Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: 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)

An incubation medium was established for the microphotometric demonstration of glutamate dehydrogenase (Gldh) in cryostat sections of the rat hippocampus which served as an exemplary brain region. The final incubation medium consisted of 100 mM L-glutamic acid monosodium salt, 5 mM NAD, 10 mM sodium azide (NaN3), 5 mM ADP, 20 mM sodium chloride, 0.15 mM phenazine methosulfate (PMS), 5 mM nitroblue tetrazolium chloride and 22% polyvinyl alcohol (PVA) in 0.05 M Hepes buffer; the final pH was 7.5. The study showed that in the histochemical demonstration of Gldh the use of relatively high PVA concentrations were necessary to avoid diffusion artefacts because Gldh seems to be only loosely bound to the mitochondrial matrix. The use of NaN3 as a blocker of the respiratory chain was indispensible, because without NaN3 most reduction equivalents were lost through the respiratory chain. With PMS as an exogenous electron carrier, the demonstrable Gldh activities increased significantly indicating that, in the case of Gldh, the endogenous NADH tetrazolium reductase was not sufficiently effective. Furthermore, it was shown that Gldh was affected by many small molecules (e.g. activation by sodium ions, inhibition by magnesium and calcium ions) so that minor variations of the incubation conditions may cause major differences in demonstrable activities.
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PMID:Microphotometric determination of enzymes in brain sections. III. Glutamate dehydrogenase. 233 53

In the presence of Mg2+, pure glutamate dehydrogenase is more reactive with NADPH than with NADH and is markedly activated by elevations in the ADP/ATP ratio or the addition of leucine. Because these are properties of glutamate dehydrogenase in mitochondria but not properties of the pure enzyme studied in the absence of Mg2+, Mg2+ could be a ligand that confers upon glutamate dehydrogenase the regulatory properties of this enzyme found in situ. In the absence of the allosteric activators ADP, leucine, or succinyl-CoA, Mg2+ is an inhibitor and increases product inhibition by alpha-ketoglutarate in the forward reaction and substrate inhibition by alpha-ketoglutarate in the reverse reaction. However, the allosteric activators convert Mg2+ from an inhibitor into an activator of the forward reaction. In the reverse reaction, ADP also converts Mg2+ from an inhibitor into an activator and leucine eliminates inhibition by Mg2+. Because Mg2+ is an inhibitor in the absence of activator that also increases inhibition by alpha-ketoglutarate, whereas in the presence of activator Mg2+ has no effect or is itself an activator, Mg2+ magnifies the effect of the activator, and magnification increases with increases in the concentration of alpha-ketoglutarate. Leucine and its analog 2-aminobicyclo (2.2.1) heptane 2-carboxylic acid (BCH) have almost identical effects on both human and bovine glutamate dehydrogenase in both the presence and absence of Mg2+. However, advantages of BCH over leucine as a potential pharmacological activator of glutamate dehydrogenase are that BCH is not metabolized and, unlike leucine, BCH does not inhibit ornithine transcarbamylase. Isoleucine and valine alone have little effect on human glutamate dehydrogenase, but isoleucine slightly inhibits the enzyme in the presence of leucine.
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PMID:Regulation of glutamate dehydrogenase by Mg2+ and magnification of leucine activation by Mg2+. 235 6

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

The effects of several natural products extracted from the leaves of Stevia rebaudiana on rat liver mitochondria were investigated. The compounds used were stevioside (a non-caloric sweetener), steviolbioside, isosteviol and steviol. Total aqueous extracts of the leaves were also investigated. S. rebaudiana natural products inhibited oxidative phosphorylation, ATPase activity NADH-oxidase activity, succinate-oxidase activity, succinate dehydrogenase, and L-glutamate dehydrogenase. The ADP/O ratio was decreased. Substrate respiration (state II respiration) was increased at low concentrations (up to 0.5 mM) and inhibited at higher concentrations (1 mM or more). In uncoupled mitochondria, inhibition of substrate respiration was the only effect observed. Net proton ejection induced by succinate and swelling induced by several substrates were inhibited. Of the compounds investigated, the sweet principle stevioside was less active. It was concluded that, in addition to the inhibitory effects, S. rebaudiana natural products may also act as uncouplers of oxidative phosphorylation. The possible physiologic consequences of the ingestion of stevioside and S. rebaudiana aqueous extracts are discussed.
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PMID:Effects of Stevia rebaudiana natural products on rat liver mitochondria. 285 11

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


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