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PubMed1967

Immunochemical studies on glutamate dehydrogenase and on two mutant forms of the protein.

Studies on the active product of pepsin digestion of rabbit immunoglobulin G (IgG), F(ab')(2) produced from antiserum against Neurospora glutamate dehydrogenase, showed it to behave in the same way, in both antienzyme and precipitation experiments, as homologous IgG. It is proposed that the inhibition of glutamate dehydrogenase by this antibody preparation is by the same mechanism as proposed for IgG and F(ab), a change in the configuration of the catalytic site brought about by antibodies. Antibodies prepared against two mutant proteins behaved in antienzyme studies in the same way as antibodies prepared against the wild-type protein. It is thought therefore that the antigenic sites on the mutant proteins which initiate the production of neutralizing antibodies were not affected by the mutation which had changed the catalytic properties of the mutant proteins.

Animals↗

PubMed1978

[Organ specific spectra of the glutamate dehydrogenase of corn, Zea mays L].

The characteristic spectra of glutamate dehydrogenase in the endosperm, embryo and pericarp of the developing seed, in the organs of the seedling (scutellum, root, mesocotyle, coleoptyle, leaf and root parenchyma of stem and ear) appear to be due to the differential activity of the genes controlling the enzyme synthesis. The type of spectrum of the initial tissue is preserved in the in vitro culture. The nature of organ specificity is discussed with respect to supposed mechanism of the formation of GDH isozyme spectrum.

Genes↗

PubMed1995

Two soluble forms of glutamate dehydrogenase isoproteins from bovine brain.

Two soluble forms of novel glutamate dehydrogenase isoproteins, designated GDH I and GDH II, have been purified from bovine brain. GDH I and GDH II were separated on a hydroxyapatite column and eluted by a step gradient at different phosphate concentrations (30 mM and 50 mM for GDH I and GDH II, respectively). The preparations were homogeneous on SDS/PAGE. GDH I and GDH II showed similarity in their molecular sizes and are composed of six identical subunits having a molecular size of 57,500 Da. Differences between the biochemical properties of GDH I and GDH II, such as N-terminal amino acid sequences of intact and tryptic-digested enzymes, kinetic parameters, optimum pH and heat stability, were extensively examined in both reductive amination of alpha-oxoglutarate and oxidative deamination of glutamate. The different effects of ADP on GDH isoproteins were also studied under various conditions. These results indicate that GDH I and GDH II, isolated from bovine brain, are novel and distinct polypeptides.

Adenosine Diphosphate↗

PubMed1990

Leukocyte glutamate dehydrogenase and CSF amino acids in late onset ataxias.

Leukocyte glutamate dehydrogenase (GDH) activity was measured in 11 healthy control subjects, 16 neurological controls, 12 patients with dominant late onset ataxia, 15 with sporadic late onset ataxia and 8 with alcoholic cerebellar ataxia. Serum hexosaminidase activity was also determined in ataxic patients. Concentrations of free amino acids were determined in the lumbal CSF of 16 neurological controls, 8 patients with late onset ataxia and 5 with alcoholic ataxia. Mean total GDH activity was reduced significantly in dominant (p less than 0.05) and sporadic (p less than 0.01) cerebellar ataxia, while the heat-labile form was decreased significantly (p less than 0.01) only in sporadic ataxia. All GDH activities were within normal range in patients with alcoholic ataxia. The serum hexosaminidase activities were also within reference range in all patient groups. The CSF concentrations of alanine, glycine, methionine and valine were significantly elevated and those of GABA and glutamate were normal in patients with late onset ataxia as compared to neurological controls. The most significant (p less than 0.01) increase was found for methionine. The amino acid levels of patients with alcoholic ataxia did not differ from those of the controls. The results suggest that GDH activity is only partially decreased in some ataxic patients and that altered amino acid metabolism may be reflected in the CSF.

Adult↗

PubMed1990

Leukocyte glutamate dehydrogenase and CSF amino acids in late onset ataxias.

