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

ABSTRACT: 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.

SUBMITTER: McCarthy AD

PROVIDER: S-EPMC1163355 | biostudies-other | 1981 Oct

REPOSITORIES: biostudies-other

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Ox glutamate dehydrogenase. Comparison of the kinetic properties of native and proteolysed preparations.

Project description:Kinetic constants were determined for commercially available samples of ox liver glutamate dehydrogenase, which had previously been shown to have suffered limited proteolysis during preparation, with a range of substrates and effectors. These were compared with the values obtained with enzyme preparations purified in such a way as to prevent this proteolysis from occurring [McCarthy, Walker & Tipton (1980) Biochem. J. 191, 605-611]. The Km values and maximum velocities determined with different substrates revealed little difference between the two preparations although the proteolysed enzyme had lower Km values for NH4+ and glutamate when the activities were determined with NADPH and NADP+ respectively. This preparation was more sensitive to inhibition by Cl- ions but less sensitive to inhibition by high concentrations of the substrate NADH. The two preparations also differed in their sensitivities to allosteric effectors, with the proteolysed enzyme being the less sensitive to inhibition by GTP. At high concentrations of NADH, this preparation was also more sensitive to activation by ADP and ATP.

| S-EPMC1152591 | biostudies-other

Purification of glutamate dehydrogenase from ox brain and liver. Evidence that commercially available preparations of the enzyme from ox liver have suffered proteolytic cleavage.

Project description:1. A rapid procedure, involving ion-exchange chromatography on DEAE-cellulose and affinity chromatography on GTP-Sepharose, was used to purify glutamate dehydrogenase from ox brain and liver. 2. Preparations purified in this way differed from those of the ox liver enzyme that were obtained from commercial suppliers in their mobilities on polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate. This difference appears to result from the occurrence of limited proteolysis during the preparation of the latter enzyme samples. 3. N-Terminal sequence analysis showed the presence of four amino acid residues in the enzyme prepared in this study that were not present in those obtained from the commercial sources and which have not been reported in previous studies on the sequence of the ox liver enzyme. 4. A preliminary examination of the enzyme prepared in this way indicated that the Michaelis constants for the substrates are similar to those obtained from the commercial preparation, but that the response to allosteric effectors was modified.

| S-EPMC1162252 | biostudies-other

A peptide containing a reactive lysyl group from ox liver glutamate dehydrogenase.

Project description:1. Inhibition of ox liver glutamate dehydrogenase with N-(N'-acetyl-4[(35)S]-sulphamoylphenyl)maleimide (ASPM) is more specific at pH7.3 than at pH6.9. At pH7.3 inhibition accompanies the incorporation at 1 mole of ASPM residues into about 53000g. of protein. 2. Digestion of the modified protein with chymotrypsin and trypsin yields a unique radioactive peptide. 3. Acid hydrolysis of 1 mole of this peptide yields 1 mole of N(in)-succin-2-yl-lysine. The in-amino group of a lysyl residue is thus the site of modification of the protein. 4. The sequence containing the modified lysyl residue is: [Formula: see text] where Asx respresents either aspartic acid or asparagine.

| S-EPMC1187597 | biostudies-other

Purification and properties of glutamate synthase and glutamate dehydrogenase from Bacillus megaterium.

Project description:Bacillus megaterium N.C.T.C. no. 10342 exhibits glutamate synthetase (EC 2.6.1.53) and glutamate dehydrogenase (EC 1.4.1.4) activities. Concentrations of glutamate synthase were high when the bacteria were grown on 3mM-NH4Cl and low when they were grown on 100mM-NH4Cl, whereas glutamate dehydrogenase concentrations were higher when the bacteria were grown on 100mM-NH4Cl than on 3mM-NH4Cl. Glutamate synthase and glutamate dehydrogenase were purified to homogeneity from B. megaterium grown in 10mM-glucose/10mM-NH4Cl. The purified enzymes had mol.wts. 840000 and 270000 for glutamate synthase and glutamate dehydrogenase respectively. The Km values for substrates with NADPH and coenzyme were (glutamate synthase activity shown first) 9 micron and 360 micron for 2-oxoglutarate, 7.1 micron and 8.7 micron for NADPH, and 0.2 mM for glutamine and 22 mM for NH4Cl, similar values to those of enzymes from Escherichia coli. Glutamate synthase contained NH3-dependent activity (different from authentic glutamate dehydrogenase), which was enhanced 4-fold during treatment at pH 4.6 NH3-dependent activity was generally about 2% of the glutamine-dependent activity. Amidination of glutamate synthase by the bi-functional cross-linking reagent dimethyl suberimidate inactivated glutamine-dependent glutamate synthase activity, but increased NH3-dependent activity. A cross-linked structure of mol.wt. approx 200000 was the main product formed.

| S-EPMC1185747 | biostudies-other

The purification and properties of ox liver short-chain acyl-CoA dehydrogenase.

