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)

Inhibition of ADP phosphorylation by both glycolysis and mitochondria in P388D1 cells exposed to H2O2 is described. Net glucose uptake and lactate production were inhibited by oxidant exposure (ED50 = 50-100 microM). Glycolysis was specifically inactivated at the glyceraldehyde-3-phosphate dehydrogenase step by three independent mechanisms: (a) direct inactivation of the intracellular enzyme (ED50 approximately equal to 100 microM); (b) reduction of the intracellular concentration and redox potential of its nicotinamide cofactors; and (c) a cytosolic pH shift further from the enzyme optima. Consistent with inhibition of glycolysis at the glyceraldehyde-3-phosphate dehydrogenase step, a rise in the intracellular concentration of glyceraldehyde 3-phosphate, dihydroxyacetone phosphate, and fructose 1,6-bisphosphate was observed. The calculated combined inhibition of glyceraldehyde-3-phosphate dehydrogenase activity could be reasonably correlated with the depression in glycolytic flux rate with the appropriate modeling. The steady-state contribution by mitochondria to the total intracellular ATP pool was indirectly determined by the use of various metabolic inhibitors and was found to rapidly decline following exposure to 300-800 microM H2O2. The inhibition of ADP phosphorylation appeared to be related more to the direct inhibition of the ATPase-synthase complex rather than to the diminished capacity of the respiratory chain for coupled electron transport. Both the estimated rates of ADP phosphorylation by glycolysis and mitochondria and the estimated rate of ATP hydrolysis by ongoing metabolism were utilized to model the approximate decline in intracellular ATP expected at 15-min exposure to various H2O2 concentrations. Theoretical calculations and the measured intracellular ATP status were in good agreement. Oxidant exposure for 15 min resulted in dose-dependent killing of the cells (ED50 = 500 microM), indicating a close correlation between H2O2-mediated loss of intracellular ATP and cell viability. The possible contribution of impaired energy homeostasis during oxidant-mediated injury to the process of cell dysfunction and death is discussed.
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PMID:Mechanisms of oxidant-mediated cell injury. The glycolytic and mitochondrial pathways of ADP phosphorylation are major intracellular targets inactivated by hydrogen peroxide. 333 86

Utilizing glutathione ethyl ester (GSH-EE), the glutathione (GSH) level of lens epithelial cells can be increased as much as 1.9-fold. The epithelial cells maintain the additional GSH in the reduced form. This system was utilized to examine the relative effectiveness of cells with elevated GSH to withstand H2O2 insult. Three parameters were investigated, 86Rb accumulation, a measure of membrane function, ATP levels, an indication of overall metabolism and glyceraldehyde-3-phosphate dehydrogenase (GPD) activity, indicating intracellular enzyme susceptibility to oxidative insult. Under oxidative stress, much of the GSH is in the oxidized form but upon removal of the stress, rapidly returns to the reduced state. However, a loss of approximately 20% in GSH equilibrium levels has been consistently observed. Elevated GSH does not significantly increase the cells' ability to withstand or recover from oxidative stress. Indeed, elevated GSH was found to be somewhat deleterious, causing a decreased ability to recover from oxidative insult. However, in the case of GPD, a significant protection of activity was observed. The overall conclusion is that elevating intracellular GSH concentration does not increase the cells' overall ability to withstand oxidative damage.
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PMID:Does elevated glutathione protect the cell from H2O2 insult? 366 67

