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:1.2.1.13 (glyceraldehyde-3-phosphate dehydrogenase)
6,511 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The fluorescence of the natural coenzyme, NADH, is used to monitor the environment of the nicotinamide moiety at the active centre of rabbit muscle glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12). Changes of the fluorescence quantum yield and polarization of a small amount of NADH, totally bound by an excess of enzyme, show that at half-saturation of the oligomer with NAD a conformational change is induced which affects the active centre regions of the remaining subunits. This conformational transition is not effected by adenosine diphosphoribose, suggesting that the binding of the nicotinamide moiety of NAD to two subunits is essential for the change of tertiary structure of the remaining subunits that causes the observed changes of the fluorescence properties of the ADH "tracer probe". It is suggested that this conformational transition of the oligomer is responsible for the major decrease of affinity for NAD which occurs at half-saturation, and possibly for the activation by NAD+ of the reductive dephosphorylation reaction catalysed by the enzyme. It is also suggested, by analogy with haemoglobin, that the molecular basis of the negative cooperativity may be the creation of additional intersubunit bonds during the binding of the first two NAD molecules to the tetramer, and a change from a "relaxed" quaternary structure to a "tense" structure at half-saturation.
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PMID:Conformational changes of glyceraldehyde-3-phosphate dehydrogenase induced by the binding of NAD. A unified model for positive and negative cooperativity. 17 91

The binding of oxidized and reduced coenzyme (NAD+ and NADH) to 3-phosphoglyceroyl-glyceraldehyde-3-phosphate dehydrogenase has been studied spectrophotometrically and fluorimetrically. The binding of NAD+ to the acylated sturgeon enzyme is characterized by a significant quenching of the enzyme fluorescence (about 25%) and the induction of a difference spectrum in the ultraviolet absorbance region of the enzyme. Both of these spectroscopic properties are quantitatively distinguishable from those of the corresponding binary enzyme-NAD+ complex. Binding isotherms estimated by gel filtration of the acylated enzyme are in close agreement to those obtained by spectrophotometric and fluorimetric titrations. Up to four NAD+ molecules are bound to the enzyme tetramer. No anticooperativity can be detected in the binding of oxidized coenzyme, which is well described on the basis of a single class of four binding sites with a dissociation constant of 25 muM at 10 degrees C, pH 7.0. The binding of NADH to the acylenzyme has been characterized spectrophotometrically. The absorption band of the dihydronicotinamide moiety of the coenzyme is blue-shifted to 335 nm with respect to free NADH. In addition, a large hypochromicity (23%) is observed together with a significant increase of the bandwidth at half height of this absorption band. This last property is specific to the acylenzyme-DADH complex, since it disappears upon arsenolysis of the acylenzyme. The binding affinity of NADH to the acylated enzyme has been estimated by performing simultaneous spectrophotometric and fluorimetric titrations of the NADH appearance upon addition of NAD+ to a mixture of enzyme and excess glyceraldehyde 3-phosphate. In contrast to NAD+, the reduced coenzyme NADH appears to be relatively strongly bound to the acylated enzyme, the dissociation constant of the acylenzyme-NADH complex being estimated as 2.0 muM at 25 degrees C. In addition a large quenching of the NADH fluorescence (about 83%) is observed. The comparison of the dissociation constants of the coenzyme-acylenzyme complexes and the corresponding Michaelis constants suggests a reaction mechanism of the enzyme in which significant formation and dissociation of NAD+-acylenzyme and NADH-acylenzyme complexes occur. Under physiological conditions the activity of the enzyme can be regulated by the ratio of oxidized and reduced coenzymes. Possible reasons for the lack of anticooperativity in coenzyme binding to the acylated form of the enzyme are discussed.
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PMID:Specific interactions of 3-phosphoglyceroyl-glyceraldehyde-3-phosphate dehydrogenase with coenzymes. 17 14

