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)

Recombinant Clostridium difficile glutamate dehydrogenase (L-glutamate:NAD oxidoreductase, EC 1.4.1.2) was purified 177-fold to electrophoretic homogeneity with a 62% recovery through a four-step procedure involving gel filtration and ion-exchange and dye affinity chromatography. The approximate molecular weights of the native enzyme by gel filtration and subunits by sodium dodecyl sulfate-polyacrylamide gel electrophoresis were consistent with a hexameric structure for the purified enzyme. The enzyme-catalyzed glutamate oxidation was an NAD-dependent sequential process in which NADP could not be substituted as coenzyme. Several dinucleotide analogs of NAD structurally altered in either the pyridine or the purine moiety were observed to function as coenzymes when substituted for NAD. Nicotinamide mononucleotide did not serve as a coenzyme for glutamate oxidation. Product inhibition by NADH was competitive with respect to NAD. In deadend inhibition studies, adenosine diphosphoribose was shown to be an effective coenzyme-competitive inhibitor.
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PMID:Purification and characterization of Clostridium difficile glutamate dehydrogenase. 842 83

In Saccharomyces cerevisiae, carbon and nitrogen metabolisms are connected via the incorporation of ammonia into glutamate; this reaction is catalyzed by the NADP-dependent glutamate dehydrogenase (NADP-GDH) encoded by the GDH1 gene. In this report, we show that the GDH1 gene requires the CCAAT box-binding activator (HAP complex) for optimal expression. This conclusion is based on several lines of evidence: (1) overexpression of GDH1 can correct the growth defect of hap2 and hap3 mutants on ammonium sulfate as a nitrogen source, (ii) Northern (RNA) blot analysis shows that the steady-state level of GDH1 mRNA is strongly lowered in a hap2 mutant, (iii) expression of a GDH1-lacZ fusion is drastically reduced in hap mutants, (iv) NADP-GDH activity is several times lower in the hap mutants compared with that in the isogenic wild-type strain, and finally, (v) site-directed mutagenesis of two consensual HAP binding sites in the GDH1 promoter strongly reduces expression of GDH1 and makes it HAP independent. Expression of GDH1 is also regulated by the carbon source, i.e., expression is higher on lactate than on ethanol, glycerol, or galactose, with the lowest expression being found on glucose. Finally, we show that a hap2 mutation does not affect expression of other genes involved in nitrogen metabolism (GDH2, GLN1, and GLN3 encoding, respectively, the NAD-GDH, glutamine synthetase, and a general activator of several nitrogen catabolic genes). The HAP complex is known to regulate expression of several genes involved in carbon metabolism; its role in the control of GDH1 gene expression, therefore, provides evidence for a cross-pathway regulation between carbon and nitrogen metabolisms.
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PMID:The CCAAT box-binding factor stimulates ammonium assimilation in Saccharomyces cerevisiae, defining a new cross-pathway regulation between nitrogen and carbon metabolisms. 860 56

Cochineal (C), a scarlet material extracted from the powdered pregnant insect, Dactylopius Coceus Costa, is used as a color food additive in the form of aluminum lakes. A 13 week subchronic toxicity study was conducted to investigate the effects of simultaneous administration of C and aluminum potassium sulfate (A). Male and female Wistar rats (5-weeks-old, 15 rats/group) were given diets containing 0.75%A and 0.75%C (1.5%AC), 1.5%A and 1.5%C (3%AC), 3%C alone or 3%A alone. The following results were obtained. 1) No toxic symptoms or death occurred in any treated group. Body weight gain in male rats of the 3%A group decreased significantly. 2) Serum levels of phospholipids, triglycerides (TG) and total cholesterol in male rats and TG in female rats fed 3%C, 3%A or 3%AC were significantly decreased at the 13th week. The serum level of glutamate dehydrogenase (GIDH) in male rats treated with 1.5% or 3%AC was increased at the 4th week but no difference from control was observed at the 13th week. 3) No histopathological changes attributable to A and/or C administration were observed. In this 13-week oral toxicity study, no dose-dependent synergistic effects of simultaneous administration of C and A were found except for an increase in serum GIDH.
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PMID:[A 13-week toxicity study of simultaneous administration of cochineal and aluminum potassium sulfate in rats]. 885 2