Sepharose-bound tetrameric, dimeric and monomeric forms of yeast glyceraldehyde-3-phosphate dehydrogenase were prepared, as well as immobilized hybrid species containing (by selective oxidation of an active center cysteine residue with H2O2) one inactivated subunit per tetramer or dimer. The catalytic properties of these enzyme forms were compared in the forward reaction (glyceraldehyde-3-phosphate oxidation) and reverse reaction (1,3-bisphosphoglycerate reductive dephosphorylation) under steady-state conditions. In the reaction of glyceraldehyde-3-phosphate oxidation, immobilized monomeric and tetrameric forms exhibited similar specific activities. The hybrid-modified dimer contributed on half of the total activity of a native dimer. The tetramer containing one modified subunit possessed 75% of the activity of an unmodified tetramer. In the reaction of 1,3-bisphosphoglycerate reductive dephosphorylation, the specific activity of the monomeric enzyme species was nearly twice as high as that of the tetramer, suggesting that only one-half of the active centers of the oligomer were acting simultaneously. Subunit cooperativity in catalysis persisted in an isolated dimeric species. The specific activity of a monomer associated with a peroxide-inactivated monomer in a dimer was equal to that of an isolated monomeric species and twice as high as that of a native immobilized dimer. The specific activity of subunits associated with a peroxide-inactivated subunit in a tetramer did not differ from that of a native immobilized tetramer; this indicates that interdimeric interactions are involved in catalytic subunit cooperativity. A complex was formed between the immobilized glyceraldehyde-3-phosphate dehydrogenase and soluble phosphoglycerate kinase. Three monomers of phosphoglycerate kinase were bound per tetramer of the dehydrogenase and one per dimer. Evidence is presented that if the reductive dephosphorylation of 1,3-bisphosphoglycerate proceeds in the phosphoglycerate kinase - glyceraldehyde-3-phosphate dehydrogenase complex, all active sites of the latter enzyme act independently, i.e. subunit cooperativity is abolished.
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PMID:Yeast glyceraldehyde-3-phosphate dehydrogenase. Evidence that subunit cooperativity in catalysis can be controlled by the formation of a complex with phosphoglycerate kinase. 388 24

The relative effectiveness of oxidizing (.OH, H2O2), ambivalent (O2-) and reducing free radicals (e- and CO2-) in causing damage to membranes and membrane=bound glyceraldehyde-3-phosphate dehydrogenase of resealed erythrocyte ghosts has been determined. The rates of damage to membrane-bound glyceraldehyde-3-phosphate dehydrogenase (R(enz)) were measured and the rates of damage to membranes (R(mb)) were assessed by measuring changes in permeability of the resealed ghosts to the relatively low molecular weight substrates of glyceraldehyde-3-phosphate dehydrogenase. Each radical was selectively isolated from the mixture produced during gamma-irradiation, using appropriate mixtures of scavengers such as catalase, superoxide dismutase and formate. .OH, O2- and H2O2 were approximately equally effective in inactivating membrane-bound glyceraldehyde-3-phosphate dehydrogenase, while e- and CO2- were the least effective. R(enz) values of O2- and H2O2 were 10-times and of .OH 15-times that of e-. R(mb) values were quite similar for e- and H2O2 (about twice that of O2-), while that of .OH was 3-times that of O2-. Hence, with respect to R(mb): .OH greater than e- = H2O2 greater than O2-, and with respect to R(enz): .OH greater than O2- = H2O2 much greater than e-. The difference between the effectiveness of the most damaging and the least damaging free radicals was more than 10-fold greater in damage to the enzyme than to the membranes. Comparison between H2O2 added as a chemical reagent and H2O2 formed by irradiation showed that membranes and membrane-bound glyceraldehyde-3-phosphate dehydrogenase were relatively inert to reagent H2O2 but markedly susceptible to the latter.
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PMID:The relative effectiveness of .OH, H2O2, O2-, and reducing free radicals in causing damage to biomembranes. A study of radiation damage to erythrocyte ghosts using selective free radical scavengers. 626 Jan 72

A site-directed mutant, D179N, in the gene encoding Phanerochaete chrysosporium manganese peroxidase isozyme 1 (mnp1), was created by overlap extension, using polymerase chain reaction. The mutant gene was expressed in P. chrysosporium under the control of the glyceraldehyde-3-phosphate dehydrogenase promoter. The mutant manganese peroxidase (MnP) was purified, and its spectra and MW were very similar to those of the wild-type enzyme. Steady-state kinetic analysis of MnP D179N revealed that the Km for the substrate MnII was approximately 50-fold greater than the corresponding Km for the wild-type recombinant enzyme (3.7 mM versus approximately 70 microM). Likewise, the kcat value for MnII oxidation of the mutant protein was only 1/265 of that for the wild-type enzyme. By comparison, the apparent Km for H2O2 of MnP D179N was similar to the corresponding value of the wild-type MnP. The first-order rate constant for MnP D179N compound II reduction by MnII was approximately 1/200 of that for the wild-type enzyme. The equilibrium dissociation constant (KD) for MnP D179N compound II reduction by MnII was approximately 100-fold greater than the KD for the wild-type compound II. In contrast, the second-order rate constant for p-cresol reduction of the mutant compound II was similar to that of the wild-type enzyme. These results also suggest that the mutation affects the binding of MnII to the enzyme and, consequently, the rate of compound II reduction by MnII. In contrast, the mutation apparently does not have a significant effect on H2O2 cleavage during compound I formation or on p-cresol reduction of compound II.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:The manganese binding site of manganese peroxidase: characterization of an Asp179Asn site-directed mutant protein. 765 16

