Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:1.4.3.11 (glutamate dehydrogenase)
4,437 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We recently described a preferential reduction of the secretory response to nutrient secretagogues (glucose; leucine plus glutamine) in islets maintained in culture after in vitro exposure to streptozotocin (SZ). The present study is an attempt to further clarify the biochemical mechanisms behind this defective insulin response. Mouse pancreatic islets were collagenase isolated and, after 4-5 days in culture, exposed during 30 min at 37 C to 1.8 mM SZ or vehicle alone (controls). The islets were subsequently cultured for 7 days in medium RPMI 1640 plus 10% calf serum, before the enzymatic and metabolic studies were performed. The activities of the glycolytic enzymes, hexokinase, glucokinase, and glyceraldehyde 3-phosphate dehydrogenase, were similar in the control and SZ-exposed islets. The relative amount of cytosolic and mitochondria-bound hexokinase was also unaffected by SZ. However, there was a 30-40% decrease in the activity of NAD+- and NADP+-dependent glutamate dehydrogenase and glutamate-aspartate transaminase in the SZ-treated islets. This coincided with a 40% decrease in L-[U-14C]glutamine oxidation in the SZ-treated islets. The D-glucose catabolism was further examined in the presence of D-[5-3H] and D-[6-14C] glucose. There was no difference between control and SZ islets in terms of glucose utilization at either 1.7 or 16.7 mM glucose. The oxidation of D-[6-14C]glucose was nevertheless decreased by more than 50% in SZ islets incubated at 16.7 mM (but not 1.7 mM) glucose. Altogether, these converging observations suggest a perturbation of distal regulatory processes, apparently at the mitochondrial level, in the D-glucose and L-glutamine catabolism of SZ-exposed islets. Whether this reflects a primary action of SZ on the islet mitochondria, or an inhibitory effect of SZ on the synthesis of mitochondrial enzymes, as a result of nuclear DNA damage, remains to be elucidated.
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PMID:Defective catabolism of D-glucose and L-glutamine in mouse pancreatic islets maintained in culture after streptozotocin exposure. 296 23

The activity of glutamate dehydrogenase (NADP+) (EC 1.4.1.4; NADP-GDH) of Saccharomyces cerevisiae is decreased under conditions in which intracellular ammonia concentrations increases. A high internal ammonia concentration can be obtained (a) by increasing the ammonium sulphate concentration in the culture medium, and (b) by growing the yeast either in acetate + ammonia media, where the pH of the medium rises during growth, or in heavily buffered glucose + ammonia media at pH 7.5. Under these conditions cellular oxoglutarate concentrations do not vary and changes in NADP-GDH activity appear to provide a constant rate of oxoglutarate utilization. The following results suggest that the decrease in NADP-GDH activity in ammonia-accumulating yeast cells is brought about by repression of synthesis: (i) after a shift to high ammonium sulphate concentrations, the number of units of activity per cell decreased as the inverse of cell doubling; and (ii) the rate of degradation of labelled NADP-GDH was essentially the same in ammonia-accumulating yeast cells and in controls, whereas the synthesis constant was much lower in the ammonia-accumulating cells than in the controls.
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PMID:Regulation by ammonium of glutamate dehydrogenase (NADP+) from Saccharomyces cerevisiae. 299 45