Leukocyte glutamate dehydrogenase (GDH) activity was measured in 11 healthy control subjects, 16 neurological controls, 12 patients with dominant late onset ataxia, 15 patients with sporadic late onset ataxia and 8 with alcoholic cerebellar ataxia. Serum hexosaminidase activity was also determined in ataxic patients. Concentrations of free amino acids were determined in the lumbal CSF of 16 neurological controls, 8 patients with late onset ataxia and 5 with alcoholic ataxia. Mean total GDH activity was reduced significantly in dominant (p less than 0.05) and sporadic (p less than 0.01) cerebellar ataxia, while the heat-labile form was decreased significantly (p less than 0.01) only in sporadic ataxia. All GDH activities were within normal range in patients with alcoholic ataxia. The serum hexosaminidase activities were also within reference range in all patient groups. The CSF concentrations of alanine, glycine, methionine and valine were significantly elevated and those of GABA and glutamate were normal in patients with late onset ataxia as compared to neurological controls. The most significant (p less than 0.01) increase was found for methionine. The amino acid levels of patients with alcoholic ataxia did not differ from those of the controls. The results suggest that GDH activity is only partially decreased in some ataxic patients and that altered amino acid metabolism may be reflected in the CSF.

Adult↗

PubMed1977

Detection after electrophoresis of enzymes involved in ammonia metabolism using L-glutamate dehydrogenase as a linking enzyme.

The use of L-glutamate dehydrogenase (GLUD) as a reagent in staining mixtures to detect the isozymes of enzymes which catalyze the production of ammonia has been investigated. Methods have been devised for the electrophoresis and detection, using GLUD, of seven enzymes: cytidine deaminase, adenosine deaminase, adenosine monophosphate deaminase, arginase, argininosuccinase, D-amino acid oxidase, and D-aspartate oxidase. GLUD-linked staining methods appear to be sensitive, specific, and of general application.

AMP Deaminase↗

PubMed1985

Crystallization of an NAD+-dependent glutamate dehydrogenase from Clostridium symbiosum.

Crystals of a bacterial NAD+-dependent glutamate dehydrogenase (GDHase) have been grown over a wide range of pH values by using the hanging drop method of vapour diffusion with ammonium sulphate as the precipitant. Sodium dodecyl sulphate/polyacrylamide gel electrophoresis of this enzyme together with high pressure liquid chromatography/gel filtration, shows that this GDHase is hexameric like the GDHases of vertebrates. X-ray photographs of the crystals show that they diffract to at least 2.0 A, and an analysis of the diffraction pattern demonstrates that the hexamer is arranged in at least pseudo 32 symmetry.

Chromatography, Gel↗

PubMed1986

Glutamate dehydrogenase and its isozyme activity in olivopontocerebellar atrophy.

Evaluation of glutamate dehydrogenase (GDH) in 12 patients with olivopontocerebellar atrophy showed deficiency of the enzyme in the group as well as in each patient. The activity of total GDH was 77.7% of that in controls. We also demonstrated two components of this enzyme differentiated by their thermostabilities. The activity of the heat-labile component was remarkably reduced in patients although that of the heat-stable component showed the same magnitude as in controls. These data suggest that GDH deficiency is mainly caused by its heat-labile component deficiency, which might be related to the pathogenesis of this disease.

Aged↗

PubMed1992

Occurrence of cold-labile NAD-specific glutamate dehydrogenase in Bacillus species.

A nicotinamide adenine dinucleotide-specific glutamate dehydrogenase (NAD-GluDH; EC 1.4.1.3) inactivated by incubation at low temperatures was detected in several species of the genus Bacillus, including strains of B. cereus, B. laterosporus, B. lentus, B. panthotenicus, B. pasteurii, B. sphaericus, B. stearothermophilus, B. subtilis and B. thuringiensis. Incubation of cell-free extracts of these strains at 0 degrees C resulted in an 80-100% inactivation of NAD-GluDH activity within 120 min. The addition of 20% glycerol protected the enzyme from this inactivation in the cold. Strains of B. fastidiosus, B. licheniformis, B. macerans, B. megaterium and B. pumilus were found to lack NAD-GluDH activity.

