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At least 721 records · Page 40Linked to original sources
PubMed1984

Transformation of Neurospora crassa with the cloned am (glutamate dehydrogenase) gene.

We used DNA containing the am gene of Neurospora crassa, cloned in the lambda replacement vector lambdaL-47 (this clone is designated lambdaC-10), and plasmid vector subclones of this DNA to transform am deletion and point mutant strains. By means of subcloning, all sequences required for transformation to am prototrophy and expression of glutamate dehydrogenase have been shown to reside on a 2.5-kilobase BamHI fragment. We also characterized several am+ strains that were obtained after transformation with lambdaC-10. These strains showed Mendelian segregation of the am+ gene, although less than 50% of the transformed strains showed the normal linkage relationship of am with inl. In all cases tested, the strains had incorporated lambda DNA as well. The lambda DNA also showed a Mendelian segregation pattern. In one case, the incorporation of am DNA in a novel position was associated with a mutagenic event producing a strain with a very tight colonial morphology. In all cases in which the am+ gene had become the resident of a new chromosome, glutamate dehydrogenase was produced to only 10 to 20% of the wild-type levels.

Bacteriophage lambda↗
PubMed1986

The binding of 1,N6-ethenoNAD to bovine liver glutamate dehydrogenase: studies using the time-correlated single photon counting fluorescence technique.

The time-correlated single photon counting (TCPC) fluorescence technique has been used as a novel approach to investigate ligand-protein interaction, for the case of the binding of the fluorescent coenzyme analogue 1,N6-ethenoNAD (epsilon NAD) to bovine liver glutamate dehydrogenase in the presence of glutarate, a substrate analogue which stabilizes the complex. System calibration was performed using solutions of epsilon ADP and carefully purified epsilon NAD mixed at variable molar ratios (pH 7.0, 0.05 M sodium phosphate buffer, 20 degrees C). The fluorescence lifetimes obtained after deconvolution were 2.4 ns (for epsilon NAD) and 23 ns (for epsilon ADP), in good agreement with literature values obtained under similar conditions. epsilon NAD binds to glutamate dehydrogenase in the presence of 50 mM glutarate, with a fluorescence quantum yield enhancement factor, Q, of about 17-fold, as previously reported (Favilla, R. and Mazzini, A. (1984) Biochim. Biophys. Acta 48-57). For this system, fluorescence lifetime values were obtained after deconvolution as 2.4 ns for free epsilon NAD and 21 ns for bound epsilon NAD. These values did not vary appreciably with enzyme concentration nor with degree of saturation, thus reflecting the existence of only one spectroscopically relevant type of complex. Addition of either GTP or ADP did not affect the lifetime of epsilon NAD bound to the enzyme, but only its affinity, thus allowing calculations of binding strengths. In the case of a simple binding (i.e., in the absence of GTP) the dissociation constant of the complex could be derived from a simple relationship, in which only the ratio between the pre-exponential factors and the parameter gamma, which represents the molar fraction of epsilon NAD molecules free in solution in the open conformation, are to be taken into account. The results are in good agreement with those reported by some of us (reference above) using a steady-state fluorescence technique, which by itself is, however, unable to resolve the number of relevant species present in the system.

Adenosine Diphosphate↗
PubMed1994

Cellular and subcellular localization of hexokinase, glutamate dehydrogenase, and alanine aminotransferase in the honeybee drone retina.

Subcellular localization of hexokinase in the honeybee drone retina was examined following fractionation of cell homogenate using differential centrifugation. Nearly all hexokinase activity was found in the cytosolic fraction, following a similar distribution as the cytosolic enzymatic marker, phosphoglycerate kinase. The distribution of enzymatic markers of mitochondria (succinate dehydrogenase, rotenone-insensitive cytochrome c reductase, and adenylate kinase) indicated that the outer mitochondrial membrane was partly damaged, but their distributions were different from that of hexokinase. The activity of hexokinase in purified suspensions of cells was fivefold higher in glial cells than in photoreceptors. This result is consistent with the hypothesis based on quantitative 2-deoxy[3H]glucose autoradiography that only glial cells phosphorylate significant amounts of glucose to glucose-6-phosphate. The activities of alanine aminotransferase and to a lesser extent of glutamate dehydrogenase were higher in the cytosolic than in the mitochondrial fraction. This important cytosolic activity of glutamate dehydrogenase was consistent with the higher activity found in mitochondria-poor glial cells. In conclusion, this distribution of enzymes is consistent with the model of metabolic interactions between glial and photoreceptor cells in the intact bee retina.