Reduced pyridine nucleotide dependent glutamate synthase [L-glutamate: NADP+ oxidoreductase (transaminating); EC 1.4.1.13] was purified to homogeneity from Bacillus subtilis PCI 219. The molecular weight of the enzyme was 210,000, and the enzyme was composed of two nonidentical subunits with molecular weights of 160,000 and 56,000. The absorption and CD spectra of the enzyme indicated that the enzyme is an iron-sulfur flavoprotein. The enzyme was found to contain 1:1:7.4:8.7 mol of FMN, FAD, iron atoms, and acid-labile sulfur atoms per mol (MW 210,000). EPR measurements of the NADPH-reduced enzyme at 77K revealed the formation of a stable flavin semiquinone intermediate; however, none of the signals originating from the iron-sulfur cluster was observed. Still at 4.2K the EPR signals in the region of g = 2, which may originate from the paramagnetic iron-sulfur cluster, were clearly observed for both the isolated and dithionite-reduced states of the enzyme. The enzyme exhibited a wide coenzyme specificity, and either NADPH or NADH could be used as electron donor, although the latter was less effective. The enzyme activity was also expressed when ammonium chloride was substituted for L-glutamine. The optimum pHs for NADPH-Gln-, NADH-Gln-, and NADPH-NH3-dependent reactions were 7.8, 6.9, and 9.4, respectively. The apoenzyme exhibited substantial inactivation of the Gln-dependent activities but still retained the NH3-dependent activities. Enzyme reduction-oxidation experiments, initial velocity experiments, and product inhibition patterns revealed that both the NADPH-Gln- and NADH-Gln-dependent reactions coincided with the two-site ping-pong uni-uni bi-bi kinetic mechanism, while the NADPH-NH3-dependent reaction deviated from Michaelis-Menten kinetics. The Gln-dependent activities were inhibited by several TCA cycle members, especially L-malate and fumarate, as well as L-methionine-SR-sulfoximine, pyridoxal-5'-phosphate, and pCMB. The regulation of the glutamate synthase, glutamine synthetase [EC 6.3.1.2], and glutamate dehydrogenase [EC 1.4.1.3] activities was examined with cultures of cells grown with various nitrogen and carbon sources.
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PMID:Glutamate synthase from Bacillus subtilis PCI 219. 301 66

The glutamate dehydrogenase (gdh) gene of Escherichia coli was transferred into an ammonium assimilation deficient mutant (Asm-) of Rhizobium japonicum (CJ9) using plasmid pRP301, a broad host range derivative of RP4. Exconjugants capable of growth on ammonia as sole N-source occurred at a frequency of 6.8 X 10(-6). Assimilatory GDH (NADP+) activity was detected in the strain carrying the E. coli gdh gene and the pattern of ammonia assimilation via GDH was similar to that of the Asm+ wild type strain. However, GDH mediated ammonia assimilation was not subject to regulation by L-glutamate. Nitrogenase activity was expressed ex planta in R. japonicum CJ9 harbouring the gdh gene, however, the presence of the gdh gene did not restore symbiotic effectiveness to the CJ9 Asm- strain in nodules. The gdh plasmid was maintained in approximately 90% of the isolates recovered from soybean nodules.
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PMID:Expression and regulation of the Escherichia coli glutamate dehydrogenase gene (gdh) in Rhizobium japonicum. 351 9

D-Glucose increased the cytosolic NADH/NAD+ ratio (but not the cytosolic NADPH/NADP+ ratio), augmented O2 uptake, raised the ATP/ADP ratio, decreased 86Rb outflow, and stimulated insulin release in tumoral insulin-producing cells of the RINm5F line. L-Leucine and 4-methyl-2-oxopentanoate also stimulated insulin secretion. In the RINm5F cells, as in normal islet cells, the nonmetabolized analogue of L-leucine, 2-aminobicyclo[2,2,1]heptane-2-carboxylic acid (BCH), activated glutamate dehydrogenase, augmented L-[U-14C]glutamine oxidation, and induced a more reduced state of cytosolic redox couples. However, in sharp contrast to either its effect in normal islet cells or that of D-glucose in the tumoral cells, BCH severely decreased O2 uptake, lowered the ATP/ADP ratio, increased 86Rb outflow, and inhibited insulin release in the RINm5F cells. These findings are interpreted to support the concept that the rate of ATP generation represents an essential determinant of the secretory response of insulin-producing cells to nutrient secretagogues.
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PMID:Opposite effects of D-glucose and a nonmetabolized analogue of L-leucine on respiration and secretion in insulin-producing tumoral cells (RINm5F). 354 45

