Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:1.4.1.2 (glutamate dehydrogenase)
 4,380 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

When a 10% aqueous solution of glutaraldehyde (GA) was alkalized to pH 8.5 in borate buffer solution and heated at 60 degrees C, the ultraviolet spectrum of GA solution showed two distinct absorption maxima. The one at 280 nm with a weak absorbance ascribable to the C = O bond in the aldehyde group shifted to near 300 nm after 50 min with a slight increase in its intensity. Another maximum at 235 nm with a strong absorbance was ascribable to the C = C bond of the alpha,beta-unsaturated aldehyde group which was formed by aldol condensation reaction of GA monomer, and its absorbance increased markedly with increasing reaction time. The high performance liquid chromatography (HPLC) analysis with detection at 235 nm indicated that several GA oligomers were formed by the alkali treatment and their concentrations increased. The cross-linking ability of these oligomers was examined by immobilizing enzymes (alcohol dehydrogenase (ADH), glutamate dehydrogenase (GLDH] to an aminated polymer gel matrix by reaction with the treated GA solution. The enzyme activities increased with increasing concentration of GA oligomers. Then, the GA oligomers were isolated and used as the cross-linking agent. The activities of ADH and GLDH were 4-fold and 13-fold higher, respectively, than those obtained by using untreated GA solution, while the total amounts of immobilized enzymes were almost unchanged. These results suggest that GA oligomers may act as cross-linkers in a manner different from the generally accepted Schiff base formation reaction; a possible mechanism may involve addition reaction of an amino group to the double bond in the aldol condensate of GA.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Development of effective cross-linking method for bioactive substance--enzyme immobilization using glutaraldehyde oligomers. 251 46

Ethanol or acetaldehyde orally administered (15% and 2% respectively in drinking water) to male Wistar rats for three months induced alterations in the main liver enzymes responsible for ethanol metabolism, aspartate and alanine aminotransferases and NAD glutamate dehydrogenase. Ethanol produced a significant decrease in the activity of soluble alcohol dehydrogenase, while acetaldehyde induced alterations both in soluble and mitochondrial aldehyde dehydrogenases: soluble activity was significantly higher than in the control and ethanol-treated groups, and mitochondrial activity was significantly diminished. Both soluble aspartate and alanine aminotransferases showed pronounced increases by the chronic effect of acetaldehyde, while mitochondrial activities were practically unchanged by the effect of ethanol or acetaldehyde. Mitochondrial NAD glutamate dehydrogenase showed a rise in its activity both by the effect of chronic ethanol and acetaldehyde consumption. The level of metabolites assayed in liver extracts showed marked differences between ethanol and acetaldehyde treatment which indicates that ethanol produced a remarkable increase in glutamate, aspartate and free ammonia together with marked decrease in pyruvate and 2-oxoglutarate concentrations. Acetaldehyde consumption induced a significant decrease in 2-oxoglutarate and pyruvate concentrations. These observations suggest that ethanol has an important effect on the urea cycle enzymes, while the effect of acetaldehyde contributes to the impairment of the citric acid cycle.
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PMID:Effect of chronic ethanol or acetaldehyde on hepatic alcohol and aldehyde dehydrogenases, aminotransferases and glutamate dehydrogenase. 286 Jul 5

Usefulness of several biochemical markers for the monitoring of chronic alcoholism were studied. Among generally used markers, only gamma-GTP showed a significant difference between alcoholic and non-alcoholic liver diseases. Serum glutamate dehydrogenase (GDH) activity was significantly high in alcoholic liver disease. When the ratios of GDH to ornithine carbamyl transferase (OCT) were calculated, differences between alcoholic and non-alcoholic liver diseases became clearer without overlapping of any value. Serum desialo-transferrin was found in about 60% of the alcoholics, and disappeared by abstinence. Microheterogeneity of serum protein was also found in other glycoproteins. Serum prealbumin level was significantly high in alcoholics without severe liver disease. Acetaldehyde dehydrogenase (ALDH) activity of erythrocytes was significantly low in alcoholics, and gradually increased after abstinence. These results indicate that microheterogeneity of glycoproteins, serum prealbumin level and erythrocyte ALDH activity are good markers of alcohol abuse, and serum GDH/OCT ratio is the most sensitive marker of alcoholic liver injury. Serum gamma-GTP activity is a good marker of both conditions.
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PMID:Biochemical markers of chronic alcoholism. 286 79

