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:1.4.1.2 (glutamate dehydrogenase)
4,380 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Three isozymes of glutamate dehydrogenase (GDH) of Chlamydomonas reinhardtii, induced under different trophic and stress conditions, have been purified about 800-1000-fold to electrophoretic homogeneity. They are hexamers of Mr 266,000-269,000 as deduced from gel filtration and sedimentation coefficient data. GDH1 consisted of six identical subunits of 44 kDa each, whereas both GDH2 and GDH3 consisted of six similar-sized monomers (4 of 44 kDa and 2 of 46 kDa). Optimum pH for the three activities with each pyridine nucleotide was identical (8.5 with NADH; 7.7 with NADPH; and 9.0 with NAD+). The isozymes exhibited similar high optimum temperature values (60-62 degrees C) and isoelectric points (7.9-8.1). Activity was enhanced in vitro by Ca2+ ions and strongly inhibited by pyridoxal 5'-phosphate, KCN, o-phenanthroline and EDTA, and to a lesser extent by pHMB and methylacetimidate. In the aminating reaction the three isozymes were inhibited in a concentration-dependent process by both NADH and NADPH, with apparent Km values for NH4+ ranging from 13-53 mM; 0.36-1.85 mM for 2-oxoglutarate and 0.07-0.78 mM for NADH and NADPH. In the deaminating reaction apparent Km values ranged from 0.64-3.52 mM for L-glutamate and 0.20-0.32 for NAD+. In addition, the three isozymes exhibited a non-hyperbolic kinetics for NAD+ with negative cooperativity (n = 0.8).
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PMID:Purification and properties of three NAD(P)+ isozymes of L-glutamate dehydrogenase of Chlamydomonas reinhardtii. 154 Jun 36

The three-dimensional crystal structure of the NAD(+)-linked glutamate dehydrogenase from Clostridium symbiosum has been solved to 1.96 A resolution by a combination of isomorphous replacement and molecular averaging and refined to a conventional crystallographic R factor of 0.227. Each subunit in this multimeric enzyme is organised into two domains separated by a deep cleft. One domain directs the self-assembly of the molecule into a hexameric oligomer with 32 symmetry. The other domain is structurally similar to the classical dinucleotide binding fold but with the direction of one of the strands reversed. Difference Fourier analysis on the binary complex of the enzyme with NAD+ shows that the dinucleotide is bound in an extended conformation with the nicotinamide moiety deep in the cleft between the two domains. Hydrogen bonds between the carboxyamide group of the nicotinamide ring and the side chains of T209 and N240, residues conserved in all hexameric GDH sequences, provide a positive selection for the syn conformer of this ring. This results in a molecular arrangement in which the A face of the nicotinamide ring is buried against the enzyme surface and the B face is exposed, adjacent to a striking cluster of conserved residues including K89, K113, and K125. Modeling studies, correlated with chemical modification data, have implicated this region as the glutamate/2-oxoglutarate binding site and provide an explanation at the molecular level for the B type stereospecificity of the hydride transfer of GDH during the catalytic cycle.
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PMID:Subunit assembly and active site location in the structure of glutamate dehydrogenase. 155 82

The gene encoding the NAD(+)-dependent glutamate dehydrogenase (GDH) of Clostridium symbiosum was cloned using the polymerase chain reaction (PCR) because it could not be recovered by standard techniques. The nucleotide sequence of the gdh gene was determined and it was overexpressed from the controllable tac promoter in Escherichia coli so that active clostridial GDH represented 20% of total cell protein. The recombinant plasmid complemented the nutritional lesion of an E. coli glutamate auxotroph. There was a marked difference between the nucleotide compositions of the coding region (G + C = 52%) and the flanking sequences (G + C = 30% and 37%). The structural gene encoded a polypeptide of 450 amino acid residues and relative molecular mass (M(r) 49,295 which corresponds to a single subunit of the hexameric enzyme. The DNA-derived amino acid sequence was consistent with a partial sequence from tryptic and cyanogen bromide peptides of the clostridial enzyme. The N-terminal amino acid sequence matched that of the purified protein, indicating that the initiating methionine is removed post-translationally, as in the natural host. The amino acid sequence is similar to those of other bacterial GDHs although it has a Gly-Xaa-Gly-Xaa-Xaa-Ala motif in the NAD(+)-binding domain, which is more typical of the NADP(+)-dependent enzymes. The sequence data now permit a detailed interpretation of the X-ray crystallographic structure of the enzyme and the cloning and expression of the clostridial gene will facilitate site-directed mutagenesis.
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PMID:The glutamate dehydrogenase gene of Clostridium symbiosum. Cloning by polymerase chain reaction, sequence analysis and over-expression in Escherichia coli. 158 67