The dual pyridine nucleotide-specific glutamate dehydrogenase [EC 1.4.1.3] was purified 37-fold from Bacteroides fragilis by ammonium sulfate fractionation, DEAE-Sephadex A-25 chromatography twice, and gel filtration on Sephacryl S-300. The enzyme had a molecular weight of approximately 300,000, and polymeric forms (molecular weights of 590,000 and 920,000) were observed in small amounts on polyacrylamide gel disc electrophoresis. The molecular weight of the subunit was 48,000. The isoelectric point of the enzyme was pH 5.1. This glutamate dehydrogenase utilized NAD(P)H and NAD(P)+ as coenzymes and showed maximal activities at pH 8.0 and 7.4 for the amination with NADPH and with NADH, respectively, and at pH 9.5 and 9.0 for the deamination with NADP+ and NAD+, respectively. The amination activity with NADPH was about 5-fold higher than that with NADH. The Lineweaver-Burk plot for ammonia showed two straight lines in the NADPH-dependent reactions. The values of Km for substrates were: 1.7 and 5.1 mM for ammonium chloride, 0.14 mM for 2-oxoglutarate, 0.013 mM for NADPH, 2.4 mM for L-glutamate, and 0.019 mM for NADP+ in NADP-linked reactions, and 4.9 mM for ammonium chloride, 7.1 mM for 2-oxoglutarate, 0.2 mM for NADH, 7.3 mM for L-glutamate, and 3.0 mM for NAD+ in NAD-linked reactions. 2-Oxoglutarate and L-glutamate caused substrate inhibition in the NADPH- and NADP+-dependent reactions, respectively, to some extent. NAD+- and NADH-dependent activities were inhibited by 50% by 0.1 M NaCl. Adenine nucleotides and dicarboxylic acids did not show remarkable effects on the enzyme activities.
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PMID:Properties of glutamate dehydrogenase purified from Bacteroides fragilis. 366 55

The RS-isomers of beta-mercapto-alpha-ketoglutarate, beta-methylmercapto-alpha-ketoglutarate and beta-methylmercapto-alpha-hydroxyglutarate have been synthesized. Beta-Mercapto-alpha-ketoglutarate was a potent inhibitor, competitive with isocitrate and noncompetitive with NADP+, of the mitochondrial NADP-specific isozyme from pig heart (Ki = 5 nM; Km (DL-isocitrate)/Ki(RS-beta-mercapto-alpha-ketoglutarate) = 650) and pig liver, the cytosolic isozyme from pig liver (I0.5 = 23 nM), and the NADP-linked enzymes from yeast (Ki = 58 nM) and Escherichia coli (Ki = 58 nM) at pH 7.4 and with Mg2+ as activator. beta-Mercapto-alpha-ketoglutarate was also an effective inhibitor of NADP-isocitrate-dehydrogenase activity in intact liver mitochondria. beta-Mercapto-alpha-ketoglutarate was a much less potent inhibitor for heart NAD-isocitrate dehydrogenase (Ki = 520 nM) than for the NADP-specific enzyme. beta-Methylmercapto-alpha-ketoglutarate (I0.5 = 10 microM) was a much less effective inhibitor than the beta-mercapto derivative for heart NADP-isocitrate dehydrogenase. The beta-sulfur substituted alpha-ketoglutarates were substrates for the oxidation of NADPH by heart NADP-isocitrate dehydrogenase without requiring CO2. beta-Methylmercapto-alpha-hydroxyglutarate, the expected product of reduction of beta-methylmercapto-alpha-ketoglutarate, did not cause reduction of NADP+ but it was an inhibitor competitive with isocitrate for NADP-isocitrate dehydrogenase. The beta-sulfur substituted alpha-ketoglutarate derivatives were alternate substrates for alpha-ketoglutarate dehydrogenase and the cytosolic and mitochondrial isozymes of heart aspartate aminotransferase but had no effect on glutamate dehydrogenase or alanine aminotransferase.
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PMID:beta-Sulfur substituted alpha-ketoglutarates as inhibitors and alternate substrates for isocitrate dehydrogenases and certain other enzymes. 394 94

