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Query: EC:1.4.3.11 (glutamate dehydrogenase)
 4,437 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The effect of nucleotides: AMP, cAMP, ADP, ATP, GDP and GTP, on glutamate dehydrogenase (GDH) purified from the mealworm fat body was studied. Guanine nucleotides and ATP inhibited the enzyme strongly in both directions. GDH was partially protected from the inhibition by the addition of ADP to an assay medium. AMP and cAMP activated the enzyme slightly. The concerted effects of ADP and ATP indicate the importance of adenylate energy charge in the regulation of fat body GDH. It is suggested that GDH may play amphibolic role in the fat body and that the direction of GDH catalysed reaction is under strong influence of nucleotides. The enzyme may synthesize glutamate at high energy charge, but when the energy reserves are low, it oxidizes glutamate.
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PMID:The effect of nucleotides on glutamate dehydrogenase from the mealworm fat body. 245 16

Mitochondrial functions were investigated in permeabilized rat liver cells. For permeabilization isolated hepatocytes were treated with digitonin using a perifusion technique. After permeabilization the cell count was almost unchanged, and the mitochondrial marker enzyme, glutamate dehydrogenase, was decreased to as little as 83%. The mitochondria in permeabilized cells were functionally intact, a finding evident from a marked stimulation of respiration by ADP, inhibition by carboxyatractyloside, and stimulation by uncoupler. The ADP-stimulated and uncoupled respiration rates with succinate as substrate were comparable to those reported for isolated mitochondria, whereas the rates with NAD(+)-dependent substrates were somewhat higher. The ratios between ADP-stimulated and carboxyatractyloside-inhibited respiration rates were in the range noted for isolated mitochondria with identical substrates. Almost unchanged ADP-stimulated and carboxyatractyloside-inhibited respiration rates were found for at least 180 min after digitonin treatment. The preparation exhibited only a low extramitochondrial ATPase activity which was partially inhibited by vanadate.
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PMID:Characterization of mitochondrial functions in digitonin-permeabilized rat liver cells. 261 33

Caffeine and theophylline inhibited the activity of rat liver glutamate dehydrogenase (GDH), but not that of beef liver GDH, in forward and reverse directions of the enzyme reaction. In the forward direction, approximately 16 mM caffeine or 16 mM theophylline inhibited 50 per cent of the rat liver GDH activity (I50); while in the reverse direction, the I50 of caffeine and theophylline was 15 mM and 8 mM, respectively. The inhibition produced by caffeine was cooperative in both directions, while that of theophylline was negatively cooperative in the forward direction and non-cooperative in the reverse. However, ADP reduced the inhibitory effect of caffeine and theophylline to the extent of 40% and 80%, respectively. The Ki values obtained for caffeine and theophylline were different in the presence of various concentrations of substrates and coenzymes. Based upon these data, we presume that certain subtle changes occurring in the conformation of the rat liver GDH (probably at the ADP/NADH site) in comparison with those of the beef liver GDH may be responsible for its inhibition by caffeine and theophylline.
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PMID:Interaction of caffeine and of theophylline with liver glutamate dehydrogenase. 271 13

Changes in conformation of glutamate dehydrogenase from beef liver as a result of interactions with allosteric effectors have been demonstrated from the phosphorescence emission of tryptophan. The triplet state lifetime shows that whereas activators ADP and L-leucine enhance considerably the rigidity of the protein structure surrounding the chromophore, inhibitors GTP, Zn2+ and Ag+ act in an opposite manner increasing the flexibility of this region of the macromolecule. Such changes in dynamical structure of the protein are confirmed independently for the ADP and GTP complexes by oxygen diffusion studies. Phosphorescence lifetime measurements at various protein concentrations and with the enzyme crosslinked by glutaraldehyde demonstrate that ADP and GTP exert the same effect on the structure of the protein regardless of its degree of polymerization. The connection between changes in protein structure and regulatory function is strengthened by the finding that (1) ligands with no regulatory function (Eu3+) do not affect protein structure; (2) pairs of opposite effectors which neutralize each other's influence on catalytic activity do restore an apparent native-like structure in the enzyme. Mutual neutralization and the observation that ADP and GTP display maximum activity at partial saturation of the binding sites has been interpreted in terms of a model which assumes asymmetry in the hexameric enzyme at the trimer level. Evidence for the existence of conformational heterogeneity among the subunits of the enzyme has been provided.
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PMID:Dynamical structure of glutamate dehydrogenase as monitored by tryptophan phosphorescence. Signal transmission following binding of allosteric effectors. 273 26

