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)

Different levels of carbon dioxide (10,20, 40, 80, mM) at pH 6.7, 7.3, 7.6 and 8.5 of incubation media were studied for their effect on the intensity of binding of 14C from bicarbonate buffer by crystalline NAD(P)+-dependent glutamate dehydrogenase from the bovine liver. The effect of a variety of substrates and effectors on this process is investigated. The intensity of the radioactive label binding is shown to increase with pH of the incubation medium and carbon dioxide concentration in it. When acidifying the medium (up to pH 6.5) the enzyme under study does not bind 14C. The preincubation of the enzyme with pyridoxal-5-phosphate decreases considerably the intensity of the label binding with the protein. On the basis of these data a conclusion is drawn about the interaction of CO2 with free amino groups of the enzyme molecule by the type of the carbamate formation. GTP and NADH inhibit this process as well.
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PMID:[Binding by glutamate dehydrogenase of 14C from bicarbonate buffer]. 393 17

Two membrane-bound glutamate dehydrogenases were found in adult Dirofilaria immitis, an NAD-linked enzyme (EC 1.4.1.2) in the cytosol (C-GDH) and an enzyme equally reactive with NAD or NADP (EC 1.4.1.3) in the mitochondria (M-GDH). The cytosolic enzyme had a pH optimum of 7.8-8.0 and exhibited 30% more activity at 25 C than at 37 C (pH 8.0). The mitochondrial enzyme had a pH optimum at 8.4 and exhibited 27% more activity at 37 C than at 25 C (pH 8.4); it was also more sensitive to heat denaturation. Gel filtration of worm subfractions separated four peaks of C-GDH activity with molecular weights of approximately 610, 285, 180, and less than 100 thousand, and a single major peak of M-GDH activity with a molecular weight of about 335,000. When assayed at pH 8, 37 C, and 200 microM NADH, the Km for the substrate, alpha-ketoglutarate, was equivalent for the two enzymes, but the Km for ADP (activator) was five times greater for M-GDH. When the two enzymes were assayed at pH 8.0, 37 C, and 100 microM NADH, 1 mM ADP approximately doubled and 1 mM ATP halved the velocity observed for each enzyme with no effector present. Under these assay conditions AMP, IDP, GDP, and GTP had opposite effects on the reaction velocities for the two enzymes. When the assay conditions were changed, the effects of added purine nucleotides varied, even directionally. Addition of up to 5 mM glutamate (product) had no significant effect on C-GDH kinetics, nor on the substrate Km of M-GDH.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Dirofilaria immitis: comparison of cytosolic and mitochondrial glutamate dehydrogenases. 395 79

The ribose-modified chromophoric and fluorescent analog of ATP, 2',3'-O-(2,4,6-trinitrocyclohexadienylidene)-ATP (TNP-ATP) (Hiratsuka, T., and Uchida, K. (1973) Biochim. Biophys. Acta 320, 635-647 and Hiratsuka, T. (1976) Biochim. Biophys. Acta 453, 293-297) has been widely used as an ATP analog for various ATPases. Although the corresponding analog of GTP,2',3'-O-(2,4,6-trinitrocyclohexadienylidene)-GTP (TNP-GTP) should be useful for the study of various GTP-requiring enzymes, it is difficult to prepare TNP-GTP by the conventional method. In the present study, we succeeded in the synthesis of TNP-GTP with the use of an alternative method. The analogs of GDP, GMP, and guanyl-5'-yl imidodiphosphate (Gpp(NH)p) were also synthesized. Visible absorption and fluorescent properties of TNP-GTP, TNP-GDP, TNP-GMP, and TNP-Gpp(NH)p were quite similar to those of TNP-ATP. TNP-GTP was found to be able to replace GTP as an inhibitor for bovine liver glutamate dehydrogenase. The enzyme was inhibited by TNP-GTP to a maximum extent of 54% at saturating concentrations of the analog with a KI of 2.7 microM. TNP-Gpp(NH)p and other ribose-modified fluorescent analogs of GTP,3'-O-anthraniloyl-GTP and 3'-O-(N-methylanthraniloyl)-GTP (Hiratsuka, T. (1983) Biochim. Biophys. Acta 742, 496-508), also inhibited the enzymatic activity. Binding of TNP-GTP to the enzyme was characterized by a 5.6-fold enhancement in analog fluorescence. In the presence of NADH, the limiting fluorescence enhancement of the bound analog decreased to 2.7-fold. As determined by fluorometric titration, the maximum number of TNP-GTP binding sites on the enzyme was 1.9 mol/mol of subunit with a KD of 0.66 microM in the absence of NADH and 2.2 mol/mol of subunit with two KD values of 0.11 and 0.71 microM in the presence of NADH. These observations suggest that NADH binding increases the affinity of only 1 mol of the 2 mol of TNP-GTP bound to the enzyme. These spectroscopic and biological properties of TNP-GTP should make this analog useful as a chromophoric and fluorescent probe for studies not only of glutamate dehydrogenase but also of various GTP-requiring enzymes, which have a high specificity for the base moiety of GTP.
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PMID:A chromophoric and fluorescent analog of GTP, 2',3'-O-(2,4,6-trinitrocyclohexadienylidene)-GTP, as a spectroscopic probe for the GTP inhibitory site of liver glutamate dehydrogenase. 398 36

