Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:1.4.1.2 (glutamate dehydrogenase)
 4,380 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

1. NADP-dependent glutamate dehydrogenase (EC 1.4.1.4) extracted from nuclear fractions of Saccharomyces cerevisiae was partially purified. The final purification achieved was over 100-fold over the initial extract. 2. Cellulose acetate electrophoresis shows that the preparation is close to homogeneity and that the enzyme is slightly more anionic than cytoplasmic glutamate dehydrogenase. 3. The response of the nuclear activity to variation of pH, of inorganic phosphate and other electrolyte concentration and of the concentration of the reaction substrates has been investigated. Several differences were detected in comparison with cytoplasmic glutamate dehydrogenase.
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PMID:Purification and properties of NADP-dependent glutamate dehydrogenase from yeast nuclear fractions. 0 26

Intact cells and extracts from Spirillum lipoferum rapidly oxidized malate, succinate, lactate, and pyruvate. Glucose, galactose, fructose, acetate, and citrate did not increase the rate of O2 uptake by cells above the endogenous rate. Cells grown on NH+/4 oxidized the various substrates at about the same rate as did cells grown on N2. Added oxidized nicotinamide adenine dinucleotide generally enhanced O2 uptake by extracts supplied organic acids, whereas oxidized nicotinamide adenine dinucleotide phosphate had little effect. Nitrogenase synthesis repressed by growth of cells in the presence of NH+/4 was derepressed by methionine sulfoximine or methionine sulfone. The total glutamine synthetase activity from N2-grown cells was about eight times that from NH+/4-grown S. lipoferum; the response of glutamate dehydrogenase was the opposite. The total glutamate synthetase activity from N2-grown S. lipoferum was 1.4 to 2.6 times that from NH+/4-grown cells. The levels of poly-beta-hydroxybutyrate and beta-hydroxybutyrate dehydrogenase were elevated in cells grown on N2 as compared with those grown on NH+/4. Cell-free extracts capable of reducing C2H2 have been prepared; both Mg2+ and Mn2+ are required for good activity.
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PMID:Carbon and ammonia metabolism of Spirillum lipoferum. 1 Feb 78

Inhibition of bovine liver glutamate dehydrogenase by pyridoxal-5'-phosphate was studied by measuring the full time course of the oxidation of NADPH. Progress curves were determined before and after incubation of the enzyme with PLP in the presence of saturating concentrations of alpha-ketoglutarate and ammonium ion, at pH 7.4 and 25 degrees C. The data were fitted to the integrated Michaelis-Menten equation and an inhibition model derived. According to the model, PLP inhibits the enzyme non-competitively, by reversible formation of the complexes E--PLP and E--PLP--NADPH; the oxidation of NADPH is also inhibited by NADP+. After incubation with PLP, the dissociation constants of E--NADPH and E--NADP+ (Km and Kp) show a very definite decrease, while the maximum rate of oxidation (Vm) is increased. The inhibition constants for PLP were also computed.
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PMID:Full-time course studies of bovine liver glutamate dehydrogenase. Simulation of inhibition by pyridoxal-5'-phosphate. 1 Nov 96

Glutamate synthase was purified about 250-fold from Thiobacillus thioparus and was characterized. The molecular weight was estimated as 280,000 g/mol. The enzyme showed absorption maxima at 280, 380, and 450 nm and was inhibited by Atebrin, suggesting that T. thioparus glutamate synthase is a flavoprotein. The enzyme activity was also inhibited by iron chelators and thiolbinding agents. The enzyme was specific for reduced nicotinamide adenine dinucleotide phosphate (NADPH) and alpha-ketoglutarate, but L-glutamine was partially replaced by ammonia as the amino donor. The Km values of glutamate synthase for NADPH, alpha-ketoglutarate, and glutamine were 3.0 muM, 50 muM, and 1.1 mM, respectively. The enzyme had a pH optimum between 7.3 and 7.8. Glutamate synthase from T. thioparus was relatively insensitive to feedback inhibition by single amino acids but was sensitive to the combined effects of several amino acids. Enzymes involved in glutamate synthesis in T. thioparus were studied. Glutamine synthetase and glutamate synthase, as well as two glutamate dehydrogenases (NADH and NADPH dependent), were present in this organism. This levels of glutamate synthase and glutamate dehydrogenase were similar in T. thioparus grown on 0.7 or 7.0 mM ammonium sulfate. The sum of the activities of both glutamate dehydrogenases was only 1/25 of that of glutamate synthase under the assay conditions. It was concluded that the glutamine pathway is important for ammonia assimilation in this autotrophic bacterium.
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PMID:Purification and properties of glutamate synthase from Thiobacillus thioparus. 1 19

