogenase from the site of synthesis to mitochondria. 4. The half-life of glutamate dehydrogenase was about 5.5 days, which was somewhat longer than that of mitochondrial total protein determined in the same experiment. 5. Mitochondrial-type malate dehydrogenase was also present in the microsomal fraction. Subfractionation of smooth microsomes revealed the existence of particular light microsomal vesicles in which both glutamate dehydrogenase and malate dehydrogenase were concentrated. These vesicles may participate in intracellular transport of matrix enzymes from microsomes to mitochondria.

Animals↗
PubMed2006

Abiotic stress generates ROS that signal expression of anionic glutamate dehydrogenases to form glutamate for proline synthesis in tobacco and grapevine.

Glutamate dehydrogenase (GDH) may be a stress-responsive enzyme, as GDH exhibits considerable thermal stability, and de novo synthesis of the alpha-GDH subunit is induced by exogenous ammonium and senescence. NaCl treatment induces reactive oxygen species (ROS), intracellular ammonia, expression of tobacco (Nicotiana tabacum cv Xanthi) gdh-NAD;A1 encoding the alpha-subunit of GDH, increase in immunoreactive alpha-polypeptide, assembly of the anionic isoenzymes, and in vitro GDH aminating activity in tissues from hypergeous plant organs. In vivo aminating GDH activity was confirmed by gas chromatorgraphy-mass spectrometry monitoring of (15)N-Glu, (15)N-Gln, and (15)N-Pro in the presence of methionine sulfoximine and amino oxyacetic acid, inhibitors of Gln synthetase and transaminases, respectively. Along with upregulation of alpha-GDH by NaCl, isocitrate dehydrogenase genes, which provide 2-oxoglutarate, are also induced. Treatment with menadione also elicits a severalfold increase in ROS and immunoreactive alpha-polypeptide and GDH activity. This suggests that ROS participate in the signaling pathway for GDH expression and protease activation, which contribute to intracellular hyperammonia. Ammonium ions also mimic the effects of salinity in induction of gdh-NAD;A1 expression. These results, confirmed in tobacco and grape (Vitis vinifera cv Sultanina) tissues, support the hypothesis that the salinity-generated ROS signal induces alpha-GDH subunit expression, and the anionic iso-GDHs assimilate ammonia, acting as antistress enzymes in ammonia detoxification and production of Glu for Pro synthesis.

Amination↗
PubMed1972

The role of histidine residues in glutamate dehydrogenase.

1. Glutamate dehydrogenase was subject to rapid inactivation when irradiated in the presence of Rose Bengal or incubated in the presence of ethoxyformic anhydride. 2. Inactivation in the presence of Rose Bengal led to the photo-oxidation of four histidine residues. Oxidation of three histidine residues had little effect on enzyme activity, but oxidation of the fourth residue led to the almost total loss of activity. 3. Acylation of glutamate dehydrogenase with ethoxyformic anhydride at pH6.1 led to the modification of three histidine residues with a corresponding loss of half the original activity. Acylation at pH7.5 led to the modification of two histidine residues and a total loss of enzyme activity. 4. One of the histidine residues undergoing reaction at pH6.1 also undergoes reaction at pH7.5. 5. The presence of either glutamate or NAD(+) in the reaction mixtures at pH6.1 had no appreciable effect. At pH7.5 glutamate caused a marked decrease in both the degree of alkylation and degree of inactivation. NAD(+) had no effect on the degree of inactivation at pH7.5 but did modify the extent of acylation. 6. The normal response of the enzyme towards ADP was unaffected by acylation at pH6.1 or 7.5. 7. The normal response of the enzyme towards GTP was altered by treatment at both pH6.1 and 7.5.

Acylation↗
PubMed1984

Glutamate dehydrogenase and a proposed glutamate-aspartate pathway for citrate synthesis in rat ventral prostate.