Alanine Transaminase↗
PubMed1996

Glutathione reductase and glutamate dehydrogenase of Plasmodium falciparum, the causative agent of tropical malaria.

The use of glutathione reductase inhibitors in chemotherapy is the raison d'être for this study. Two enzymes were purified to homogeneity from the intraerythrocytic malarial parasite Plasmodium falciparum: glutathione disulfide reductase, an antioxidative enzyme, which appears to play an essential role for parasite growth and differentiation, and glutamate dehydrogenase, an enzyme not occurring in the host erythrocyte. The two proteins were copurified and separated by gel electrophoresis with yields of approximately 20%. Malarial glutathione reductase, a homodimer of 110 kDa with a pH optimum of 6.8 and a high preference for NADPH over NADH, was shown to contain FAD as its prosthetic group. The N-terminal sequence, VYDLIVIGGGSGGMA, which can be aligned with residues 20-34 of human glutathione reductase, represents the first beta strand and the diphosphate-fixing helix of the FAD domain. Glutamate dehydrogenase was confirmed as a hexamer with blocked N-termini; it is an enzyme that is highly specific for NADP and NADPH. The copurification of the proteins and the potential of P.falciparum glutathione reductase as a drug target are discussed.

Amino Acid Sequence↗
PubMed1986

Histochemical demonstration of sodium-dependent glutamate uptake in brain tissues by glutamate dehydrogenase reaction.

Using a special tetrazolium salt technique, a striking correlation was observed between Na+ concentration of the incubation medium and the formation of formazan catalyzed by glutamate dehydrogenase (GDH) in glutamatergic neuropil areas of the hippocampal formation, cerebellum, and other brain regions. Na+ concentrations of 130-150 mmol/l caused maximal formazan production. The GDH catalyzed sodium-dependent increase in formazan production is suggested to be a consequence of the sodium dependence of glutamate uptake in glutamatergic brain structures supplying the enzyme with substrate.

Animals↗
PubMed1977

[Effect of dissociating agents on Chlorella glutamate dehydrogenases].

Effect of urea beta-mercaptoethanol and guanidine--hydrochloride on two Chlorella glutamate dehydrogenases (GDH's) have been studied. Both GDH's are inactivated irreversibly by 2-3 M guanidine hydrochloride. Urea above 4 M rapidly inactivates only NH+4-induced NADP--GDH. Constitutive NAD(P)--GDH is stable in 8M urea solution at room temperature for a long time. beta-Mercaptoethanol does not effect significantly the stability of constitutive GDH in 8m urea. Urea above 1M being in reaction mixture inhibits constitutive GDH in a competitive manner to L-glutamate and uncompetitively regards to alpha-oxoglutarate. Taking this into account, one may conclude that L-glutamate and alpha-oxoglutarate seems to bind to different groups on the enzyme molecule.

Chlorella↗
PubMed1989

Effects of in vivo perfusion of glutamate dehydrogenase in the guinea pig cochlea on the VIIIth nerve compound action potential.

Glutamate is considered as the best candidate for the neurotransmission between the inner hair cell and the primary efferent neurons in the mammalian cochlea. In order to test its presence in the synapse, a degradative enzyme for glutamate, glutamate dehydrogenase (GDH) was perfused in the cochlea of guinea pigs. The intensity function of the VIIIth nerve compound action potential was recorded as a physiological test. The results show that the GDH induces a decrease in the auditory nerve responsiveness. The threshold elevation observed is dependent upon the enzyme concentration.

Action Potentials↗
PubMed1999

Amino acid residues involved in the catalytic mechanism of NAD-dependent glutamate dehydrogenase from Halobacterium salinarum.