1. The interaction of beef liver glutamate dehydrogenase with cardiolipin from both beef liver mitochondria and beef heart mitochondria, with phosphatidylcholine from both beef liver mitochondria and egg-yolk, and with beef brain phosphatidylserine was investigated by steady-state kinetic methods. 2. the phosphatidylcholine did not inhibit the enzyme under a wide range of conditions. The cardiolipins and phosphatidylserine inhibited the enzyme. The inhibition by these lipids was found to diminish with time if the lipids were prepared and the reaction was studied in either phosphate or Tris buffers, but in zwitterionic buffers these lipid brought about a rapid, reversible inhibition which remained stable with time for at least 150 min. 3. The kinetic type of the inhibition was difficult to determine because of variation between lipid sonicates. Complex mixed types of inhibition were found with cardiolipin, and with phosphatidylserine the inhibition approximated to a non-competitive interaction with Ki(app) values varying between (0.9-6.1) x 10(-6)M. 4. The extent of inhibition decreased with increasing pH and with increasing ionic strength. Basic proteins, such as cytochrome c, show a higher affinity for the anionic membranes and can dissociate the enzyme-lipid complexes. Cosonicates of the cardiolipin and phosphatidylcholine inhibited the enzyme, the extent of inhibition increasing in proportion to the amount of acidic lipid. 5. Sodium dodecylsulphate causes a time-dependent inhibition of the enzyme. The kinetics of this effect and its variation with detergent concentration were studied. 6. The relationship of these observations to the structure and function of the enzyme is discussed. It is suggested that their apparent regulation of the enzyme by oestrogens and other small molecules is due to their binding in vitro at sites on the enzyme designed for binding cardiolipin, when the enzyme is functioning in vivo. The association of the enzyme oligomer in vitro may, for similar reasons, be an artifact.
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PMID:The interaction of phospholipid membranes and detergents with glutamate dehydrogenase. 1 31

In phosphate buffer at pH 7.0, 5,5'-dithio-bis(2-nitrobenzoic acid), N-ethylmaleimide or iodoacetamide do not alter the activity of beef liver glutamate dehydrogenase. Iodoacetate, however, inactivities the enzyme irreversibility by alkylation. Combined addition of the coenzyme NADH and the substrate 2-oxoglutarate or the effector GTP protects against this inactivation. The alkylation reaction is independent of pH between pH 6-9 indicating that amino, imidazole or phenolic groups are probably not involved in this reaction. Titration of the thiol groups, after denaturation of the enzyme, revealed the loss of approximately one group per polypeptide chain. However, this is not due to the exclusive alkylation of a cysteine residue, since alkylation with iodo-[2-14C]acetic acid also labels a methionine residue. 50% of the label is incorporated into methionine-169 and only 7% into cysteine-115, the remaining radioactivity is distributed in minor quantities (4%) in several unidentified residues. A probable cause of the erroneous thiol groups titration is discussed.
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PMID:Studies of glutamate dehydrogenase. Methionine-169: the preferentially carboxymethylated residue. 1 38

Kinetic studies of pyridoxal 5'-phosphate binding to glutamate dehydrogenase (EC 1.4.1.3) has provided evidence for two specific binding sites, chemically identified as Lys 126 and Lys 333. Use of protecting ligands permitted the selective modification of only one of these lysines, and showed that (1) Lys 333 modification results in depolymerisation of the enzyme into active hexamers; (2) Lys 126-modified enzyme was 92% inactivated. The residual activity was desensitized to GTP. The inactivation process was cooperative, maximum inactivation occurring as soon as half of the Lys 126 were modified.
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PMID:Physicochemical evidence for the existence of two pyridoxal 5'-phosphate binding sites on glutamate dehydrogenase and characterization of their functional role. 2 Jan 55