Up to now, removal of sodium dodecyl sulfate (SDS) from proteins in terms of restoration of their activity was an unsolved problem. A general procedure using ceramic hydroxyapatite (HAP) chromatography was developed for the complete removal of SDS bound to soluble or membrane proteins. This procedure involves (i) the binding of the SDS-protein complexes onto the ceramic hydroxyapatite column, (ii) extensive washing of bound proteins with phosphate buffer containing a mild detergent to exchange SDS, (iii) elution of the retained protein by increasing the phosphate concentration. Using this approach, complete exchange of [35S]SDS into a nonionic detergent such as dodecyl maltoside was achieved with a 90-100% protein recovery. The efficiency of protein-bound SDS removal is very likely due to the combined effect of phosphate ions and the hydrophobic tail of nonionic detergent: acting together, they are able to displace SDS molecules from their protein-binding sites. The advantages of this HAP-mediated SDS removal method include high efficiency, rapidity, simplicity and general applicability to a wide variety of detergents and soluble or membrane proteins. Of utmost importance, SDS-treated P-glycoprotein, glutamate dehydrogenase, and lysozyme fully recovered their enzymatic activities after HAP chromatography, including lysozyme electroeluted from SDS-polyacrylamide gel electrophoresis. This demonstrates that reactivation of SDS-treated protein can be achieved, provided that SDS is completely removed under mild conditions.
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PMID:Complete removal and exchange of sodium dodecyl sulfate bound to soluble and membrane proteins and restoration of their activities, using ceramic hydroxyapatite chromatography. 917 96

The ADP-receptor on the surface of human platelets and cells of megakaryocytic lineage has been classified as P2T purinergic receptor for which ADP is an agonist and ATP is an antagonist. Although it is one of the earliest identified of the important cellular receptors, it has neither been purified nor cloned. We have developed an immunoaffinity method for rapidly identifying the platelet ADP-receptor and this method can be extended to the purification of the receptor. A polyclonal antibody to glutamate dehydrogenase (GDH) covalently modified by 5'-p-fluorosulfonylbenzoyladenosine (FSBA) recognized neither FSBA nor glutamate dehydrogenase. Immunoblot of the gel obtained by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of solubilized FSBA-labeled platelets showed the presence of a protein band at 100 kDa and this band was absent in the immunoblots of platelets that were preincubated with ADP and ATP or covalently modified by the chemically reactive ADP-affinity analogs, 2- and 8-(4-bromo-2,3-dioxobutylthio)adenosine-5'-diphosphate (2- and 8BDB-TADP) and 2-(3-bromo-2-oxopropylthio)adenosine-5'-diphosphate (2-BOP-TADP), prior to treatment with FSBA. FSBA as well as 2- and 8-BDB-TADP and 2-BOP-TADP have been previously shown to inhibit ADP-induced platelet responses by selectively and covalently modifying aggregin (100 kDa), an ADP-receptor in intact human blood platelets. The results show that polyclonal antibody to FSBA-labeled GDH is capable of recognizing FSBA-labeled aggregin on platelets and, thus, could be used to purify aggregin by immunoaffinity column chromatography. The immunoaffinity method was found to be far more sensitive than the radiochemical methods to identify aggregin previously developed in our laboratory. Since FSBA is also capable of reacting with enzymes that require ATP for their catalytic function, the polyclonal antibody may be used to identify and purify other P2-type purinergic receptors that require binding of ATP before eliciting cellular responses.
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PMID:Immunoaffinity method to identify aggregin, a putative ADP-receptor in human blood platelets. 936 34

NADP+-specific glutamate dehydrogenase (EC 1.4.1.4) was purified to homogeneity from the extremely thermophilic, strictly anaerobic, sulfate-reducing archaeon Archaeoglobus fulgidus strain 7324. The native enzyme (263 kDa) is composed of subunits of mol. mass 46 kDa, suggesting a hexameric structure. The temperature optimum for enzyme activity was > 95 degrees C. The enzyme was highly thermostable, having a half-life of 140 min at 100 degrees C. Potassium phosphate, KCl, and NaCl enhanced the thermal stability and increased the rate of activity three- to fourfold. The N-terminal 26-amino-acid sequence showed a high degree of similarity to glutamate dehydrogenases from Pyrococcus spp. and Thermococcus spp.
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PMID:Purification and properties of an extremely thermostable NADP+-specific glutamate dehydrogenase from Archaeoglobus fulgidus. 938 47

