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At least 775 records · Page 43Linked to original sources
PubMed1988

Low leukocyte glutamate dehydrogenase activity does not correlate with a particular type of multiple system atrophy.

Leucocyte glutamate dehydrogenase (GDH) activity was measured in 26 normal control subjects, 20 patients with multiple system atrophy presenting features of either olivopontocerebellar atrophy or striatonigral degeneration and in a heterogenous group of 15 patients with spinocerebellar degenerations. A broad range of GDH activity was found in all three groups. Low activity failed to correlate with a specific clinical entity. Patients followed to post-mortem examination to date have demonstrated histological features of at least three distinct morbid entities. It is concluded, contrary to earlier reports including the authors', that low leukocyte GDH activity does not identify a particular type of multiple system atrophy.

Adult↗
PubMed1986

Electron microscopic localization of glutamate dehydrogenase in rat liver mitochondria by an immunogold procedure and monoclonal and polyclonal antibodies.

Glutamate dehydrogenase (GDH) was localized in rat liver by indirect electron microscopic immunogold, using different sizes of gold particles and monoclonal and polyclonal antibodies. Using the protein A-gold technique in double immunocytochemical experiments, both antibodies, at their optimal dilutions, gave similar results. A novel assessment of the distribution of GDH was made by measurements of the number of gold particles per square micrometer of cross-sectional images of individual mitochondria. The data indicate intracellular homogeneity among mitochondria in individual parenchymal cells. The enzyme is almost absent in non-parenchymal cells. Finally, GDH was found mainly in association with the mitochondrial inner membrane.

Animals↗
PubMed1999

Detection of glutamate dehydrogenase enzyme activity in Plasmodium falciparum infection.

We describe the separation of an active glutamate dehydrogenase [GDH (NADP+)] enzyme from the plasma of patients with P. falciparum infection using columns of sepharose anti-GDH (NADP+) of Proteus spp. The activity of this enzyme was also detected in P. falciparum culture supernatant. The parasitic origin of this enzyme was suggested by western blot analysis using anti-P. falciparum culture supernatant and anti-whole parasite antibodies. The differential inhibition of the P. falciparum GDH (NADP+) indicates that some epitopes recognised by the antibodies in both preparations may be different. The determination of P. falciparum GDH (NADP+) activity could be developed into a specific technique for the diagnosis of falciparum malaria.

Animals↗
PubMed1989

Coenzyme non-specific glutamate dehydrogenase from Chlorella pyrenoidosa 82T: electron microscopic studies.

The constitutive coenzyme non-specific glutamate dehydrogenase (GDH) from Chlorella pyrenoidosa 82T was purified to homogeneity by column immunoaffinity chromatography and examined by an electron microscope. The enzyme molecule was found to be a hexameric oligomer composed of monomers arranged in three 2-point group symmetry in two layers slightly twisted round the 3-fold axis. The molecule is 8 +/- 1 nm in diameter and 10 +/- 1 nm in height. The enzyme molecules appear both to dissociate into trimers and to associate along the 3-fold axis forming linear aggregates under certain conditions. A tentative model of the Chlorella GDH molecule is proposed, which is very similar to those described for bovine liver GDH and GDH from Clostridium symbiosum.

Chlorella↗
PubMed1995

Alteration of the amino acid substrate specificity of clostridial glutamate dehydrogenase by site-directed mutagenesis of an active-site lysine residue.