Bacillus↗

PubMed1969

Desensitization of glutamate dehydrogenase by reaction of tyrosne residues.

1. The reaction of glutamate dehydrogenase with N-acetylimidazole and with tetranitromethane leads to modification of tyrosine residues. 2. Modification of 1 tyrosine residue/subunit does not affect the enzymic activity but decreases the response of the enzyme to the allosteric inhibitor, GTP. 3. The physical properties of the enzyme (sedimentation coefficient and optical rotatory dispersion) remain unaltered. 4. GTP partially protects against desensitization. 5. The diminished responses of the modified enzymes to GTP are also detected by using the fluorescence of 1-anilinonaphthalene-8-sulphonate as a conformational probe. 6. Difficulties that generally arise in chemical modifications from inhomogeneous distributions of products are discussed.

Aniline Compounds↗

PubMed1990

Inhibition of ox brain glutamate dehydrogenase by perphenazine.

Factors affecting the inhibition of ox brain glutamate dehydrogenase (GDH) by the antipsychotic drug perphenazine have been studied. Inhibition was found to be of mixed type with respect to 2-oxoglutarate and competitive towards NADH. However, the data indicate that perphenazine binds to a site distinct from the catalytic site to which NADH binds. Perphenazine also enhanced the high-substrate inhibition by these two substrates. Inhibition by perphenazine was not affected by the allosteric effector GTP but it was enhanced by increasing pH, in the range of 6.3 to 7.6, and diminished by increasing ionic strength. Low concentrations of perphenazine relieved the inhibition of GDH by phosphatidylserine and cardiolipin. However, at higher concentrations phosphatidylserine did not interfere with the inhibition by perphenazine whereas cardiolipin relieved it. The possible significance of these interactions in terms of the behaviour of this antipsychotic drug in vivo are discussed.

Animals↗

PubMed1973

The effect of modifying lysine-126 on the physical, catalytic and regulatory properties of bovine liver glutamate dehydrogenase.

1. The reaction of 4-iodoacetamidosalicylate with bovine liver glutamate dehydrogenase is dependent on pH. The pH-activity curve is bell-shaped and can be described by apparent pK values of 7.8+/-0.2 and 9.1+/-0.2. 2. Enzyme in which lysine-126 has been modified by 4-iodoacetamidosalicylate has unaltered sedimentation characteristics except when measured in the presence of GTP and NADH. 3. GTP binding to the inhibited enzyme is unaltered. However, GTP can no longer promote the binding of a second molecule of NADH, since this is already bound to the inhibited enzyme without GTP. 4. The equilibrium binding of ADP, GTP, NAD-sulphite and NADH (when measured at low concentrations) was largely unchanged by modification. 5. The number of binding sites for 2-oxoglutarate to the enzyme-NADH complex were decreased by 60% in an enzyme that has been inhibited by 70%.

Adenosine Diphosphate↗

PubMed1986

The subcellular localization of glutamate dehydrogenase (GDH): is GDH a marker for mitochondria in brain?