We describe a procedure using immobilized nicotinamide as an affinity chromatographic ligand for the binding of NAD(P)+-dependent dehydrogenases. The procedure involves preparation of nicotinamide N1-(N-(6-aminohexyl)-acetamide)-agarose and modification of the immobilized nicotinamide by the addition of a ketone or an aldehyde to form an adduct. The nicotinamide, which has no affinity for dehydrogenase, becomes a very specific ligand of dehydrogenase, which binds the ketone or the aldehyde as substrate or inhibitor. In tests, the adduct prepared with immobilized nicotinamide and sodium pyruvate bound specifically to lactate dehydrogenase (EC 1.1.1.27), whereas the adduct prepared with alpha-ketoglutarate bound to glutamate dehydrogenase (EC 1.4.1.3). This technique enables the rapid isolation of a given dehydrogenase.
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PMID:Rapid separation of dehydrogenases by affinity chromatography with new induced specificity phases. 294 22

The strictly anaerobic archaeon Thermococcus strain ES-1 was recently isolated from near a deep-sea hydrothermal vent. It grows at temperatures up to 91 degrees C by the fermentation of peptides and reduces elemental sulfur (S(o)) to H2S. It is shown here that the growth rates and cell yields of strain ES-1 are dependent upon the concentration of S(o) in the medium, and no growth was observed in the absence of S(o). The activities of various catabolic enzymes in cells grown under conditions of sufficient and limiting S(o) concentrations were investigated. These enzymes included alcohol dehydrogenase (ADH); formate benzyl viologen oxidoreductase; hydrogenase; glutamate dehydrogenase; alanine dehydrogenase; aldehyde ferredoxin (Fd) oxidoreductase; formaldehyde Fd oxidoreductase; and coenzyme A-dependent, Fd-linked oxidoreductases specific for pyruvate, indolepyruvate, 2-ketoglutarate, and 2-ketoisovalerate. Of these, changes were observed only with ADH, formate benzyl viologen oxidoreductase, and hydrogenase, the specific activities of which all dramatically increased in cells grown under S(o) limitation. This was accompanied by increased amounts of H2 and alcohol (ethanol and butanol) from cultures grown with limiting S(o). Such cells were used to purify ADH to electrophoretic homogeneity. ADH is a homotetramer with a subunit M(r) of 46,000 and contains 1 g-atom of Fe per subunit, which, as determined by electron paramagnetic resonance analyses, is present as a mixture of ferrous and ferric forms. No other metals or acid-labile sulfide was detected by colorimetric and elemental analyses. ADH utilized NADP(H) as a cofactor and preferentially catalyzed aldehyde reduction. It is proposed that, under So limitation, ADH reduces to alcohols the aldehydes that are generated by fermentation, thereby serving to dispose of excess reductant.
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PMID:Effects of elemental sulfur on the metabolism of the deep-sea hyperthermophilic archaeon Thermococcus strain ES-1: characterization of a sulfur-regulated, non-heme iron alcohol dehydrogenase. 764 2

The effects of pyridoxal 5'-phosphate (PalP) on ox liver glutamate dehydrogenase (94% inactivation by 1.8 mM reagent at pH 7 and 25 degrees C) have been compared with those of three analogues, 5'-deoxypyridoxal (96% inactivation), pyridoxal 5'-sulphate (97%) and pyridoxal 5-methylsulphonate (94%), in order to establish whether PalP acts as an affinity label for this enzyme. Like PalP and unlike pyridoxal, which is a much less potent inactivator, none of the analogues has a free 5'-OH group to cyclize with the aldehyde function. The result with 5'-deoxypyridoxal shows that a negative charge, such as that of the phosphate group, is not required for efficient inactivation. With all four reagents, addition of an excess of cysteine or lysine led to 90-100% re-activation over 3-20 h. Dialysis also caused reactivation to a similar extent. A combination of 2.15 mM NADH, 1 mM GTP and 10 mM 2-oxoglutarate gave complete protection against PalP, but only partial protection against the analogues. 5'-Deoxypyridoxal still caused 20-25% inactivation in the presence of the protection mixture. Absorbance measurements after reduction with NaBH4 show the characteristic features of a reduced Schiff's base and allowed estimation of the extent of reaction. With all the reagents the protection mixture decreased incorporation by about 1 mol/mol, but levels of incorporation without protection varied from about 2 mol/mol for PalP up to about 5 mol/mol for 5'-deoxypyridoxal. The labelling at additional sites may explain the residual inactivation in the presence of potent protecting agents.
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PMID:Is pyridoxal 5'-phosphate an affinity label for phosphate-binding sites in proteins?: The case of bovine glutamate dehydrogenase. 837 38