Glutamate dehydrogenase activity was determined in mitochondrial preparations from rat ventral prostate and rat kidney. Kinetic parameters of the ventral prostate enzyme were comparable to those for the kidney enzyme. Glutamate dehydrogenase activity in the direction of glutamate oxidative deamination was inhibited by alpha-ketoglutarate. However, the characteristics of alpha-ketoglutarate inhibition indicated that glutamate oxidation via glutamate dehydrogenase can occur at in vivo prostatic alpha-ketoglutarate levels. These results suggest that glutamate dehydrogenase activity in prostate may provide a continuous source of alpha-ketoglutarate for aspartate transamination to oxalacetate and ultimate citrate synthesis. In addition prostate mitochondria are able to couple the glutamic dehydrogenase reaction to aspartate aminotransferase. Under these conditions aspartate in the presence of glutamate and acetyl coenzyme A will result in a net synthesis of citrate. Consequently we propose an aspartate-glutamate pathway for citrate synthesis in prostate.

Acetyl Coenzyme A↗
PubMed1977

Increased susceptibility of carbamylated glutamate dehydrogenase to proteolysis.

Glutamate dehydrogenase is very susceptible to carbamylation which results in loss of activity. The effect of a number of proteolytic enzymes (pronase, trypsin and chymotrypsin) on native and carbamylated glutamate dehydrogenase was tested. In all cases, the carbamylated enzyme was at least twice as susceptible to proteolysis as the native enzyme. Antibodies were prepared against glutamate dehydrogenase and carbamylated glutamate dehydrogenase; the carbamylated enzyme was antigenically indistinguishable from the native enzyme. Preliminary experiments indicate that the carbamylated glutamate dehydrogenase is taken up by ascites tumor cells while glutamate dehydrogenase is not. It seems possible that the effects described can be extrapolated to degradation by lysosomes and to other covalently modified enzymes.

Acylation↗
PubMed1977

Nicotinamide adenine dinucleotide-specific glutamate dehydrogenase of Neurospora. IV. The COOH-terminal 669 residues of the peptide chain; comparison with other glutamate dehydrogenases.

A sequence is presented for the COOH-terminal 669 residues of the NAD-specific glutamate dehydrogenase of Neurospora crassa. Comparison of this sequence with those of the vertebrate glutamate dehydrogenases of chicken and bovine liver and with the NADP-specific enzyme of Neurospora shows some similarities in sequences around residues previously identified as important for the function of these enzymes. These are: (a) the reactive lysine residue of low pK in the NADP and the vertebrate enzymes; (b) the tyrosine residue of the NADP enzyme that is readily nitrated by tetranitromethane with inactivation, a residue protected by NADP or by NMN; and (c) the arginine residue of the NADP-enzyme that is reactive with 1,2-cyclohexanedione with inactivation. Despite these similarities, comparison of the sequence of the NAD-enzyme with those of the other glutamate dehydrogenases of known sequences revealed relatively little overall homology as determined by computer analysis.

Amino Acid Sequence↗
PubMed1992

Some properties of glutamate dehydrogenase, glutamine synthetase and glutamate synthase from Corynebacterium callunae.

Characteristics of the three major ammonia assimilatory enzymes, glutamate dehydrogenase (GDH), glutamine synthetase (GS) and glutamate synthase (GO-GAT) in Corynebacterium callunae (NCIB 10338) were examined. The GDH of C. callunae specifically required NADPH and NADP+ as coenzymes in the amination and deamination reactions, respectively. This enzyme showed a marked specificity for alpha-ketoglutarate and glutamate as substrates. The optimum pH was 7.2 for NADPH-GDH activity (amination) and 9.0 for NADP(+)-GDH activity (deamination). The results showed that NADPH-GDH and NADP(+)-GDH activities were controlled primarily by product inhibition and that the feedback effectors alanine and valine played a minor role in the control of NADPH-GDH activity. The transferase activity of GS was dependent on Mn+2 while the biosynthetic activity of the enzyme was dependent on Mg2+ as essential activators. The pH optima for transferase and biosynthetic activities were 8.0 and 7.0, respectively. In the transfer reaction, the Km values were 15.2 mM for glutamine, 1.46 mM for hydroxylamine, 3.5 x 10(-3) mM for ADP and 1.03 mM for arsenate. Feedback inhibition by alanine, glycine and serine was also found to play an important role in controlling GS activity. In addition, the enzyme activity was sensitive to ATP. The transferase activity of the enzyme was responsive to ionic strength as well as the specific monovalent cation present. GOGAT of C. callunae utilized either NADPH or NADH as coenzymes, although the latter was less effective. The enzyme specifically required alpha-ketoglutarate and glutamine as substrates.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenine Nucleotides↗