Chemical crosslinking with dimethyl pimelimidate has been used to examine the quaternary structure and conformational mobility of bovine liver glutamate dehydrogenase. Crosslinking patterns are shown to be consistent with either a stacked or staggered dimer of trimers structure of the hexamer. Crosslinking in the absence of coligands results in a small loss of activity but an almost complete loss of GTP inhibitory effects. Protection experiments show that the active site can be protected by a variety of ligand combinations, and that the loss of GTP inhibition is protected by several complexes containing either NADH or NADPH, indicating that the second coenzyme site per subunit (which preferentially binds NADH) is not involved in the protection process. A significant loss of ADP activation occurs during crosslinking which is not protected against by any combination of protecting ligands tried, including those which involve second coenzyme site binding, showing that the ADP site is functionally distinct from the GTP site and from the second coenzyme binding site. Crosslinking in the presence of protecting ligands gives similar gel patterns to those obtained in the absence of protection. Affinity chromatography experiments show that the crosslinked enzyme still binds GTP despite the loss of GTP inhibition, and hysteresis experiments show that the second coenzyme site is left functional if protected with either coenzyme. A model is presented where crosslinking affects the conformational linkage between various ligand binding sites involved in GTP inhibition rather than the sites themselves.
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PMID:Investigation of the effects of crosslinking glutamate dehydrogenase with dimethyl pimelimidate. 400 63

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.
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PMID:Ox glutamate dehydrogenase. Comparison of the kinetic properties of native and proteolysed preparations. 405 48

Explicit expressions are derived which describe the binding of a univalent ligand to equivalent and independent sites on each state of an acceptor undergoing indefinite self-association that is governed by an isodesmic equilibrium constant KI. From considerations of systems in which the same site-binding constant kA applies to all acceptor-ligand interactions, the general forms of binding curves and Scatchard plots are deduced for situations in which binding sites are either created or lost at each monomer-monomer interface. Greater generality is then introduced into the model by allowing ligand interactions with polymeric acceptor states to be governed by a site-binding constant kp that differs in magnitude from that for monomeric acceptor kA. Finally, experimental results with the glutamate dehydrogenase-GTP and lysozyme-saccharide systems are used to illustrate ways in which the present quantitative expressions may be applied to the characterization of inteactions between a ligand and an indefinitely self-associating acceptor.
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PMID:The binding of a ligand to an acceptor undergoing indefinite self-association. 409 54