The fluorescence and phosphorescence properties of the tryptophan residues in glutamate dehydrogenase were utilized to probe the conformation of the macromolecule at various states of aggregation of its subunits (hexamer, trimer, and monomer) in guanidine hydrochloride. According to the phosphorescence lifetime no gross alteration in the conformation of the protein follows from complete dissociation of the hexamer into native monomer, implying that the native fold is stabilized exclusively by intrasubunit bonding. Although modest concentrations of denaturant induce a change in configuration in the enzyme, a comparison with the macromolecule cross-linked into the hexameric form by glutaraldehyde confirms that this alteration in structure is not the result of subunit dissociation. Inhibition of catalysis by the denaturant is found to be considerably smaller than anticipated from the extent of hexamer dissociation. Furthermore, this inhibition is in no way prevented by cross-linking the enzyme in its hexameric form. This finding together with the ability of the trimer to bind the coenzyme and to undergo the characteristic structural changes induced by the effectors ADP and GTP suggests that, contrary to what is generally believed, the smallest functional unit of glutamate dehydrogenase is not the hexameric form.
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PMID:Relationship between the conformation of glutamate dehydrogenase, the state of association of its subunit, and catalytic function. 275 97

A new bifunctional affinity label, 5'-p-(fluorosulfonyl)benzoyl-8-azidoadenosine (5'-FSBAzA), has been synthesized by condensation of p-(fluorosulfonyl)benzoyl chloride with 8-azidoadenosine. 5'-FSBAzA has been characterized by elemental analysis, thin-layer chromatography, and ultraviolet and 1H NMR spectroscopy. The affinity label contains both an electrophilic fluorosulfonyl moiety and a photoactivatable azido group which are capable of reacting with several classes of amino acids found in enzymes. 5'-FSBAzA reacts with bovine liver glutamate dehydrogenase in a two-step process: a dark reaction yielding about 0.5 mol of the sulfonylbenzoyl-8-azidoadenosine (SBAzA) group bound/mol enzyme subunit by reaction of the enzyme at the fluorosulfonyl group, followed by photolysis in which 25% of the covalently bound SBAzA becomes crosslinked to the enzyme. 5'-FSBAzA-modified glutamate dehydrogenase, both before and after photolysis, retains full catalytic activity but is less sensitive to allosteric inhibition by GTP, to activation by ADP, and to inhibition by 1 mM NADH. These results suggest the modification in the dark reaction of a regulatory nucleotide binding site. Photoactivation of the covalently bound reagent may have general applicability in relating modified amino acids which are close to each other in the region of the purine nucleotide binding sites of glutamate dehydrogenase and other proteins.
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PMID:5'-p-fluorosulfonyl)benzoyl-8-azidoadenosine: a new bifunctional affinity label for nucleotide binding sites in proteins. 281 2

A rapid enzymatic assay method for ammonia was developed by using glutamine synthetase from glutamate-producing bacteria together with pyruvate kinase, lactate dehydrogenase, and NADH. The time required for determination of 25 nmol of ammonia was 5 min with 1 unit of glutamine synthetase, as opposed to 14-30 min with 1 unit of glutamate dehydrogenases from various sources. The present method was used to determine ammonia in serum, microbiol-culture broth, and waste water. The method can be modified for spectrophotometry in the visible region by substituting pyruvate oxidase, peroxidase, and appropriate chromogens for lactate dehydrogenase and NADH. With 4-aminoantipyrine (4AA) and phenol, and with 4AA and N-ethyl-N-2-hydroxyethyl-m-toluidine as chromogens, the sensitivity of ammonia determination was 0.65 and 1.7 times that with glutamate dehydrogenase, respectively. The present method was also applicable to the continuous detection of the activity of some ammonia-forming enzymes such as guanase, adenosine deaminase, and urease and to the determination of 0.5-30 microM ATP-ADP after some modification of the mixture.
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PMID:A rapid assay method for ammonia using glutamine synthetase from glutamate-producing bacteria. 288 29