The synthesis of an affinity adsorbent, 8-(6-aminohexyl)aminoadenosine 2'-phosphate-Sepharose 4B, is described. The assembly of the 2'-AMP ligand and the hexanediamide spacer arm was synthesized in free solution before its attachment to the Sepharose matrix. This adsorbent retarded the hexameric NADP-specific glutamate dehydrogenase of Neurospora crassa, showing a capacity for this enzyme similar to that of comparable coenzyme-analogue adsorbents for other dehydrogenases. The enzyme was eluted either at pH 6.8 in a concentration gradient of NADP+, or at pH 8.5 in the presence of NADP+ in concentration gradients of either dicarboxylates or NaCl. Anomalous effects of dicarboxylates in facilitating elution are discussed. 2'-AMP and its derivatives, 8-bromoadenosine 2'-phosphate and 8-(l-aminohexyl)aminoadenosine 2'-phosphate, which were used in the synthesis of the adsorbent, all acted as enzyme inhibitors competitive with NADP+. The chromatographic properties of the wild-type enzyme were compared with those of mutationally modified variants containing defined amino acid substitutions. This approach was used to assess the biospecificity of adsorption and elution and the contribution of non-specific binding. The adsorbent showed a low capacity for the enzyme from mutant am1 (Ser-336 replaced by Phe), a variant that has a localized defect in NADP binding, but an otherwise almost normal conformation, suggesting that non-specific interactions are at most weak. The enzyme from mutant am3, a variant modified in a conformational equilibrium, was fully retarded by the adsorbent, but showed a significantly earlier elution position than the wild-type enzyme. This is consistent with measurements in free solution that showed the am3 enzyme to have a higher Ki for 2'-AMP than the wild-type enzyme. The enzyme from mutant am19 was eluted as two distinct peaks at both pH 6.8 and 8.5. The adsorbent was used to separate hybrid hexamers constructed in vitro by a freeze-thaw procedure from pairs of purified variants. Several chromatographically distinct peaks of differing enzymological properties were purified from each hybridization mixture in quantities of up to a few milligrams, and represented distinct species of hybrid hexamers differing in subunit ratio.
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PMID:Affinity chromatography of the Neurospora NADP-specific glutamate dehydrogenase, its mutational variants and hybrid hexamers. 2 28

Yeast cells growing on mineral medium plus ammonia and glucose contained high levels of nicotinamide adenine dinucleotide phosphate-dependent glutamate dehydrogenase activity, as measured in crude extracts. After suspension of cells in fresh medium lacking glucose, there was a loss of the glutamate dehydrogenase activity. Loss of activity was inhibited by 2,4-dinitrophenol, sodium azide, iodoacetic acid, and cycloheximide. The enzyme activity was restored when glucose was added back to the medium, and this recovery was fully prevented in the presence of cycloheximide.
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PMID:Effect of glucose starvation on the nicotinamide adenine dinucleotide phosphate-dependent glutamate dehydrogenase of yeast. 2 40

The nicotinamide adenine dinucleotide phosphate-dependent glutamate dehydrogenase (NADP-GDH) from the food yeast Candida utilis was found to be rapidly inactivated when cultures were starved of a carbon source. The addition of glutamate or alanine to the starvation medium stimulated the rate of inactivation. Loss of enzyme activity was irreversible since the reappearance of enzyme activity, following the addition of glucose to carbon-starved cultures, was blocked by cycloheximide. A specific rabbit antibody was prepared against the NADP-GDH from C. utilis and used to quantitate the enzyme during inactivation promoted by carbon starvation. The amount of precipitable antigenic material paralleled the rapid decrease of enzyme activity observed after transition of cells from NH(4) (+)-glucose to glutamate medium. No additional small-molecular-weight protein was precipitated by the antibody as a result of the inactivation, suggesting that the enzyme is considerably altered during the primary steps of the inactivation process. Analysis by immunoprecipitation of the reappearance of enzyme activity after enzyme inactivation showed that increase of NADP-GDH activity was almost totally due to de novo synthesis, ruling out the possibility that enzyme activity modulation is achieved by reversible covalent modification. Enzyme degradation was also measured during steady-state growth and other changes in nitrogen and carbon status of the culture media. In all instances so far estimated, the enzyme was found to be very stable and not normally subject to high rates of degradation. Therefore, the possibility that inactivation was caused by a change in the ratio of synthesis to degradation can be excluded.
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PMID:Evidence for the degradation of nicotinamide adenine dinucleotide phosphate-dependent glutamate dehydrogenase of Candida utilis during rapid enzyme inactivation. 2 41


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