1. The effect of NADH and the non-competitive inhibitor GTP on the optical-rotatory-dispersion properties of glutamate dehydrogenase has been studied. 2. Analysis of the data in terms of the a(0) and b(0) parameters of the Moffitt-Yang equation indicates that a conformational change is induced either by NADH or by GTP in the presence of small amounts of NADH. 3. Sedimentation measurements under comparable conditions showed that the enzyme reversibly dissociates into sub-units but that this dissociation is only secondary to the conformational changes. 4. Fluorescence measurements showed that the binding constant of NADH and the number of binding sites on the enzyme increased in the presence of GTP. 5. This is confirmed by studies of fluorescence polarization, which in addition showed that the movement of NADH on the enzyme surface is more restricted in the presence of GTP. 6. The relation of these results to possible regulatory mechanisms is discussed.
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PMID:Conformational changes and the regulation of glutamate-dehydrogenase activity. 428 81

1. The interaction of 1-anilinonaphthalene-8-sulphonate with ox liver glutamate dehydrogenase was examined. 2. The fluorescence of the dye is enhanced 100-fold on binding. 3. A further enhancement is observed when NADH and GTP are added to the enzyme. 4. By using this property of the dye to measure conformational equilibria in the enzyme the effects of coenzyme, inhibitors, enzyme concentration, ionic strength and pH on the allosteric transitions were studied. 5. GTP and NADH interact with the enzyme in a heterotropic manner. 6. The rate of the structural transition brought about by GTP and NADH is biphasic with half-lives of 34 and 200msec. 7. The relation of these observations to regulatory mechanisms is discussed.
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PMID:1-Anilinonaphthalene-8-sulphonate, a fluorescent conformational probe for glutamate dehydrogenase. 430 11

1. Modification with 2,4,6-trinitrobenzenesulphonic acid was studied for its effect on the structure, activity and response to regulatory effectors of ox liver glutamate dehydrogenase. 2. The modification affected amino groups only, and the relative reactivities of the amino groups of the enzyme are described. 3. A biphasic inactivation of the enzyme was observed and analysis of the course of inactivation and of modification showed that the rapid reaction of one amino group/subunit leads to loss of 80% of the enzymic activity. 4. NADH retarded the inactivation by 2,4,6-trinitrobenzenesulphonic acid, the protection increasing with NADH concentration. This, together with the previous observation, suggests that the rapidly reacting group is essential for the activity of the enzyme. 5. The effects of modification on the optical-rotatory-dispersion and sedimentation behaviour of the enzyme were studied. 6. The enzyme's response to the allosteric effector GTP was rapidly lost on modification, whereas its response to ADP was unaffected. Comparison of the inactivation and desensitization suggests that the reactive amino group is essential for both activity and GTP response, and that only a completely unmodified enzyme oligomer responds fully to GTP. 7. The merits of chemical-modification studies of large enzymes are discussed critically in connexion with the interpretation of these results.
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PMID:Chemical modification of glutamate dehydrogenase by 2,4,6-trinitrobenzenesulphonic acid. 430 31

An affinity labeling reagent for the estrogenic-binding site of bovine liver L-glutamate dehydrogenase (EC 1.4.1.3) was prepared by conversion of diethylstilbestrol to its alkylating analogue, bromoacetyldiethylstilbestrol. Under standard assay conditions, the analogue acted as a reversible allosteric ligand with regulatory activity much like that of diethylstilbestrol. However, incubation of the enzyme with the alkylating agent in the presence of DPNH resulted in a permanent decrease in glutamate (X form) and an increase in alanine (Y form) activities, and in covalent attachment of diethylstilbestrol in the ratio of 1 mol per subunit (of particle weight 52,000). The brominated analogue behaved as an affinity label that mimicked the allosteric effects of diethylstilbestrol. Diethylstilbestrol protection of the enzyme against alkylation by bromoacetylated sterol suggested competition for the same binding site, while ADP protection indicated a shift of protein equilibrium into the X form. The diethylstilbestrol-enzyme compound was desensitized (relative to the native enzyme) to allosteric reagents such as ADP and GTP. The results were consistent with conformational freezing of the modified protein molecule into the Y form.
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PMID:Covalent attachment of diethylstilbestrol to glutamate dehydrogenase: implications for allosteric regulation. 432 3


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