Oxidative Deamination Of Glutamate Research Articles

The pathway of glutamate metabolism in rat brain mitochondria.

1. The pathway of glutamate metabolism in non-synaptic rat brain mitochondria was investigated by measuring glutamate, aspartate and ammonia concentrations and oxygen uptakes in mitochondria metabolizing glutamate or glutamine under various conditions. 2. Brain mitochondria metabolizing 10mm-glutamate in the absence of malate produce aspartate at 15nmol/min per mg of protein, but no detectable ammonia. If amino-oxyacetate is added, the aspartate production is decreased by 80% and ammonia production is now observed at a rate of 6.3nmol/min per mg of protein. 3. Brain mitochondria metabolizing glutamate at various concentrations (0-10mm) in the presence of 2.5mm-malate produce aspartate at rates that are almost stoicheiometric with glutamate disappearance, with no detectable ammonia production. In the presence of amino-oxyacetate, although the rate of aspartate production is decreased by 75%, ammonia production is only just detectable (0.3nmol/min per mg of protein). 4. Brain mitochondria metabolizing 10mm-glutamine and 2.5mm-malate in States 3 and 4 were studied by using glutamine as a source of intramitochondrial glutamate without the involvement of mitochondrial translocases. The ammonia production due to the oxidative deamination of glutamate produced from the glutamine was estimated as 1nmol/min per mg of protein in State 3 and 3nmol/min per mg of protein in State 4. 5. Brain mitochondria metabolizing 10mm-glutamine in the presence of 1mm-amino-oxyacetate under State-3 conditions in the presence or absence of 2.5mm-malate showed no detectable aspartate production. In both cases, however, over the first 5min, ammonia production from the oxidative deamination of glutamate was 21-27nmol/min per mg of protein, but then decreased to approx. 1-1.5nmol/min per mg. 6. It is concluded that the oxidative deamination of glutamate by glutamate dehydrogenase is not a major route of metabolism of glutamate from either exogenous or endogenous (glutamine) sources in rat brain mitochondria.

Glutamate dehydrogenase from Bacillus subtilis PCI 219. I. Purification and properties.

Bacillus subtilis PCI 219 has a single glutamate dehydrogenase (GDH) [EC 1.4.1.3] with dual coenzyme specificity [for NAD(H) and NADP(H)]. The enzyme was purified 800-fold from crude extracts of B. subtilis from the post-exponential phase of growth and showed one significant protein band on gel electrophoresis. This band was determined, by activity staining, to have all the GDH nucleotide specificities. Its molecular weight was estimated to be 250,000+/-20,000 by gel filtration, and 270,000+/-30,000 by zone centrifugation in a sucrose density gradient. Polyacrylamide gel electrophoresis in sodium dodecyl sulfate showed that GDH has a subunit size of about 57,000. The pI of GDH was found to bepH 3.7 by isoelectric focusing. GDH exhibited nonlinear kinetics in the reduction of NAD+, and in the reverse direction, the substrate, NH4+, was strongly inhibitory at high concentrations. Purine nucleotides did not affect the activity. The oxidative demination of glutamate was significantly inhibited by the metabolites oxaloacetate and citrate, which acted as allosteric effectors of this enzyme,inhibiting the reaction in one direction. The pH optimum of each of the activities of GDH and the stability of GDH are also reported.

Properties of apoglutamate synthase and comparison with glutamate dehydrogenase.

Glutamate synthase from Escherichia coli K-12 exhibits NH3-dependent activity. NH3-dependent activity is increased approximately 5-fold in apoglutamate synthase lacking flavin and non-heme iron. Whereas glutamine plus 2-oxoglutarate have the capacity to reoxidize the chemically reduced flavoenzyme, no such reoxidation is obtained with 2-oxoglutarate plus NH3. These results establish that the glutamine- and NH3-dependent syntheses of glutamate occur by different pathways of electron transfer from NADPH. The NH3-dependent activity of native and apoglutamate synthase exhibits similar catalytic properties. Some properties of apoglutamate synthase are similar to those of glutamate dehydrogenase. These properties include pH optima for synthesis and oxidative deamination of glutamate, inactivation by alkylating reagents and p-mercuribenzoate, an enhanced rate of inactivation by alkylating reagents and p-mercuribenzoate at low pH, 2-oxoglutarate protection against inactivation by p-mercuribenzoate, and reactivation of p-mercuribenzoate-treated enzyme by 2-mercaptoethanol. 2-Oxoglutarate protects against alkylation of glutamate synthase by iodo [1-14C]acetamide and reduces incorporation of methyl [1-14C]carboxamide into the small subunit of the enzyme.

Fast-kinetic studies of the oxidative deamination of glutamate catalyzed by glutamate dehydrogenase.

The release of protons, one of the products of the reaction, in the oxidative deamination of glutamate by glutamate dehydrogenase, has been followed in a stopped‐flow spectrophotometer by measuring the absorbance change of an indicator dye. The results obtained at pH 7.8 and 15 °C, in 0.05 M Na2SO4, with NADP+ as coenzyme, indicate that the liberation of the protons occurs after the electron transfer but before the release of NADPH, i.e. before the steady state; the results obtained in the same conditions, but in presence of the inhibitor GTP, indicate moreover that it occurs also before the liberation of 2‐oxoglutarate.The oxidative deamination of glutamate has in addition been studied in the presence of equi‐molar amounts of the reduced coenzyme NADPH. The elimination of the presteady‐state step when all the enzyme is in the form of the enzyme · NADPH · glutamate complex supports the hypothesis that the rate‐limiting step in the overall mechanism is the dissociation of NADPH and the regeneration of the enzyme from this ternary complex.