A cold-labile glutamate dehydrogenase (GDH, EC 1.4.1.3) has been purified to homogeneity from the crude extracts of Azospirillum brasilense. The purified enzyme shows a dual coenzyme specificity, and both the NADPH and NADH-dependent activities are equally cold-sensitive. The enzyme is highly specific for the substrates 2-oxoglutarate and glutamate. Kinetic studies with GDH indicate that the enzyme is primarily designed to catalyse the reductive amination of 2-oxoglutarate. The NADP+-linked activity of GDH showed Km values 2.5 X 10(-4) M and 1.0 X 10(-2) M for 2-oxoglutarate and glutamate respectively. NAD+-linked activity of GDH could be demonstrated only for the amination of 2-oxoglutarate but not for the deamination of glutamate. The Lineweaver-Burk plot with ammonia as substrate for NADPH-dependent activity shows a biphasic curve, indicating two apparent Km values (0.38 mM and 100 mM) for ammonia; the same plot for NADH-dependent activity shows only one apparent Km value (66 mM) for ammonia. The NADPH-dependent activity shows an optimum pH from 8.5 to 8.6 in Tris/HCl buffer, whereas in potassium phosphate buffer the activity shows a plateau from pH 8.4 to 10.0. At high pH (greater than 9.5) amino acids in general strongly inhibit the reductive amination reaction by their competition with 2-oxoglutarate for the binding site on GDH. The native enzyme has a Mr = 285000 +/- 20000 and appears to be composed of six identical subunits of Mr = 48000 +/- 2000. The GDH level in A. brasilense is strongly regulated by the nitrogen source in the growth medium.
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PMID:NADPH/NADH-dependent cold-labile glutamate dehydrogenase in Azospirillum brasilense. Purification and properties. 395 1

The glutamate dehydrogenase catalyzed reduction of delta 1-pyrroline-2-carboxylic acid (PCA; an alpha-imino acid) with reduced nicotinamide adenine dinucleotide phosphate (NADPH) to give L-proline and NADP+ is employed as a model for the redox step of the corresponding enzyme-catalyzed reductive amination of alpha-ketoglutarate. We demonstrate the reversibility of the model reaction and measure its equilibrium constant. The pH profiles for the model reactions show that the active substrates are the N-protonated imino acid in one direction and the proline anion with a neutral amino group in the other. The V/K value for the imino acid reduction is enhanced by a group Z of pK = 8.6 in the enzyme-NADPH complex, while that for the proline reaction is unaffected by any such group in the enzyme-NADP+ complex. The following conclusions emerge from a comparison of the pH dependence of the rates for the model reactions with that for the oxidative deamination of L-glutamate [Rife, J. E., & Cleland, W. W. (1980) Biochemistry 19, 2328]. The N-protonated form of alpha-iminoglutarate and the conjugate base of glutamate are the active substrates. The redox step is not sensitive to the protonation state of the groups that catalyze the hydrolysis of bound alpha-iminoglutarate. The group Z, which facilitates the PCA reaction, plays no role in the binding of alpha-ketoglutarate. We propose a chemical mechanism for the glutamate reaction where an unprotonated enzyme group of pK = 5.2 in enzyme-NADPH catalyzes the conversion of the alpha-iminoglutarate to the carbinolamine.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Reversible reduction of an alpha-imino acid to an alpha-amino acid catalyzed by glutamate dehydrogenase: effect of ionizable functional groups. 399 79

Kinetic constants were determined for commercially available samples of ox liver glutamate dehydrogenase, which had previously been shown to have suffered limited proteolysis during preparation, with a range of substrates and effectors. These were compared with the values obtained with enzyme preparations purified in such a way as to prevent this proteolysis from occurring [McCarthy, Walker & Tipton (1980) Biochem. J. 191, 605-611]. The Km values and maximum velocities determined with different substrates revealed little difference between the two preparations although the proteolysed enzyme had lower Km values for NH4+ and glutamate when the activities were determined with NADPH and NADP+ respectively. This preparation was more sensitive to inhibition by Cl- ions but less sensitive to inhibition by high concentrations of the substrate NADH. The two preparations also differed in their sensitivities to allosteric effectors, with the proteolysed enzyme being the less sensitive to inhibition by GTP. At high concentrations of NADH, this preparation was also more sensitive to activation by ADP and ATP.
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PMID:Ox glutamate dehydrogenase. Comparison of the kinetic properties of native and proteolysed preparations. 405 48


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