Two soluble forms of brain glutamate dehydrogenase isoproteins were inactivated by pyridoxal 5'-phosphate. Restoration of catalytic activity can be accomplished by dialysis and addition of an excess of cysteine or lysine. Spectral evidence is presented to indicate that the inactivation proceeds through Schiff base formation with amino groups of the enzyme. Inactivation became irreversible after reduction with NaBH4 and the NaBH4-reduced enzyme showed a characteristic absorption peak at 325 nm. Using spectral titration at 325 nm, the stoichiometry was 2 mol/mol of GDH subunit without protection and 1 mol/mol with protection, indicating the complete masking of one mol of lysine. The results with analogs of pyridoxal 5'-phosphate show that the aldehyde group, but not the phosphate group, is required for efficient inactivation.
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PMID:Modification of brain glutamate dehydrogenase isoproteins with pyridoxal 5'-phosphate. 911 50

An enzymatic assay was developed for the spectrophotometric determination of glycolate in urine and plasma. Glycolate was first converted to glyoxylate with glycolate oxidase, and the glyoxylate formed was condensed with phenylhydrazine. The glyoxylate phenylhydrazone formed was then oxidized with K(3)Fe(CN)(6) in the presence of excess phenylhydrazine, and A(515) of the resulting 1, 5-diphenylformazan was measured. Since glycolate oxidase also acts on glyoxylate and L-lactate, the incubation of samples with glycolate oxidase was carried out in 120-170 mM Tris-HCl (pH 8.3) to obtain glyoxylate as its adduct with Tris. The pyruvate formed from lactate was removed by subsequent brief incubation with alanine aminotransferase in the presence of L-glutamate, and alpha-ketoglutarate formed was converted back to L-glutamate by glutamate dehydrogenase and an NADPH generating system. Thus the specificity of the assay relies principally on the substrate specificity of glycolate oxidase, and high sensitivity is provided by the high absorbance of 1,5-diphenylformazan at 515-520 nm. Plasma was deproteinized with perchloric acid, and then neutralized with KOH. Plasma and urine samples were then incubated with approximately 5 mM phenylhydrazine, and then treated with stearate-deactivated activated charcoal to remove endogenous keto and aldehyde acids as their phenylhydrazones. The normal plasma glycolate and urinary glycolate/creatinine ratio for adults determined by this method are approximately 8 microM and approximately 0.036, respectively.
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PMID:A spectrophotometric method for the determination of glycolate in urine and plasma with glycolate oxidase. 1073 95

It has been suggested that reactive lysine residue(s) may play an important role in the catalytic activities of glutamate dehydrogenase (GDH). There are, however, conflicting views as to whether the lysine residues are involved in Schiff's base formation with catalytic intermediates, stabilization of negatively charged groups or the carbonyl group of 2-oxoglutarate during catalysis, or some other function. We have expanded on these speculations by constructing a series of cassette mutations at Lys130, a residue that has been speculated to be responsible for the activity of GDH and the inactivation of GDH by pyridoxal 5'-phosphate (PLP). For these studies, a 1557-bp gene that encodes human GDH has been synthesized and inserted into Escherichia coli expression vectors. The mutant enzymes containing Glu, Gly, Met, Ser, or Tyr at position 130, as well as the wild-type human GDH encoded by the synthetic gene, were efficiently expressed as a soluble protein and are indistinguishable from that isolated from human and bovine tissues. Despite an approximately 400-fold decrease in the respective apparent Vmax of the Lys130 mutant enzymes, apparent Km values for NADH and 2-oxoglutarate were almost unchanged, suggesting the direct involvement of Lys130 in catalysis rather than in the binding of coenzyme or substrate. Unlike the wild-type GDH, the mutant enzymes were unable to interact with PLP, indicating that Lys130 plays an important role in PLP binding. The results with analogs of PLP suggest that the aldehyde moiety of PLP, but not the phosphate moiety, is required for efficient binding to GDH.
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PMID:Cassette mutagenesis of lysine 130 of human glutamate dehydrogenase. An essential residue in catalysis. 1138 22