Project description:The FAD-containing short-chain acyl-CoA dehydrogenase was purified from ox liver mitochondria by using (NH4)2SO4 fractionation, DEAE-Sephadex A-50 and chromatofocusing on PBE 94 resin. The enzyme is a tetramer, with a native Mr of approx. 162 000 and a subunit Mr of 41 000. Short-chain acyl-CoA dehydrogenases are usually isolated in a green form. The chromatofocusing step in the purification presented here partially resolved the enzyme into a green form and a yellow form. In the dye-mediated assay system, the enzyme exhibited optimal activity towards 50 microM-butyryl-CoA at pH 7.1. Kinetic parameters were also determined for a number of other straight-chain acyl-CoA substrates. The u.v.- and visible-absorption characteristics of the native forms of the enzyme are described, together with complexes formed by addition of butyryl-CoA, acetoacetyl-CoA and CoA persulphide.

| S-EPMC1153367 | biostudies-other

Kinetic properties of the partially purified pyruvate dehydrogenase complex of ox brain.

Project description:The properties of a purified preparation of the pyruvate dehydrogenase complex from ox brain have been compared with those of a similar preparation from ox kidney. A broad pH optimum around 7.8, similar dependence on ionic strength, and independence of the nature of the buffer anions or cations characterized preparations from both tissues. Michaelis constants for the binding of pyruvate, thiamin pyrophosphate, NAD(+) and CoA were also similar. Enzyme from both tissues was inhibited by NADH, by copper and other heavy metals, by high concentrations of tricarboxylic acid-cycle intermediates, and by preincubation with ATP. Acetyl-CoA itself did not appear to inhibit these preparations, although some commercial preparations of acetyl-CoA did contain an inhibitor. Although oxaloacetate and alpha-oxobutyrate were weak inhibitors, a number of other alpha-oxo acids including phenylpyruvate did not inhibit. The properties of the pyruvate dehydrogenase complex from brain and kidney appeared similar.

| S-EPMC1177436 | biostudies-other

The kinetic mechanism of ox liver glutamate dehydrogenase in the presence of the allosteric effector ADP. The oxidative deamination of L-glutamate.

Project description:In steady-state kinetic studies of ox liver glutamate dehydrogenase in 0.11 M-potassium phosphate buffer, pH7, at 25 degrees C, the concentration of ADP was varied from 0.5 to 1000 microM. Inhibition was observed except when the concentrations of both glutamate and coenzyme were high, when activation was seen. With NAD+ or NADP+ as coenzyme, 200 microM-ADP was sufficient to saturate the enzyme with respect to the major effect of this nucleotide. In the presence of 210 microM-ADP, widely varied concentrations of coenzyme give linear Lineweaver-Burk plots, in marked contrast with results obtained previously for kinetics without ADP. This has allowed evaluation for the reaction with NAD+, NADP+ and acetylpyridine-adenine dinucleotide (315 microM-ADP in the last case) of all four initial rate parameters, i.e. the phi coefficients in the equation: (Formula: see text) where A is coenzyme and B is glutamate. The relative constancy of phi B and of phi AB/phi A with the different coenzymes point to a compulsory-order mechanism with glutamate as the leading substrate. This conclusion, though unexpected, agrees well with various previous observations on the binding of oxidized coenzyme.

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GeneChip expression profiling of Glud1 (glutamate dehydrogenase 1) transgenic mice

Project description:Glud1 (glutamate dehydrogenase 1) transgenic mice release more excitatory neurotransmitter glutamate to synaptic cleft throughout lifespan and show signs of accelerated aging. Here we compared transcriptomic profiles of these animals to their wild-type counterparts. The hippocampus was used for the analysis. Keywords: transgenic analysis Three Glud1 transgenic mice vs. three age-matched wide-type mice. Age: 9-month-old. Tissue: hippocampus.

2010-07-12 | E-GEOD-11419 | biostudies-arrayexpress

GeneChip expression profiling of Glud1 (glutamate dehydrogenase 1) transgenic mice

2010-07-12 | GSE11419 | GEO

Kinetic properties of highly purified preparations of sheep liver cytoplasmic aldehyde dehydrogenase.

Project description:The kinetic properties of highly purified preparations of sheep liver cytoplasmic aldehyde dehydrogenase (preparations that had been shown to be free from contamination with the corresponding mitochondrial enzyme) were investigated with both propionaldehyde and butyraldehyde as substrates. At low aldehyde concentrations, double-reciprocal plots with aldehyde as the variable substrate are linear, and the mechanism appears to be ordered, with NAD+ as the first substrate to bind. Stopped-flow experiments following absorbance and fluorescence changes show bursts of NADH production in the pre-steady state, but the observed course of reaction depends on the pre-mixing conditions. Pre-mixing enzyme with NAD+ activates the enzyme in the pre-steady state and we suggest that the reaction mechanism may involve isomeric enzyme--NAD+ complexes. High concentrations of aldehyde in steady-state experiments produce significant activation (about 3-fold) at high concentrations of NAD+, but inhibition at low concentrations of NAD+. Such behaviour may be explained by postulating the participation of an abortive complex in product release. Stopped-flow measurements at high aldehyde concentrations indicate that the mechanism of reaction under these conditions is complex.

| S-EPMC1158276 | biostudies-other

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