Two residues, K89 and S380, thought to interact with the gamma-carboxyl group of the substrate L-glutamate, have been altered by site-directed mutagenesis of clostridial glutamate dehydrogenase (GDH). The single mutants K89L and S380V and the combined double mutant K89L/S380V were constructed. All three mutants were satisfactorily overproduced in soluble form. However, only the K89L mutant was retained by the dye column normally used in purifying the wild-type enzyme. All three mutant enzymes were purified to homogeneity and tested for substrate specificity with 24 amino acids. The single mutant S380V showed no detectable activity. The alternative single mutant K89L showed an activity towards L-glutamate that was decreased nearly 2000-fold compared with wild-type enzyme, whereas the activities towards the monocarboxylic substrates alpha-aminobutyrate and norvaline were increased 2- to 3-fold. A similar level of activity was obtained with methionine (0.005 U/mg) and norleucine (0.012 U/mg), neither of which give any activity with the wild-type enzyme under the same conditions. The double mutant showed decreased activity with all substrates compared with the wild-type GDH. In view of its novel activities, the K89L mutant was investigated in greater detail. A strictly linear relationship between reaction velocity and substrate concentration was observed up to 80 mM L-methionine and 200 mM L-norleucine, implying very high Km values. Values of kcat/Km for L-methionine and L-norleucine were 6.7 x 10(-2) and 0.15 s-1 M-1, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1998

Insights into the molecular basis of salt tolerance from the study of glutamate dehydrogenase from Halobacterium salinarum.

A homology-based modeling study on the extremely halophilic glutamate dehydrogenase from Halobacterium salinarum has been used to provide insights into the molecular basis of salt tolerance. The modeling reveals two significant differences in the characteristics of the surface of the halophilic enzyme that may contribute to its stability in high salt. The first of these is that the surface is decorated with acidic residues, a feature previously seen in structures of halophilic enzymes. The second is that the surface displays a significant reduction in exposed hydrophobic character. The latter arises not from a loss of surface-exposed hydrophobic residues, as has previously been proposed, but from a reduction in surface-exposed lysine residues. This is the first report of such an observation.

Amino Acid Sequence↗
PubMed1996

Refolding pathway and association intermediates of glutamate dehydrogenase from the hyperthermophile Pyrococcus furiosus.

The denaturation and renaturation processes of the hexameric glutamate dehydrogenase from the hyperthermophilic archaeon Pyrococcus furiosus have been investigated using guanidinium chloride as denaturant. The enzyme is highly stable and the transition midpoint for guanidinium chloride denaturation is 6.1 M. The recovery of enzyme structure occurs after dilution of the denaturant at 20 degrees C through the formation of structured monomers. Concentration of the structured monomers leads to the formation of higher association states with a tertiary structure different from that of the native enzyme. Activity is observed only in the presence of the hexamers, although a heating step at 70 degrees C is required to fully reactivate the hexamer formed at 20 degrees C. The refolding process and the intermediate(s) were studied by activity assay, spectroscopic methods, size-exclusion chromatography, and ultracentrifugation analysis.

Archaea↗
PubMed1999

An enhancer element located downstream of the major glutamate dehydrogenase gene of Bacillus subtilis.

The rocG gene of Bacillus subtilis, encoding a catabolic glutamate dehydrogenase, is transcribed by SigL (sigma(54))-containing RNA polymerase and requires for its expression RocR, a member of the NtrC/NifA family of proteins that bind to enhancer-like elements, called upstream activating sequences (UAS). Unlike the case for other sigma(54)-dependent genes, rocG has no UAS; instead, its expression depends on a sequence located 1.5 kilobases downstream of the rocG promoter, beyond the end of the rocG coding region. The same sequence also serves as the UAS for the downstream rocABC operon and can activate rocG if moved upstream of its promoter. Furthermore, the activating sequence can be moved as far as 15 kilobases downstream of the rocG promoter and still retain partial activity.

Arginine↗
PubMedDate not supplied

NAD(+)-specific glutamate dehydrogenase of Neurospora crassa: cloning, complete nucleotide sequence, and gene mapping.

The NAD(+)-specific glutamate dehydrogenase (NAD-GDH) of the filamentous fungus Neurospora crassa is a tetrameric enzyme, regulated by catabolite repression. The amino acid sequence of this enzyme had been published several years ago. With the object of investigating the molecular mechanism of catabolite repression, the nucleotide sequence of genomic clones containing the coding region, along with 5'- and 3'-flanking noncoding segments of the NAD-GDH transcription unit, was obtained. The gdh structural gene was shown to code for a polypeptide of 1047 residues, with a calculated molecular mass of 118,280 daltons. The coding sequence is interrupted by two short introns located close to the N- and C-terminal domains of the polypeptide. Consensus intron boundaries and internal splice sequences resemble closely those of other N. crassa genes. A comparison of the amino acid sequence deduced from the nucleotide sequence with the previously published sequence showed several discrepancies between the two. Nucleotide sequence corresponding to a gap in the amino acid sequence was located in the genomic clone. Genetic mapping by restriction fragment length polymorphism analysis localized the gdh gene close to the loci trp-1 and con-7 on the right arm of linkage group III.