The kinetic method and selective chemical modification have been used in studies of the kinetic manifestations of active site interactions in D-glyceraldehyde-3-phosphate dehydrogenase (GAP dehydrogenase). The reactions of glyceraldehyde and glyceraldehyde-3-phosphate oxidation were studied in the absence of substrate excess. In support of the data obtained previously it was shown that only a part of the tightly bound NAD molecules can be reduced after substrate addition. "Partial reducibility" is observed at various degrees of saturation of the enzyme with NAD involving a single NAD molecule per tetrametric enzyme. These facts can hardly be explained by assumption of functional non-equivalence of active sites, whether induced by coenzyme or preexisting in the apoenzyme. It was proven by selective alkylation of the catalytic SH groups that "partial reducibility" is due to the circumstance that equilibrium in the system under investigation is established at nearly equal NAD and NADH concentrations. A plot of initial reaction rates versus NAD concentration (at non-saturating substrate concentrations) gives S-shaped curves; this is explained by considerable enzyme activation upon saturation of the fourth site with coenzyme. After modification of three active sites with iodoacetate the S-shape of the curve disappeared. This fact leads to the conclusion that active site interactions are required for formation of the S-shaped curves. The activity of a single site functioning in the modified enzyme reached values equal to those of the active sites in the native enzyme in the fully activated state. A model is proposed which can explaine the variations in mode of enzyme activation in the native and modified states. It is suggested that the surroundings of all four SH groups must be altered in order to activate the enzyme; such changes can be induced either by alkylation of the SH groups or by NAD binding. Evidence is presented that important functional properties of GAP dehydrogenase cannot be elucidated at low enzyme concentrations and with excess of substrates: three active sites are saturated under such conditons and practically inactive, and the fourth site obeys Michaelis - Menten kinetics.
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PMID:[Kinetic manifestations of the interaction of active centers in swine skeletal muscle D-glyceraldehyde-3-phosphate dehydrogenase]. 18 4

Kinetic analysis of the reactivation in vitro of glyceraldehyde-3-phosphate dehydrogenase from yeast in the presence of NAD+ suggested that transconformation reactions of inactive monomers and their subsequent association to native tetramers are responsible for the sigmoidal relaxations [R. Rudolph et al. (1977) Eur. J. Biochem. 81, 563-570]. Comparison with the reactivation behaviour in the absence of coenzyme was not feasible at this stage due to the instability of the apoenzyme. In the present study, solvent conditions were established which allowed both apoenzyme and holoenzyme to exhibit high stability. The apoenzyme is stable in phosphate buffer; but if excess NAD+ and phosphate are present (both of which stabilize the enzyme if applied separately), destabilization occurs. Protection of functional groups against oxidation by addition of a reducing agent and by degassing and preventing contact with air, increase the stability. Only partial stabilization can be achieved in the presence of NADH. Comparing the kinetics of reactivation in the presence and absence of coenzymes shows that both oxidized and reduced coenzyme enhance the rate of reactivation significantly, and to the same extent. The kinetic effect of coenzyme binding to the refolding polypeptide chain is discussed in terms of the stabilization of intermediates or end products of reconstitution on the one hand, and acceleration of folding and association reactions, on the other.
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PMID:Influence of coenzyme on the refolding and reassociation in vitro of glyceraldehyde-3-phosphate dehydrogenase from yeast. 22 31

Specific reaction of Cys-149 with 3,3,3-trifluorobromacetone allows one to probe symmetry relation between the active center regions of tetrameric glyceraldehyde-3-phosphate dehydrogenase by 19F nuclear magnetic resonance (nmr) techniques. Nmr titration studies in the pH range of greatest enzymic activity reveal the existence of species with an (alphaalpha')2 structure; this symmetry is not induced by the coenzyme. Addition of NADH to the ketone-labeled protein causes the enzymic reduction of the ligand in a stereospecific manner and is used to demonstrate the functionality of residues other than Cys-149 that are essential for catalysis. The interpretation of chemical shift characteristics found for the trifluoroacetonyl group together with the kinetics of its reduction allows the derivation of a dynamic model for the enzymic structure which may contribute to understanding of the half-of-the-sites phenomenon.
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PMID:19F nuclear magnetic resonance studies of structure and function relationships in trifluoroacetonylated rabbit muscle glyceraldehyde-3-phosphate dehydrogenase. 23 79

Glycerolization of freshly collected human erythrocytes leads to a reduction in ATP levels which return to their original values following deglycerolization. The reduction in ATP levels is largely prevented by pyruvate but not by inosine and glucose, singly or in combination. This implies that the ATP lesion associated with glycerolization reflects mainly a decrease in the NAD/NADH ratio and accordingly a reduced glyceraldehyde-3-phosphate dehydrogenase activity. 2,3-DPG levels were not influenced by glycerolization and deglycerolization. Erythrocytes depleted of ATP in the presence of glycerol can have their ATP levels repleted by either pyruvate or inosine, but require the presence of both compounds for maintenance during a post-rejuvenation incubation period. This indicates that the prolonged presence of glycerol influences both the NAD/NADH ratio and the carbon-containing intermediates of glycolysis. Glycerol did not influence ATP repletion of erythrocytes stored at 4 degrees C for 20 days by inosine in combination with pyruvate, but substantially decreased the repletion of their 2,3-DPG levels.
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PMID:Influence of glygerol on ATP and 2,3-DPG levels of human erythrocytes. 46 14