The NAD-dependent glutamate dehydrogenase (GDH) (EC 1.4.1.2) from Laccaria bicolor was purified 410-fold to apparent electrophoretic homogeneity with a 40% recovery through a three-step procedure involving ammonium sulfate precipitation, anion-exchange chromatography on DEAE-Trisacryl, and gel filtration. The molecular weight of the native enzyme determined by gel filtration was 470 kDa, whereas sodium dodecyl sulfate-polyacrylamide gel electrophoresis gave rise to a single band of 116 kDa, suggesting that the enzyme is composed of four identical subunits. The enzyme was specific for NAD(H). The pH optima were 7.4 and 8.8 for the amination and deamination reactions, respectively. The enzyme was found to be highly unstable, with virtually no activity after 20 days at -75 degrees C, 4 days at 4 degrees C, and 1 h at 50 degrees C. The addition of ammonium sulfate improved greatly the stability of the enzyme and full activity was still observed after several months at -75 degrees C. NAD-GDH activity was stimulated by Ca2+ and Mg2+ but strongly inhibited by Cu2+ and slightly by the nucleotides AMP, ADP, and ATP. The Michaelis constants for NAD, NADH, 2-oxoglutarate, and ammonium were 282 &mgr;M, 89 &mgr;M, 1.35 mM, and 37 mM, respectively. The enzyme had a negative cooperativity for glutamate (Hill number of 0.3), and its Km value increased from 0.24 to 3.6 mM when the glutamate concentration exceeded 1 mM. These affinity constants of the substrates, compared with those of the NADP-GDH of the fungus, suggest that the NAD-GDH is mainly involved in the catabolism of glutamate, while the NADP-GDH is involved in the catalysis of this amino acid. Copyright 1997 Academic Press. Copyright 1997 Academic Press
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PMID:Purification and characterization of the NAD-dependent glutamate dehydrogenase in the ectomycorrhizal fungus laccaria bicolor (Maire) orton 945 44

The structural differences between two types of glutamate dehydrogenase (GDH) isoproteins (GDH I and GDH II), homogeneously isolated from bovine brain, were investigated using a biosensor technology and monoclonal antibodies. A total of seven monoclonal antibodies raised against GDH II were produced, and the antibodies recognized a single protein band that comigrates with purified GDH II on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot. Of seven anti-GDH II monoclonal antibodies tested in the immunoblot analysis, all seven antibodies interacted with GDH II, whereas only four antibodies recognized the protein band of the other GDH isoprotein, GDH I. When inhibition tests of the GDH isoproteins were performed with the seven anti-GDH II monoclonal antibodies, three antibodies inhibited GDH II activity, whereas only one antibody inhibited GDH I activity. The binding affinity of anti-GDH II monoclonal antibodies for GDH II (K(D) = 1.0 nM) determined using a biosensor technology (Pharmacia BIAcore) was fivefold higher than for GDH I (K(D) = 5.3 nM). These results, together with epitope mapping analysis, suggest that there may be structural differences between the two GDH isoproteins, in addition to their different biochemical properties. Using the anti-GDH II antibodies as probes, we also investigated the cross-reactivities of brain GDHs from some mammalian and an avian species, showing that the mammalian brain GDH enzymes are related immunologically to each other.
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PMID:Different antigenic reactivities of bovine brain glutamate dehydrogenase isoproteins. 1021 98

Anoxic, fresh-water enrichment cultures to oxidize different organosulfonates were set up with nitrate, ferric iron or sulfate as electron acceptors. Pure cultures were easily obtained with two naturally occurring sulfonates, cysteate (2-amino-3-sulfopropionate) and taurine (2-aminoethanesulfonate), under nitrate-reducing conditions. These two sulfonates were also oxidized during reduction of iron(III), though isolation of pure cultures was not successful. One nitrate-reducing cysteate-oxidizing bacterium, strain NKNCYSA, was studied in detail. It was identified as Paracoccus pantotrophus. Eighteen sulfonates were tested, and the organism degraded cysteate, taurine, isethionate (2-hydroxyethanesulfonate), sulfoacetate or 3-aminopropanesulfonate with concomitant reduction of nitrate, presumably to molecular nitrogen. The carbon skeleton of these substrates was converted to cell material and, presumably, CO2. The amino group was released as ammonia and the sulfono moiety was recovered as sulfate. Cell-free extracts of P. pantotrophus NKNCYSA contained constitutive L-cysteate:2-oxoglutarate aminotransferase (EC 2.6.1.-) and glutamate dehydrogenase (EC 1.4.1.4). Taurine:pyruvate aminotransferase, in contrast, was inducible.
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PMID:Anaerobic oxidations of cysteate: degradation via L-cysteate:2-oxoglutarate aminotransferase in Paracoccus pantotrophus. 1037 31

Recombinant glutamate dehydrogenase from a hyperthermophilic archaeon, Thermococcus profundus, was crystallized in the presence of both polyethylene glycol 8000 and lithium sulfate. Four types of crystals having different morphologies appeared in the crystallization trials; however, only one type was suitable for X-ray crystal structure analysis. The crystal belonged to the monoclinic space group P2(1) and the unit-cell parameters were a = 112.99, b = 163.70, c = 133.07 A, beta = 113.46 degrees at 110 K. The calculated V(M) value of 3.42 A(3) Da(-1) was acceptable when one hexamer of the enzyme, which was the physiological functional unit, occupied a crystallographic asymmetric unit. X-ray diffraction intensity data were collected to a resolution of 2.25 A with good statistics at the BL44B2 beamline of SPring-8.
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PMID:Crystallization and preliminary x-ray diffraction studies of hyperthermostable glutamate dehydrogenase from Thermococcus profundus. 1053 94


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