Amino Acid Sequence↗
PubMed1994

The catalytic role of aspartate in the active site of glutamate dehydrogenase.

A putative catalytic aspartyl residue, Asp-165, in the active site of clostridial glutamate dehydrogenase has been replaced with serine by site-directed mutagenesis. The mutant enzyme is efficiently overexpressed in Escherichia coli as a soluble protein and can be successfully purified by the dye-ligand chromatographic procedure normally employed for the wild-type enzyme. By several criteria, including circular dichroism spectrum, sulphydryl reactivity with Ellman's reagent, crystallization and mobility in non-denaturing electrophoresis, the enzyme appears to be correctly folded. NAD+ protects the D165S mutant against modification by Ellman's reagent, suggesting unimpaired binding of coenzyme. In standard assays the specific activity is decreased 10(3)-fold in the reductive amination reaction and 10(5)-fold for oxidative deamination. Kinetic studies show that apparent Km values for NADH and 2-oxoglutarate are almost unchanged. The large reduction in the reaction rate coincides with a weakening of the affinity for ammonium ion (Km > 300 mM, compared with 60 mM for the wild-type). The data are entirely consistent with the direct involvement of D165 in catalysis rather than in the binding of coenzyme or 2-oxoglutarate.

Amino Acid Sequence↗
PubMed2002

Single amino acid substitution (G456A) in the vicinity of the GTP binding domain of human housekeeping glutamate dehydrogenase markedly attenuates GTP inhibition and abolishes the cooperative behavior of the enzyme.

Human glutamate dehydrogenase (GDH) exists in two isoforms encoded by the GLUD1 and GLUD2 genes, respectively. Although the two enzymes share in their mature form all but 15 of their 505 amino acids, they differ markedly in their allosteric regulation. To identify the structural basis for these allosteric characteristics, we performed site-directed mutagenesis on the human GLUD1 gene at sites that differ from the GLUD2 gene using a cloned GLUD1 cDNA. Results showed that substitution of Ala for Gly-456, but not substitution of His for Arg-470 or Ser for Asn-498, renders the enzyme markedly resistant to GTP inhibition (IC(50) = 2.80 microm) as compared with the wild type GLUD1-derived GDH (IC(50) = 0.19 microm). The G456A mutation abolished the cooperative behavior of the enzyme, as revealed by the GTP inhibitory curves. The catalytic and kinetic properties of the G456A mutant and its activation by ADP were comparable with those of the wild type GDH. Gly-456 lies in a very tightly packed region of the GDH molecule, and its replacement by Ala may lead to steric clashes with neighboring amino acids. These, in turn, may affect the conformational state of the protein that is essential for the allosteric regulation of the enzyme by GTP.

Adenosine Diphosphate↗
PubMed1987

A transfer nuclear Overhauser effect study of coenzyme binding to distinct sites in binary and ternary complexes in glutamate dehydrogenase.

The oxidized coenzyme NAD binds to two sites per subunit of bovine liver glutamate dehydrogenase with equal affinity in the absence of dicarboxylic acid coligands. In the presence of glutarate or 2-oxoglutarate, the affinity to one site is unchanged, but the affinity to the other (presumed to be the active site) is considerably increased and now requires two dissociation constants to describe its saturation. A combination of transfer nuclear Overhauser effects (TRNOE) together with an examination of the slopes of TRNOE time dependence indicates that while NAD is bound in a syn conformation at both binding sites, NADP (which binds only to the active site) is bound in a syn-anti mixture. The existence of N6 to N3' and N6 and N2' and N1' to N3' NOE's with NAD suggests that the two coenzyme binding sites are located near enough to allow intermolecular NOE's. In the presence of 2-oxoglutarate where only binding to the active site is effectively observed, the conformation of either coenzyme is syn. Modeling studies using the distance estimates from the TRNOE results suggest that the nicotinamide ribose approximates a 3'-endo conformation. The absence of evidence for intermolecular NOE's under these conditions indicates that while the active and regulatory NAD sites per subunit are in close proximity, the six active sites per hexamer are located greater than 5 A apart.