Studies on the levels of glutamate dehydrogenase (GDH), glutamine synthetase, and glutamate synthase were carried out as a function of temperature, nutritional conditions, and the morphological (yeast or mycelium) form of Benjaminiella poitrasii. Since both NAD- and NADP-dependent GDH activities were found in B. poitrasii, the quantitative relation between these two enzymes expressed as the NADP-GDH/NAD-GDH activity ratio (GDH ratio) was studied to evaluate its possible role in the morphogenesis. In the yeast-to-mycelium transition, a decrease in the GDH ratio occurred (between 1 and 2 h) and germ tube formation could be observed only at 3 h. Under similar sets of experimental conditions, exogenous addition 1.0 mM of alpha-ketoglutarate delayed germ tube emergence (4 h) compared with the control. On the other hand, in the presence of 1.0 mM glutamate an earlier onset of the germ tube formation was noted. The morphological (monomorphic) mutants, Y-2 and Y-5, showed a high GDH ratio and maintained the yeast morphology.
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PMID:Significance of NADP/NAD glutamate dehydrogenase ratio in the dimorphic behavior of Benjaminiella poitrasii and its morphological mutants. 159 24

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.
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PMID:Physiological and genetic analysis of the carbon regulation of the NAD-dependent glutamate dehydrogenase of Saccharomyces cerevisiae. 165 57

Release of the endogenous transmitter, glutamate, was measured from individual cone photoreceptors using a microfluorometric technique. The assay for glutamate was conducted within the lumen of a suction pipette, and was based on the fluorometric measure of the production of NADH from NAD+. This reaction was catalyzed by glutamate dehydrogenase contained in the pipette. Upon introduction of glutamate to the pipette, an increase in the NADH fluorescence was observed, representing the stoichiometric conversion of glutamate to NADH. The fluorescent signal was quantified, allowing an estimate of glutamate release from a single cone upon depolarization. The release observed was elicited upon depolarization of the cell with extrinsic current, and was detectable simultaneous with stimulation of the cell. Depolarization-induced release of endogenous glutamate was from the synaptic pedicle of the cell, and this release decreased with subsequent stimulations. The decrease in the release could be briefly reversed by an increase in the depolarization current used, or by allowing the cell to rest for several minutes.
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PMID:Application of a fluorometric method to measure glutamate release from single retinal photoreceptors. 167 56

Previously, the synthesis and validation of [32P]2N3NAD+ as an active site directed photoaffinity probe for glutamate dehydrogenase (GDH) was reported (8). This report shows that 2N3NAD+ is also an effective probe for the NAD+ binding site of lactate dehydrogenase (LDH). With the appropriate photolabeling procedures and immobilized boronate column chromatography the active site peptides of GDH and LDH involved in the adenine base binding domain have been isolated and sequenced. With both GDH and LDH a single photolabeled peptide, which contained the majority of the photoinserted radiolabel, was isolated. Additionally, these peptides had UV spectra that were markedly different from the nonphotolabeled peptides. The modified peptide from GDH corresponded to Cys270 through Lys289. Both sequencing and compositional analysis identified Glu275 as the site of photoinsertion. Sequencing of this peptide aborted at Glu275 after five rounds of analysis, indicating that insertion was blocking further progress. Compositional analysis showed that the entire sequence from residues 270 to 289 was present except that the single Glu residue was missing. This is interpreted as indicating that the photoinsertion is into the polypeptide backbone at the Glu site. The peptide isolated from LDH corresponded to Asp82 through Arg90. Sequencing of this peptide could be completed throughout with only the round at Tyr83 giving no identifiable residue. Compositional analysis of this peptide was in agreement with the peptide from Asp82 to Arg90 with the exception that the single Tyr residue was missing. This indicates that the photoinsertion is into the tyrosine side chain. This data was found to be in agreement with X-ray crystallographic results identifying the NAD(+)-binding domains.
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PMID:Identification of peptides in the adenine ring binding domain of glutamate and lactate dehydrogenase using 2-azido-NAD+. 168 10