Linoleic acid, a polyunsaturated fatty acid, is a constituent of margosa oil which has been implicated as a cause of Reye's syndrome (RS) in infants. Increased concentrations of polyunsaturated fatty acids have been found in sera from patients with RS. Isolated rat liver mitochondria exposed to the peroxidized (but not unperoxidized) methyl esters of linoleic (C18:2) or linolenic (C18:3) acids showed decreases in state 3 and uncoupled respiratory rates and in respiratory control and ADP/O ratios. In addition, they caused mitochondrial swelling as demonstrated spectrophotometrically. Between the two, the peroxidized methyl ester of linolenic acid was more toxic and was capable of inducing high amplitude swelling ultrastructurally similar to that seen in the hepatocytes of RS victims. The ability of rat liver mitochondria to oxidize glutamate was inversely related to the peroxide concentration in the medium. This accords with the reports of reduced glutamic dehydrogenase activities in the livers of both patients with Reye's syndrome and rats treated with margosa oil.
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PMID:Effects of peroxidized polyunsaturated fatty acids on mitochondrial function and structure: pathogenetic implications for Reye's syndrome. 290 Jun 17

The activation of glutamate dehydrogenase (L-glutamate: NAD(P)+ oxidoreductase (deaminating), EC 1.4.1.3) by L-leucine has been studied. Apparently homogeneous preparations from ox liver and brain were found to respond similarly. Commercially obtained preparations of the enzyme, which had suffered limited proteolysis during the purification procedure, were shown to behave similarly to preparations which had not suffered such proteolysis when the effects of L-leucine on the oxidative deamination reaction were studied using either NAD+ or NADP+ as the coenzyme. There was also no significant difference in the responses when the reductive reaction was determined with NADPH or with 40 microM NADH. At higher concentrations of NADH (160 microM) the unproteolysed preparations were activated by L-leucine to a considerably greater extent than those which had suffered limited proteolysis. These results accord with the greater sensitivity of the former preparations to inhibition by high concentrations of NADH and the relief of such inhibition by L-leucine. This amino acid was also found to relieve the inhibition of the enzyme by GTP, resulting in an apparent increase in the activation observed in the presence of this nucleotide. In contrast, under the conditions used in this work, the apparent degree of activation by L-leucine was found to be decreased in the presence of the activators ATP or ADP. The presence of high concentrations of NADH (200 microM) potentiated the high substrate inhibition by 2-oxoglutarate, and L-leucine significantly reduced this effect. The effects of L-leucine on the activity of glutamate dehydrogenase thus appear to be composed of a direct effect on the activity of the enzyme together with a relief of high substrate inhibition. The effects of GTP and 2-oxoglutarate in potentiating inhibition by NADH can account for their effects in enhancing the apparent activation by L-leucine. The marked differences in the responses of proteolysed and unproteolysed preparations of the enzyme result from the effects of proteolysis in decreasing the sensitivity to high concentrations of NADH.
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PMID:Activation of glutamate dehydrogenase by L-leucine. 292 20

Bovine liver glutamate dehydrogenase reacts covalently with 2-(4-bromo-2,3-dioxobutylthio)adenosine 5'-monophosphate (2-BDB-TAMP) with incorporation of 1 mol reagent/mol enzyme subunit and loss of one of the two ADP sites of native enzyme [S. P. Batra and R. F. Colman, J. Biol. Chem. 261, 15565-15571 (1986)]. Incorporation of reagent is prevented specifically by ADP. The modified enzyme has now been digested with trypsin. The nucleotidyl peptide has been purified by chromatography on phenylboronate-agarose, followed by reverse-phase HPLC. On the basis of amino acid composition following acid hydrolysis, and gas-phase sequencing, the modified tryptic peptide was established as Ala-Gln-His-Ser-Gln-His-Arg, corresponding to amino acids 80-86 of the known glutamate dehydrogenase primary structure. The evidence presented indicates that the target amino acid attacked by 2-BDB-TAMP is histidine-82 and that this residue is located within the high-affinity ADP-activating site of glutamate dehydrogenase. In the course of this work, it was found that the positions of Gln84 and His85 had been reported as reversed in the revised sequence of bovine liver glutamate dehydrogenase [J. H. Julliard and E. L. Smith, J. Biol. Chem. 254, 3427-3438 (1979)]. Three additional corrections are here reported in the amino acid sequence of the native enzyme on the basis of gas-phase sequencing of other peptides purified by HPLC: Asp168 (not Asn); His221-Gly222 (not Gly-His); and Glu355 (not Gln).
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PMID:Identification of histidyl peptide labeled by 2-(4-bromo-2,3-dioxobutylthio)adenosine 5'-monophosphate in an ADP regulatory site of glutamate dehydrogenase. 293 Jan 90


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