Animals↗
PubMed1988

Induction, isolation, and some properties of the NADPH-dependent glutamate dehydrogenase from the nonheterocystous cyanobacterium Phormidium laminosum.

The level of the NADPH-dependent glutamate dehydrogenase activity (EC 1.4.1.4) from nitrate-grown cells of the thermophilic non-N2-fixing cyanobacterium Phormidium laminosum OH-1-p.Cl1 could be significantly enhanced by the presence of ammonium or nitrite, as well as by L-methionine-DL-sulfoximine and other sources of organic nitrogen (L-Glu, L-Gln, and methylamine). The enzyme was purified more than 4,400-fold by ultracentrifugation, ion-exchange chromatography, and affinity chromatography, and at 30 degrees C it showed a specific activity of 32.9 mumol of NADPH oxidized per min per mg of protein. The purified enzyme showed no aminotransferase activity and catalyzed the amination of 2-oxoglutarate preferentially to the reverse catabolic reaction. The enzyme was very specific for its substrates 2-oxoglutarate (Km = 1.25 mM) and NADPH (Km = 64 microM), for which hyperbolic kinetics were obtained. However, negative cooperativity (Hill coefficient h = 0.89) and [S]0.5 of 18.2 mM were observed for ammonium. The mechanism of the aminating reaction was of a random type with independent sites. The purified enzyme showed its maximal activity at 60 degrees C (Ea = 5.1 kcal/mol [21.3 kJ/mol]) and optimal pH values of 8.0 and 7.5 when assayed in Tris hydrochloride and potassium phosphate buffers, respectively. The native molecular mass of the enzyme was about 280 kilodaltons. The possible physiological role of the enzyme in ammonia assimilation is discussed.

Ammonia↗
PubMed2003

A polymerase chain reaction (PCR) assay specific for Streptococcus suis based on the gene encoding the glutamate dehydrogenase.

Polymerase chain reaction (PCR) primers that flank a 688-bp segment within the glutamate dehydrogenase gene (gdh) of Streptococcus suis type 2 could amplify efficiently the DNA of all 306 (100%) clinical S. suis isolates tested (pigs, n=305; human, n=1) encompassing all serotypes obtained from diverse organs, and geographic origins. When DNA from other bacteria were used as templates for amplification, no product was detected indicating specificity of the primers. Multiplex PCR was developed using the gdh gene primer pair and primers that targeted the gene encoding S. suis capsular biosynthesis (cps). This strategy enabled the detection of strains belonging to serotypes 1/2, 1, 2, 7, and 9, respectively. Using the multiplex-PCR technique, 12 out of 14 (86%) isolates that were previously identified as non-typable S. suis (based on biochemical reactions and serology) gave positive PCR results of which four were positive for serotype 7, three for serotype 2, and five for S. suis strains that belong to other serotypes. Retest results of all 14 isolates by several veterinary laboratories were identical with PCR and confirmed that the two non-PCR reactive isolates belonged to strains of other streptococcal species. These results indicated that PCR improved species determination and can thus be used as a reliable species-specific molecular diagnostic reagent for the accurate identification of S. suis isolates and a serotype-specific method for the detection of strains of serotypes 1/2, 1, 2, 7, and 9, respectively. The PCR method therefore has potential clinical and epidemiological applications.

DNA Primers↗
PubMed1992

Effect of altered acid-base balance and of various agonists on levels of renal glutamate dehydrogenase mRNA.