We analyzed the upstream region of the GDH2 gene, which encodes the NAD-linked glutamate dehydrogenase in Saccharomyces cerevisiae, for elements important for the regulation of the gene by the nitrogen source. The levels of this enzyme are high in cells grown with glutamate as the sole source of nitrogen and low in cells grown with glutamine or ammonium. We found that this regulation occurs at the level of transcription and that a total of six sites are required to cause a CYC1-lacZ fusion to the GDH2 gene to be regulated in the same manner as the NAD-linked glutamate dehydrogenase. Two sites behaved as upstream activation sites (UASs). The remaining four sites were found to block the effects of the two UASs in such a way that the GDH2-CYC1-lacZ fusion was not expressed unless the cells containing it were grown under conditions favorable for the activity of both UASs. This complex regulatory system appears to account for the fact that GDH2 expression is exquisitely sensitive to glutamine, whereas the expression of GLN1, coding for glutamine synthetase, is not nearly as sensitive.
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PMID:Role of the complex upstream region of the GDH2 gene in nitrogen regulation of the NAD-linked glutamate dehydrogenase in Saccharomyces cerevisiae. 168 1

Steady-state kinetic properties of glutamate dehydrogenase from Clostridium symbiosum are reported. Rates with NADP(H) are over three hundred times lower than with NAD(H) under identical conditions. The 3-acetyl pyridine and 6-deamino adenine analogues of NAD+, on the other hand, are used almost as well as NAD+ itself. Amino acid specificity is very tight at both pH 7 and pH 9. The best alternative substrate of those tested, L-alpha-amino-gamma-nitraminobutyrate, gave only 0.5% of the rate seen with glutamate. With 400 microM NAD+ a 160-fold variation of the glutamate concentration gave a linear Eadie plot apart from slight inhibition at the highest concentrations. With 40 mM L-glutamate and varied [NAD+], the Eadie plot appeared linear between 1.6 microM and 60 microM and again between 60 microM and 2000 microM, but the slopes of the two lines differed by a factor of 8.4. This striking pattern is not attributable to impurities in the coenzyme or to changes in the state of aggregation of the enzyme. For the high concentration range (greater than 60 microM NAD+), the presence of all four linear terms in the reciprocal form of the initial rate equation indicates a sequential mechanism. Similar measurements made for APAD+ and dnNAD+ show no sign of non-linearity in the Eadie plot over the wide concentration ranges explored. In the reductive amination direction, with NADH as coenzyme, linear reciprocal plots were obtained for all three substrates. Systematic variation of concentrations led via primary, secondary and tertiary plots to all eight possible initial-rate parameters in a linear reciprocal initial-rate equation. Compulsory-order and enzyme-substitution mechanisms appear to be excluded, and a random route to the central complex seems the only possibility compatible with the results.
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PMID:Functional studies of a glutamate dehydrogenase with known three-dimensional structure: steady-state kinetics of the forward and reverse reactions catalysed by the NAD(+)-dependent glutamate dehydrogenase of Clostridium symbiosum. 176 63

In adult male and female rat liver, the activity of NAD(+)-and NADP(+)-dependent glutamate dehydrogenase (GDH) was microquantitatively measured in tissue samples of 50-150 ng, microdissected continuously along the sinusoidal length. Total activity of GDH with NAD+ as co-factor was found to be higher by a ratio of about 1:2.3 than with NADP+. All intra-acinar enzyme profiles, irrespective of sex, showed an increasing gradient of GDH activity from the periportal beginning to the perivenous end. These findings are at variance with the immunohistochemical localization of GDH in rat liver. The microquantitative GDH profiles with higher perivenous values could indicate a more pronounced glutamine synthesis in Zone 3 of the liver acinus.
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PMID:Microquantitative analysis of the intra-acinar profiles of glutamate dehydrogenase in rat liver. 185 59


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