A reduction in myocardial oxygen supply during ischemia, not only leads to reduced aerobic ATP production but does not stimulate glycolytic ATP synthesis. The residual aerobically synthesized ATP comes primarily from continued inefficient (i.e., compared to glucose in terms of moles of ATP produced per mole of O2 consumed) oxidation of fatty acids. This leads to elevated tissue levels of long chain fatty acyl-CoA and fatty acyl-carnitine. Both are potentially cell damaging metabolic intermediates. Restriction of glycolysis is due to inhibition of glyceraldehyde-3-phosphate dehydrogenase by accumulated metabolites, such as H+, lactate and NADH. The reduced production of ATP leads to decreased levels of high energy phosphate stores which in turn may impair myocardial mechanical function.
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PMID:Energy metabolism in the ischemic heart. 55 21

The stress of chronic hypobaric hypoxia present at high altitudes induces a series of adaptive changes in the intermediate metabolism in erythrocytes of high-altitude natives. Aymaras of the high Andean Plateau are shown to have within erythrocytes: (a) increased activity of NADH2 (GAPDH) generating stages, (b) decreased activity of NADH2 (LDH) consuming steps, (c) significantly increased methaemoglobin content, and (d) a large increase in the level of reduced glutathione. These alterations occur also in persons of the same ethnic group residing at low altitude. There is, however, only a moderate elevation of classic haematological parameters (erythrocyte count, haemoglobin and haematocrit) in highland natives. The functional implications of these metabolite changes are discussed with respect to regulation of erythrocyte metabolism.
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PMID:Methaemoglobin and erythrocyte reducing systems in high-altitude natives. 58 58

Initial rate studies at pH 7.6 with three aldehydes, product inhibition patterns with NADH and dead-end inhibition with adenosine diphosphoribose show that the kinetic mechanism of glyceraldehyde-3-phosphate dehydrogenase from rabbit muscle cannot be ordered, and support an enzyme-substitution mechanism. Deviations from Michaelis-Menten behaviour are consistent with negative interactions in the binding of NAD+ and instability of the species E(NAD)3 and E(NAD)4. Inhibition with large concentrations of phosphate and arsenate indicates competition for a binding site for glyceraldehyde 3-phosphate, and is not found with glyceraldehyde as substrate.
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PMID:Kinetic studies of glyceraldehyde-3-phosphate dehydrogenase from rabbit muscle. 62 82

Human erythrocyte ghosts depleted of glyceraldehyde-3-phosphate dehydrogenase are used as specific high-affinity adsorbents for the purification of glyceraldehyde-3-phosphate dehydrogenase from mouse muscle, liver, kidney and brain. On incubation with the crude tissue homogenates, the depleted ghosts bind glyceraldehyde-3-phosphate dehydrogenase, aldolase, and a few other proteins. Washing the incubated ghosts several times with 5 mM phosphate buffer(pH 8.0) removed several of the non specifically bound proteins. Aldolase can be eliminated from the membrane by incubating the ghosts for 30 min in 5 mM phosphate buffer (pH 8.0)/2mM fructose 1,6-biphosphate, and then washing with the same solution. Glyceraldehyde-3-phosphate dehydrogenase can then be specifically eluted from the ghosts by incubating them with 2 mM NADH in 5mM phosphate buffer (pH 8.0). Although the enzyme from brain appears to bind less strongly to the ghosts it was possible, using this procedure, to purify glyceraldehyde-3-phosphate dehydrogenase from all the tissues investigated. The purified enzyme exhibits high specific activity and migrates as a single band (during SDS polyacrylamide gel electrophoresis) which corresponds to a protomer molecular weight of 37 000.
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PMID:Use of glyceraldehyde-3-phosphate dehydrogenase-depleted human erythrocyte ghosts as specific high affinity adsorbents for the purification of glyceraldehyde-3-phosphate dehydrogenase from various tissues. 71 58


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