Glutamate dehydrogenase (GDH, EC 1.4.1.2) has long been used as a marker for mitochondria in brain and other tissues, despite reports indicating that GDH is also present in nuclei of liver and dorsal root ganglia. To examine whether GDH can be used as a marker to differentiate between mitochondria and nuclei in the brain, we have measured GDH by enzymatic activity and on immunoblots in rat brain mitochondria and nuclei which were highly enriched by density-gradient centrifugation methods. The activity of GDH was enriched in the nuclear fraction as well as in the mitochondrial fraction, while the activities of other "mitochondrial" enzymes (fumarase, NAD-isocitrate dehydrogenase and pyruvate dehydrogenase complex) were enriched only in the mitochondrial fraction. Immunoblots using polyclonal antibodies against bovine liver GDH confirmed the presence of GDH in the rat brain nuclear and mitochondrial fractions. The GDH in these two subcellular fractions had a very similar molecular weight of 56,000 daltons. The mitochondrial and nuclear GDH differed, however, in their susceptibility to solubilization by detergents and salts. The mitochondrial GDH could be solubilized by extraction with low concentrations of detergents (0.1% Triton X-100 and 0.1% Lubrol PX), while the nuclear GDH could be solubilized only by elevated concentrations of detergents (0.3% each) plus KCl (greater than 150 mM). Our results indicate that GDH is present in both nuclei and mitochondria in rat brain. The notion that GDH may serve as a marker for mitochondria needs to be re-evaluated.

Animals↗

PubMed1989

Interaction of caffeine and of theophylline with liver glutamate dehydrogenase.

Caffeine and theophylline inhibited the activity of rat liver glutamate dehydrogenase (GDH), but not that of beef liver GDH, in forward and reverse directions of the enzyme reaction. In the forward direction, approximately 16 mM caffeine or 16 mM theophylline inhibited 50 per cent of the rat liver GDH activity (I50); while in the reverse direction, the I50 of caffeine and theophylline was 15 mM and 8 mM, respectively. The inhibition produced by caffeine was cooperative in both directions, while that of theophylline was negatively cooperative in the forward direction and non-cooperative in the reverse. However, ADP reduced the inhibitory effect of caffeine and theophylline to the extent of 40% and 80%, respectively. The Ki values obtained for caffeine and theophylline were different in the presence of various concentrations of substrates and coenzymes. Based upon these data, we presume that certain subtle changes occurring in the conformation of the rat liver GDH (probably at the ADP/NADH site) in comparison with those of the beef liver GDH may be responsible for its inhibition by caffeine and theophylline.

Adenosine Diphosphate↗

PubMed1986

Kinetics of pressure-induced inactivation of bovine liver glutamate dehydrogenase.

Kinetics of pressure-induced denaturation of bovine liver glutamate dehydrogenase (EC 1.4.1.3) were investigated in the pressure range 1.8-2.8 kbar by observing the residual activity after the pressure-release and the scattered light intensity during the incubation at high pressure. The residual activity decreased exponentially with the incubation time, whereas the scattered light intensity showed a bimodal profile indicating parallel aggregation and dissociation reactions. The latter suggested that two kinds of aggregates were formed during the incubation under pressure. The observed first-order rate constant for the inactivation, k obs, showed a minimum around 30 degrees C. These experimental results were interpreted in terms of the following reaction scheme; (formula; see text) where N represents the enzyme entity with native structure, D1 the partially denatured intermediate, D2 the irreversibly denatured state, and A1 and A2 the two kinds of aggregates, one of which (A1) is reversibly formed at an early stage of the incubation under high pressure. The apparent activation volume for the inactivation reaction was estimated to be delta V*app = -113 +/- 5 cm3 X mol-1 from the pressure dependence of k obs. The effect of coenzyme, NAD+, on the pressure-induced inactivation was also studied. The inactivation was retarded by the presence of the coenzyme, whereas the apparent activation volume for the holoenzyme (delta V*app = -104 +/- 2 cm3 X mol-1) did not differ significantly from that for the apoenzyme.

Animals↗

PubMed1999

In vitro and in vivo study of glutamate dehydrogenase encapsulated into mouse erythrocytes by a hypotonic dialysis procedure.

