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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)

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

The effect of pH and temperature on the capacity and binding of Bacillus stearothermophilus, alcohol dehydrogenase and phosphofructokinase to N6-(6-aminohexyl)-5'-AMP-Sepharose has been examined. Specific elution from the substituted AMP-Sepharose was examined using a variety of cofactors, fragments of cofactors and substrates. A purification scheme for each enzyme on the substituted AMP-Sepharose using nucleotides and gradients of pH and salt is presented. Interestingly, elevated temperature increased the affinity of both enzymes for N6-(6-aminohexyl)-5'-AMP-Sepharose, however, the Michaelis constant for nucleotide determined at various temperatures remained constant. The effect of pH and salt concentration on the binding of B. stearothermophilus glyceraldehyde-3-phosphate dehydrogenase to 6-aminohexanoyl-NAD+-Sepharose was also examined; raising the pH above 7.5 lowers the capacity of the matrix and the effect of a range of ammonium sulphate concentrations on the adsorption of the enzyme was examined. A specific purification of glyceraldehyde-3-phosphate dehydrogenase from partially purified extracts of this organism was achieved.
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PMID:Affinity chromatography on immobilised nucleotides. Some applications to the purification of thermophilic dehydrogenases and kinases. 24 Jun 92

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

Failure of glycolysis to increase sufficiently to supply optimal levels of energy production in ischemic heart muscle is due in part to the cummulative restrainst of acidosis on rate-limiting enzymes, particularly glyceraldehyde-3-phosphate dehydrogenase. In an effort to modify this inhibition and salvage jeopardized myocardium, treatment with excess levels of pyruvate and tromethamine (Tris), designed to buffer intracellular hydrogen ion accumulations and improve the oxidation-reduction ratio, NAD+/NADH, was tested in 59 swine hearts in two separate preparations of global and regional ischemia. Global ischemia, per se, caused hemodynamic deterioration and shortened survival time (44.3 +/- 3.1 minutes). Myocardial oxygen consumption, fatty acid oxidation, and glucose uptake were all significantly (P less than 0.001) reduced as were estimates of glycolysis and tissue stores of creatine phosphate and ATP (P less than 0.01). Although treatment with Tris alone was inconclusive, administrations of pyruvate (40-50 mM) buffered with Tris (added directly into the coronary perfusate) effected an improvement in mechanical function and a significant prolongation in survival time (56.9 +/- 2.6 minutes. P less than 0.01). Glycogenolysis was enhanced and levels of key glycolytic intermediates were reduced, suggesting an acceleration of glycolytic flux. Excess levels of pyruvate (1.52 +/- 0.48 mumol/ml of coronary perfusate) provided added substrate for oxidation and led to a greater than 5-fold incrase in rates of pyruvate decarboxylation as compared to untreated ischemic hearts...
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PMID:Effects of treatment with pyruvate and tromethamine in experimental myocardial ischemia. 95 68

The reaction of sulfhydryl groups of glyceraldehyde-3-phosphate dehydrogenase from rabbit and pig muscles with a large molar excess of 5,5'-dithiobis(2-nitrobenzoate) (Nbs2) shows three-phasic pseudo-first-order kinetics. Since the fastest reaction between active cysteine-149 and Nbs2 is apparently biphasic, half-of-the-sites reactivity towards Nbs2 is suggested. Further sulfhydryl groups become reactive as an effect of conformational changes in the protein molecule after formation of a mixed disulfide on cysteine-149. In the presence of 40 mM borate the reaction is biphasic only, and two sulfhydryl groups per subunit react very quickly. The bound NAD+ is only partially released even after a long reaction with Nbs2. It was demonstrated that the two NAD+ binding sites with the highest dissociation constants have no significant effect on the reaction between cysteine-149 and Nbs2.
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PMID:Kinetic studies of the reactivity of the sulfhydryl groups of glyceraldehyde-3-phosphate dehydrogenase. 96 68

Both H2O2 (IC50 = 70 microM) and HOCl (IC50 = 8.5 microM) inhibited mitogen-induced MNL proliferation in a dose-dependent manner. This was found to be due to a depletion of intracellular ATP by at least two distinct mechanisms. HOCl and high concentrations (greater than 100 microM) of H2O2 inhibit ATP generation via sulfhydryl group oxidation on the active site of the glyceraldehyde-3-phosphate dehydrogenase (G3PDH) enzyme of the glycolytic pathway. On the other hand, low H2O2 concentrations cause ATP depletion by an activation of the DNA repair enzyme, poly(ADP-ribose)polymerase (pADPRP), leading to consumption of NAD+, an essential cofactor for G3PDH. The anti-oxidants ascorbate and cysteine protected MNL against the anti-proliferative effects of HOCl. Similar results were achieved with the HOCl-mediated inhibition of ATP production and G3PDH activity. However, ascorbate was unable to protect against H2O2-mediated inhibition of MNL functions, while cysteine protected against the inhibitory effects on ATP production and G3PDH activity, induced by this oxidant.
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PMID:Biochemical mechanisms of hydrogen peroxide- and hypochlorous acid-mediated inhibition of human mononuclear leukocyte functions in vitro: protection and reversal by anti-oxidants. 132 47