Effect of X-Rays on the Regulatory Functions of Glutamate Dehydrogenase from Beef Liver

Glutamate dehydrogenase from beef liver was irradiated with X-rays in solution in the presence of air. The ability of the enzyme to catalyze the oxidative deamination of glutamate and the reductive amination of pyruvate was inactivated with the same yield (G = 0.3 per protomer). The radiation inactivation of the modifying effects of ADP, GTP, sodium chloride, sulfhydryl blocking and substrate inhibition was measured. When assayed with glutamate as substrate, the different allosteric functions had the same or higher radiosensitivity than the catalytic activity. The most radiosensitive function was the substrate inhibition which was destroyed at a yield 3-4 times that of the catalytic activity. When measured with pyruvate as substrate only the GTP inhibition was more sensitive than the catalytic activity. The ADP and the sodium chloride inhibition was less radiosensitive. Thus, the allosteric effect of each particular effector showed widely different radiosensitivity when the enzyme was assayed with the two...

Activation of glutamate dehydrogenase from Blastocladiella emersonii by AMP

The activation by AMP of glutamate dehydrogenase ( l-glutamate:NAD + oxidoreductase (deaminating), EC 1.4.1.2) from Blastocladiella emersonii has been studied by stopped flow technique and steady state kinetic methods. The results demonstrate that the activation involves a bimolecular reaction. When the oxidative deamination of glutamate was measured the rate constant for the activation was 2·10 3 M −1·sec −1 at pH 8. The rate constant increased strongly at decreasing pH. NAD + and glutamate did not affect the rate constant for the activation of the enzyme by AMP. Measurements of the kinetic parameters of the enzyme show that the activation is associated with an increase in K m for NAD +, NADH and α-ketoglutarate, while K m for ammonia decreases. The K m for glutamate was not affected by AMP. Increasing concentrations of all substrates, except glutamate resulted in a decrease in the binding constant for AMP. The results suggest that the activating action of AMP is mediated through a conformational change in the protein.

AMP Like contamination in commercial NAD preparations

AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL No abstract is available for this article. References 1 H.B. LéJohn, S. Jackson, J. Biol. Chem., 243, (1968), 3447– 2 H.B. LéJohn, Biochem. Biophys. Res. Commun., 32, (1968), 278– 3 H.B. LéJohn, J. Biol. Chem., 243, (1968), 5126– 4 J. Teipel, D.E. Koshland Jr., Biochemistry, 8, (1969), 4656– 5 O.H. Lowry, N.J. Rosebrough, A.L. Farr, R.J. Randall, J. Biol. Chem., 193, (1951), 265– 6 Y.P. Lee, J. Biol. Chem., 227, (1957), 999– 7 O.H. Lowry, J.V. Passoneau, M.K. Rock, J. Biol. Chem., 236, (1961), 2756– 8 K. Dalziel, J. Biol. Chem., 238, (1963), 1538– 9 A.D. Winer, J. Biol. Chem., 239, (1964), PC 3598– Citing Literature Volume18, Issue1October 15, 1971Pages 70-72 ReferencesRelatedInformation

Unidirectional Inhibition of Glutamate Dehydrogenase by Metabolites: A Possible Regulatory Mechanism

Abstract A variety of metabolites and chelating agents have been found to elicit a unique form of inhibition on an NAD-specific glutamate dehydrogenase from Blastocladiella emersonii. When measuring the initial rate of the reaction, the oxidative deamination of glutamate was completely inhibited by the metabolites citrate, isocitrate, fructose 1,6-diphosphate, and fumarate and by the nonmetabolites ATP and ethylenediaminetetraacetate. (ATP may be regarded as a metabolite in some reactions involving its transformation to ADP and AMP.) The rate of the reductive amination of α-ketoglutarate reaction was not affected by any of the inhibitors except ATP. The phenomenon of unidirectional inhibition in these initial velocity studies does not consider the microscopic reversibility of the reaction. Divalent cations, AMP, and ADP reversed this inhibition. AMP and ADP, in addition, served as allosteric effectors of this enzyme, activating the reaction in both directions of assay. ATP, on the other hand, participated as a negative allosteric effector. The inhibitory effects of the metabolites and EDTA were dependent on pH. All of the metabolic inhibitors are important intermediates which are involved in the production of energy in the tricarboxylic acid cycle and in glycolysis. A scheme that incorporates these findings into a regulatory network connecting glycolysis and the tricarboxylic acid cycle is formulated.

Oxidative deamination of glutamate and transdeamination through glutamate

Rat liver mitochondria are able to produce ammonia from glutamate at a steady rate under the conditions described. A system generating ADP from ATP is necessary for the continual deamination of glutamate. Maximum stimulation is reached with a hexokinase-glucose system in the presence of 0.2 m m ATP. The phosphorlyation control ratio (ammonia produced in the presence of hexokinase and glucose divided by ammonia produced in their absence) is of the order of four. Mitochondria incubated with glutamate at 38 ° for 30 minutes in the medium plus 0.5 m m ATP, retain the ability to increase their rate of glutamate deamination on the subsequent addition of hexokinase and glucose; they lose this ability if incubated for 20 or 40 minutes at 38 ° as a sucrose suspension. 2,4-Dinitrophenol increases the rate of ammonia production from glutamate and thus abolishes a rate-limiting step. Ammonia is produced from glutamate at both 25 ° and 38 ° but at a faster rate at the higher temperature. The phosphorylation control ratio in terms of ammonia is greater at 38 ° than at 25 °. Transdeamination of alanine and aspartate by rat liver mitochondria may be mediated by the addition of α-ketoglutarate or glutamate. Excess α-ketoglutarate (greater than 0.5 m m) inhibits the production of ammonia from alanine and aspartate.