Glutamate dehydrogenase (GDH) has been encapsulated into mouse erythrocytes by a hypotonic dialysis/isotonic resealing method. Although a low GDH entrapment yield was achieved (3.8%), this percentage appeared sufficient enough to metabolize high quantities of ammonia. Carrier cell recovery yield was 56%. Due to the decrease in cell volume and haemoglobin content, constant mean cell haemoglobin concentration (MCHC) values were obtained. The osmotic fragility curves (OFC) indicated that dialyzed/resealed-RBCs are more resistant to hypotonic haemolysis than native-RBCs. The successful in vitro ammonia degradation by GDH-RBCs was reflected in its total disappearance from the incubation medium at around 48 h. In contrast, initial ammonia levels were not affected during the incubation in the presence of native-RBCs and remained constant. Two different methods were used for the preparation of hyperammonaemic mice model. Since the intraperitoneal (i.p.) administration of ammonium acetate produced high ammonia levels that lasted only a few minutes, the i.p. administration of urease was chosen, given that it generated elevated ammonia levels for longer periods of time. Hyperammonaemic mice quickly removed high levels of circulating ammonia in the presence of GDH-RBCs, whereas in the presence of native-RBCs ammonia was slowly metabolized. These results suggest that loaded GDH-erythrocytes can be used as a potential carrier systems for the in vivo removal of high levels of ammonia from blood.

Ammonia↗

PubMed1974

Slow conformational changes of a Neurospora glutamate dehydrogenase studied by protein fluorescence.

1. The NADP-dependent glutamate dehydrogenase of Neurospora crassa undergoes slow reversible structural transitions, with half-times in the order of a few minutes, between active and inactive states. The inactive state of the enzyme, which predominates at pH values below 7.0, has an intrinsic tryptophan fluorescence 25% lower than that of the active state, which predominates at pH values above 7.6. The inactive state can be activated either by an increase in pH or by addition of activators such as succinate. 2. The kinetics of the slow transitions that follow activating and inactivating rapid changes in conditions have been monitored by measurements of protein fluorescence. The results show that the slow reversible conformational change detected by the change in fluorescence is the rate-limiting process for enzyme activation and inactivation. 3. In both directions this conformational change follows apparent first-order kinetics and the rate constant is independent of protein concentration. These kinetics and published measurements of molecular weight are indicative of an isomerization process. 4. In both directions the changes show a large energy of activation and a large positive entropy of activation, consistent with a considerable disturbance of conformation in the transition state. 5. Comparisons of the fluorescence emission spectra of the active and inactive states indicate that the difference in fluorescence is produced by quenching, possibly intramolecular, in the inactive conformation. Iodide ions cause similar quenching. 6. In some mutationally altered forms of the enzyme comparable but modified conformational changes can be followed by protein fluorescence.

Enzyme Activation↗

PubMed1986

Abnormal platelet glutamate dehydrogenase activity and activation in dominant and nondominant olivopontocerebellar atrophy.

Glutamate dehydrogenase (GDH) activity and its allosteric modulation by purine nucleotides were studied in platelet preparations from 4 patients with a nondominant form of adult-onset olivopontocerebellar atrophy (OPCA) and in affected and nonaffected members of two families with a dominant form of OPCA. A partial deficiency of GDH activity (40 to 50% of control values) was present in 3 patients with nondominant OPCA and in 2 patients, father and son, with a dominant form of OPCA. Platelet GDH from these patients and controls was regularly inactivated by 2 mM guanosine-5'-triphosphate (GTP) and simulated one- to twofold by 2 mM adenosine-5'-diphosphate (ADP). In the presence of 0.2% Triton X-100, the activating effect of ADP was enhanced four- to sixfold. The partial deficiency in maximum catalytic activity observed in these patients persisted under all conditions used for enzyme assay. In affected members, but not in one unaffected member of another family with a dominant type of OPCA, GDH activity was in the control range but was not activated by ADP in either the presence or absence of Triton. These results suggest that there may be at least two possible alterations of GDH in patients with OPCA: one which decreases the maximum catalytic activity and one which impairs the regulatory properties of the enzyme. Furthermore, this study suggests that platelet GDH determination in patients with OPCA may provide a simple and useful tool to classify these disorders and to understand the basic pathophysiological mechanisms involved.

Adolescent↗