The pH dependence of kinetic parameters for a competitive inhibitor (glutarate) was determined in order to obtain information on the chemical mechanism for NAD-dependent glutamate dehydrogenase from Halobacterium salinarum. The maximum velocity is pH dependent, decreasing at low pHs giving a pK value of 7.19+/-0.13, while the V/K for l-glutamate at 30 degrees C decreases at low and high pHs, yielding pK values of 7.9+/-0.2 and 9.8+/-0.2, respectively. The glutarate pKis profile decreases at high pHs, yielding a pK of 9. 59+/-0.09 at 30 degrees C. The values of ionization heat calculated from the change in pK with temperature are: 1.19 x 10(4), 5.7 x 10(3), 7 x 10(3), 6.6 x 10(3) cal mol-1, for the residues involved. All these data suggest that the groups required for catalysis and/or binding are lysine, histidine and tyrosine. The enzyme shows a time-dependent loss in glutamate oxidation activity when incubated with diethyl pyrocarbonate (DEPC). Inactivation follows pseudo-first-order kinetics with a second-order rate constant of 53 M-1min-1. The pKa of the titratable group was pK1=6.6+/-0.6. Inactivation with ethyl acetimidate also shows pseudo-first-order kinetics as well as inactivation with TNM yielding second-order constants of 1.2 M-1min-1 and 2.8 M-1min-1, and pKas of 8.36 and 9.0, respectively. The proposed mechanism involves hydrogen binding of each of the two carboxylic groups to tyrosyl residues; histidine interacts with one of the N-hydrogens of the l-glutamate amino group. We also corroborate the presence of a conservative lysine that has a remarkable ability to coordinate a water molecule that would act as general base.

Amino Acids↗
PubMed1988

Pyruvate carboxylase, phosphate-dependent glutaminase and glutamate dehydrogenase in the developing rat small intestinal mucosa.

The activities of glutaminase and glutamate dehydrogenase in the small intestinal mucosa of infant rats were found to increase at the time of weaning. Pyruvate carboxylase activity, on the other hand, was very high during the suckling period and decreased to negligible values at weaning. It is suggested that gluconeogenesis in the infant mucosa occurs primarily via oxaloacetate and not via alpha-ketoglutarate.

Aging↗
PubMed1983

Beef liver glutamate dehydrogenase: effects of partial proteolysis with chymotrypsin.

1. After chymotryptic digestion of bovine glutamate dehydrogenase, the assay conditions determine whether activation or inhibition is observed. 2. The major fragments appear to remain physically associated. 3. Responses to both GTP and ADP are altered. Inhibition by GTP at pH 7 and 8 is almost abolished. 4. Out of various ligand combinations tested, GTP and NADH together provide the best protection against all the proteolytic effects.

Adenosine Diphosphate↗
PubMed1982

Serum glutamate dehydrogenase is not a reliable marker of liver cell necrosis in alcoholics.

Serum glutamate dehydrogenase (EC 1.4.1.3.) activity was measured in 73 hospital patients who had a history of chronic alcohol abuse and who all had a liver biopsy performed. High levels of serum GDH activity occurred in those patients with recent excess alcohol consumption independently of the underlying liver histology, and did not discriminate between those patients with and those without alcoholic hepatitis.

Ethanol↗
PubMed1992

Guanosine 5'-O-[S-(3-bromo-2-oxopropyl)]thiophosphate: a new reactive purine nucleotide analog labeling Met-169 and Tyr-262 in bovine liver glutamate dehydrogenase.

A new guanosine nucleotide has been synthesized and characterized: guanosine 5'-O-[S-(3-bromo-2-oxopropyl)]thiophosphate (GMPSBOP), with a reactive functional group which can be placed at a position equivalent to the pyrophosphate region of GTP. This new analog is negatively charged at neutral pH and is similar in size to GTP. GMPSBOP has been shown to react with bovine liver glutamate dehydrogenase with an incorporation of 2 mol of reagent/mol of subunit. The modification reaction desensitizes the enzyme to inhibition by GTP, activation by ADP, and inhibition by high concentrations of NADH, but does not affect the catalytic activity of the enzyme. The rate constant for reaction of GMPSBOP with the enzyme exhibits a nonlinear dependence on reagent concentration with KD = 75 microM. The addition to the reaction mixture of alpha-ketoglutarate, GTP, ADP, or NADH alone results in little decrease in the rate constant, but the combined addition of 5 mM NADH with 0.4 mM GTP or with 10 mM alpha-ketoglutarate reduces the reaction rate approximately 6-fold. GMPSBOP modifies peptides containing Met-169 and Tyr-262, of which Tyr-262 is not critical for the decreased sensitivity of the enzyme toward allosteric ligands. The presence of 0.4 mM GTP plus 5 mM NADH protects the enzyme against reaction at both Met-169 and Tyr-262, but yields enzyme with 1 mol of reagent incorporated/mol of subunit which is modified at an alternate site, Met-469. In the presence of 0.2 mM GTP + 0.1 mM NADH, protection against modification of Tyr-262, but only partial protection against labeling of Met-169, is observed. In contrast, the presence of 10 mM alpha-ketoglutarate + 5 mM NADH protect only against reaction with Met-169. The results suggest that GMPSBOP reacts at the GTP-dependent NADH regulatory site [Lark, R. H., & Colman, R. F. (1986) J. Biol. Chem. 261, 10659-10666] of bovine liver glutamate dehydrogenase, which markedly affects the sensitivity of the enzyme to GTP inhibition. The reaction of GMPSBOP with Met-169 is primarily responsible for the altered allosteric properties of the enzyme.