Rat kidney contains 3.5-kb and 2.8-kb mRNAs that encode for glutamate dehydrogenase (GDH). The levels of both mRNAs are increased gradually after onset of chronic metabolic acidosis and reach a maximum induction of 2.5-fold after 7 days. In contrast, during recovery from chronic acidosis, the levels of the GDH mRNAs are returned to normal within 1 day. The development of an acute metabolic acidosis causes a more rapid induction of GDH mRNA. This increase occurs after a 7-h lag and plateaus after 18 h at a level that is threefold greater than normal. A very similar profile was observed after the transfer of LLC-PK-F+ cells from normal medium to an acidic medium containing 10 mM bicarbonate and adjusted to pH 6.9. However, the transfer of cells from acidic to normal medium caused an immediate and rapid [half-life (t) = 1 h] decrease in GDH mRNA. The apparent half-lives of GDH mRNA were measured by treating cells grown in normal (t = 4 h) and acidic media (t = 12 h) with actinomycin D. Thus, increased stability may account for the induction of GDH mRNA that occurs during growth in response to acidosis. The levels of GDH mRNA are independently affected by changes in medium pH or bicarbonate concentration. The levels of GDH mRNA are also increased by treating cells with adenosine 3',5'-cyclic monophosphate, epinephrine, triiodothyronine, or retinoic acid, whereas treatment with angiotensin II, vasopressin, phorbol 12-myristate 13-acetate, or cycloheximide did not produce an increase. The inductive effect of dexamethasone, which is observed in vivo, is not reproduced in the LLC-PK-F+ cells.

Acid-Base Equilibrium↗
PubMed1983

L-leucine methyl ester stimulates insulin secretion and islet glutamate dehydrogenase.

Column perifusion of collagenase-isolated mouse pancreatic islets was used to study the dynamics of insulin release in experiments lasting for several hours. The methyl esters of L-leucine and L-arginine were synthesized. Whereas L-arginine methyl ester (L-arginine OMe) had no effect, L-leucine OMe stimulated the release of insulin. The effect of L-leucine OMe was maximal at 5 mmol/liter. Whereas the Km for glucose-stimulated insulin release was unaffected by 1 mmol/liter L-leucine OMe, the maximal release of D-glucose was increased by the amino acid derivative that appeared more effective than L-leucine. L-Leucine OMe was also a potent stimulus of insulin release from the perfused mouse pancreas. In the presence of 10 mmol/liter L-glutamine, 1 mmol/liter L-leucine OMe induced a 50- to 75-fold increase in insulin release. A similar stimulatory effect was also observed in column-perifused RIN 5F cells, a cloned rat islet tumor cell line. A twofold increase in islet glutamate dehydrogenase activity was induced by 5 mmol/liter L-leucine OMe, a larger effect than that of L-leucine (P less than 0.02), whereas L-arginine OMe had a small inhibitory effect. We conclude that L-leucine OMe is a potent stimulus of insulin secretion and that its effect on the beta-cells may be exerted by activating islet glutamate dehydrogenase.

1-Methyl-3-isobutylxanthine↗
PubMed1977

Glutamate dehydrogenases from tissues of the ribbed mussel Modiolus demissus: ADP activation and possible physiological significance.

Glutamate dehydrogenase (E.C. 14.1.3) was localized in the mitochondria from heart, gill, mantle and hepatopancreas of this euryhaline bivalve mollusc. Activity levels were low (0.1-0.4 mumoles/min/g wet weight) in all tissues when assayed in the glutamate forming direction. Partially purified gill mitochondrial GDH was most active at pH 8.5. The rate in the glutamate deaminating direction was 10-20% of the rate in the glutamate forming direction. ADP at apparent Ka concentrations of micrometer (glutamate formation) and 170 micrometer (glutamate deamination) enhanced GDH activity, 8- and 4-fold respectively. GDH, in vivo, is probably in the activated form and appears to function in glutamate synthesis rather than ammonia formation. However, based on the low activities obtained, the role of GDH in salinity induced amino acid synthesis seems minimal.

Adenosine Diphosphate↗