The molecular basis of thermal stability of globular proteins is a highly significant yet unsolved problem. The most promising approach to its solution is the investigation of the structure-function relationship of homologous enzymes from mesophilic and thermophilic sources. In this context, D-glyceraldehyde-3-phosphate dehydrogenase has been the most extensively studied model system. In the present study, the most thermostable homolog isolated so far is described with special emphasis on the stability of the enzyme under varying solvent conditions. D-Glyceraldehyde-3-phosphate dehydrogenase from the hyperthermophilic eubacterium Thermotoga maritima is an intrinsically thermostable enzyme with a thermal transition temperature around 110 degrees C. The amino acid sequence, electrophoresis, and sedimentation analysis prove the enzyme to be a homotetramer with a gross structure similar to its mesophilic counterparts. The enzyme in the absence and in the presence of its coenzyme, NAD+, exhibits no drastic structural differences except for a 3% change in sedimentation velocity reflecting slight alterations in the quaternary structure of the enzyme. At low temperature, in the absence of denaturants, neither "cold denaturation" nor subunit dissociation are detectable. Guanidinium chloride and pH-dependent deactivation precede the decrease in fluorescence emission and ellipticity, suggesting a complex denaturation mechanism. An up to 3-fold activation of the enzyme at low guanidinium concentration may be interpreted in terms of a compensation of the tight packing of the thermophilic enzyme at low temperature. Under destabilizing conditions, e.g. moderate concentrations of chaotropic agents, low temperature favors denaturation. The effect becomes important in reconstitution experiments after preceding guanidinium denaturation; the reactivation yield at low temperature drops to zero, whereas between 35 and 80 degrees C reactivation exceeds 80%. Shifting the temperature from approximately 0 degrees C to greater than or equal to 30 degrees C releases a trapped tetrameric intermediate in a fast reaction. Concentration-dependent reactivation experiments prove renaturation of the enzyme to involve consecutive folding and association steps. Reconstitution at room temperature yields the native protein, in spite of the fact that the temperature of the processes in vitro and in vivo differ by more than 60 degrees C.
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PMID:Stability and reconstitution of D-glyceraldehyde-3-phosphate dehydrogenase from the hyperthermophilic eubacterium Thermotoga maritima. 136 31

1. Binding of Zn2+ and Cu2+ ions to GAPDHs from bovine heart and rabbit muscle resulted in a partial loss of enzymatic activity of both enzymes, in a time and metal ion concentration dependent manner. Cu2+ ions caused a much larger decrease of the activity than Zn2+ ions. 2. Addition of NAD+ or EDTA to either enzyme resulted in a protective effect on GAPDH activity. A similar protective effect was observed following addition of 2-mercaptoethanol to the enzyme solution. 3. The association constant for GAPDH-Zn2+ complex, calculated from equilibrium dialysis data, was 0.9 x 10(4) M-1 for the bovine heart GAPDH and 1.3 x 10(4) M-1 for the rabbit muscle enzyme. The association constant for GAPDH-Cu2+ complex was the same for both enzymes, 11.3 x 10(4) M-1. 4. Equilibrium dialysis data also revealed that in either enzyme the specific sites, binding the metal ions, are identical or very similar, and independent from each other. They are situated in the most conserved part of the enzyme molecule. 5. Some zinc was found in GAPDH preparations from bovine heart. It is discussed if Zn2+ ions could have a kind of modulation effect on GAPDH activity.
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PMID:Interaction of Zn2+ and Cu2+ ions with glyceraldehyde-3-phosphate dehydrogenase from bovine heart and rabbit muscle. 142 32

Auto-ADP-ribosylation of the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GraPDH) has recently been demonstrated to be dramatically stimulated in the presence of nitric oxide. In order to obtain insight into the sequence of events leading to ADP-ribosylation of GraPDH, we studied the target amino acid, the nucleotide cofactor requirement, pH dependency and the stoichiometry of the reaction. Basal as well as stimulated ADP-ribose transfer is inhibited by the SH-group alkylating reagent, N-ethylmaleimide. Furthermore, the radiolabel of auto-[32P]ADP-ribosylated GraPDH is removed by treatment with HgCl2, suggesting an ADP-ribose-cysteine bond. Several indirect and direct mechanistic considerations point to NAD+ as the only cofactor for the ADP-ribosylation reaction, excluding the possibility of a reaction sequence involving a NAD-glycohydrolase(s) followed by nonenzymatic ADP-ribose transfer to GraPDH. Optimal ADP-ribosylations were carried out at alkaline pH values using 10 microM free NAD+ as the sole nucleotide cofactor. Bovine serum albumin with an S-nitrosylated SH group can serve as a model of ADP-ribose transfer from NAD+ and suggests that the nitric-oxide-modified SH group (S-nitrosylated SH group) is a prerequisite for the reaction.
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PMID:Characterization of a nitric-oxide-catalysed ADP-ribosylation of glyceraldehyde-3-phosphate dehydrogenase. 144 79

Nitric oxide and nitric oxide-generating agents like 3-morpholinosydnonimine (SIN-1) stimulate the mono-ADP-ribosylation of a cytosolic, 39-kDa protein in various tissues. This protein was purified from human platelet cytosol by conventional and fast protein liquid chromatography techniques. N-terminal sequence analysis identified the isolated protein as the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Nitric oxide stimulates the auto-ADP-ribosylation of GAPDH in a time and concentration-dependent manner with maximal effects after about 60 min. Associated with ADP-ribosylation is a loss of enzymatic activity. NAD(+)-free enzyme is not inhibited by SIN-1, indicating the absolute requirement of NAD+ as the substrate of the ADP-ribosylation reaction. Inhibition of the glycolytic enzyme GAPDH may be relevant as a cytotoxic effect of NO complementary to its inhibitory actions on iron-sulfur enzymes like aconitase and electron transport proteins of the respiratory chain.
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PMID:Nitric oxide causes ADP-ribosylation and inhibition of glyceraldehyde-3-phosphate dehydrogenase. 151 18


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