Affinity Labels↗
PubMed1989

Normal cerebellar glutamate dehydrogenase protein in spinocerebellar degeneration.

Immunochemical analyses (Western blots) of cerebellar homogenates for glutamate dehydrogenase (GDH) from patients with spinocerebellar degeneration and control subjects were conducted. Four patients with autosomal dominant Joseph disease type of spinocerebellar degeneration, one patient with autosomal dominant olivopontocerebellar degeneration and four control subjects were studied. GDH was of the same molecular weight and amount in all patients and control subjects. These data together with normal GDH activity from these same homogenates published previously support the view that GDH is not involved in the pathogenesis of these types of dominantly inherited spinocerebellar degeneration.

Adult↗
PubMedDate not supplied

Coimmobilization of urease and glutamate dehydrogenase in electrochemically prepared polypyrrole-polyvinyl sulfonate films.

Immobilization of urease and glutamate dehydrogenase enzymes in electrochemically prepared polypyrrole-polyvinyl sulfonate films (PPY-PVS) was carried out using physical adsorption and electrochemical entrapment techniques. Detailed studies on optimum pH, Fourier transform infrared spectroscopy, cyclic voltammetry, and scanning electron microscopy of the enzymes in the immobilized state were conducted. The value of the apparent Michaelis-Menten constant was experimentally determined to be 2.5 and 2.7 for physically adsorbed and electrochemically entrapped urease in PPY-PVS films, respectively.

Biosensing Techniques↗
PubMed1984

Pathology of olivopontocerebellar atrophy with glutamate dehydrogenase deficiency.

We report the neuropathologic findings in the first patient with recognized glutamate dehydrogenase (GDH) deficiency to come to postmortem examination. He had progressive cerebellar ataxia beginning at age 21. He died at age 47 of pulmonary emboli. Postmortem examination revealed pancerebellar, olivary, and mild pontine atrophy, demyelination of the posterior columns, degeneration of anterior horn and dorsal root ganglion cells, and reduction of myelinated fibers in the sural nerve. In addition, there was neuronal storage of lipopigment diffusely throughout the CNS and the autonomic neurons, with cell distention, atrophy, and loss in selected areas.

Atrophy↗
PubMed1981

Sedimentation properties of native and proteolysed preparations of ox glutamate dehydrogenase.

The concentration-dependent aggregation behaviour of purified ox liver and brain glutamate dehydrogenase preparations was compared with that of commercially-obtained preparations of the liver enzyme, which have recently been shown to have suffered proteolytic cleavage. Although there were no significant differences in these effects, the presence of 3 mM-GTP and 3 mM-NADH had markedly different effects on the two types of preparation. In this situation, at higher protein concentrations the commercially obtained preparations existed in a higher degree of aggregation than those which had not suffered proteolysis. Studies of the effects of GTP and NADH concentrations on the sedimentation coefficients at a fixed enzyme concentration suggested these effects to be largely due to differences in the affinities of the two preparations for nucleotides.

Animals↗
PubMed1995

Expression and in vitro assembly of recombinant glutamate dehydrogenase from the hyperthermophilic archaeon Pyrococcus furiosus.

The gdhA gene, encoding the hexameric glutamate dehydrogenase (GDH) from the hyperthermophilic archaeon Pyrococcus furiosus, was expressed in Escherichia coli by using the pET11-d system. The recombinant GDH was soluble and constituted 15% of the E. coli cell extract. The N-terminal amino acid sequence of the recombinant protein was identical to the sequence of the P. furiosus enzyme, except for the presence of an initial methionine which was absent from the enzyme purified from P. furiosus. By molecular exclusion chromatography we showed that the recombinant GDH was composed of equal amounts of monomeric and hexameric forms. Heat treatment of the recombinant protein triggered in vitro assembly of inactive monomers into hexamers, resulting in increased GDH activity. The specific activity of the recombinant enzyme, purified by heat treatment and affinity chromatography, was equivalent to that of the native enzyme from P. furiosus. The recombinant GDH displayed a slightly lower level of thermostability, with a half-life of 8 h at 100 degrees C, compared with 10.5 h for the enzyme purified from P. furiosus.

Amino Acid Sequence↗