Conditions to induce and parameters to evaluate sublethal oxidative stress of cultured human fibroblasts have been investigated in the attempt to identify markers for a more accurate quantification of cell injury. Sublethal oxidative stress was obtained by treating fibroblasts with 0.5 mM H2O2 in DMEM plus 5% FCS for times not exceeding 60 min. Under these conditions cells remained viable throughout long-term incubation, showing no appreciable release of cytosolic enzymes into the medium. On the contrary, exposures of fibroblasts to 0.5 mM H2O2 for times > 60 min induced a lethal cell injury which was fully expressed 2 days later by massive monolayer wasting and leakage of cytosolic components. Early metabolic effects of sublethal stress consisted of a rapid and significant fall of both ATP and NAD+ pools. Concomitantly, there was a moderate increase (about threefold) in both ADP-ribosyl transferase activity and free [Ca2+]i, while the specific activity of glyceraldehyde-3-phosphate dehydrogenase was partially decreased upon treatment. Oxidative injury also caused delayed effects consisting of a large depression of both protein and DNA synthesis. However, while the former was partially restored within 10 days of incubation, the latter remained severely impaired, as encountered in a growth-arrested population. Microfilaments of H2O2-treated cells appeared to be morphologically altered due to partial fragmentation of cytoskeleton actin which, however, was still maintained in the polymerized form as F-actin. Moreover, sublethally injured fibroblasts exhibited a reduced adhesiveness to plastic once they were detached and reseeded into new dishes. Relative adhesion efficiencies (number of adherent cells at 16 h as a percentage of seeded cells) were found to correlate inversely with times of exposure to H2O2. This finding allowed the identification of a biological parameter which showed itself to be very sensitive to oxidative stress and was also useful for developing an assay to grade sublethal injury to fibroblasts.
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PMID:Induction, effects, and quantification of sublethal oxidative stress by hydrogen peroxide on cultured human fibroblasts. 784 83

Chemical oxidants can induce the covalent binding of low molecular weight thiols to reactive sulfhydryls on proteins (S-thiolation). We found that stimulation of the respiratory burst of human blood monocytes resulted in S-thiolation of several proteins, most prominently one of 38 kDa. This purified protein was identified as glyceraldehyde-3-phosphate dehydrogenase (GAPDH) by enzyme activity, immunoblotting, and amino acid analysis. After stimulation of the respiratory burst, S-thiolation of GAPDH gradually increased, and cytosol GAPDH activity decreased; so that at 60 min, GAPDH activity was reduced by approximately 40%. Activity was restored by the addition of the sulfhydryl-reducing agent dithioerythritol. H2O2 appeared to be particularly important in mediating S-thiolation during the respiratory burst. Exposure of monocytes to H2O2 induced concentration-dependent S-thiolation of GAPDH and a concomitant decrease in enzyme activity. The addition of respiratory burst stimuli to lymphocytes, which lack a full respiratory burst, had no effect on GAPDH S-thiolation or activity; but H2O2 induced S-thiolation of lymphocyte GAPDH and inhibition of enzyme activity. Stimulation of monocytes from three patients with chronic granulomatous disease resulted in no respiratory burst, S-thiolation of GAPDH, or inactivation of GAPDH activity. The thiols covalently bound to purified S-thiolated GAPDH were removed by dithioerythritol and were identified as glutathione and cysteine; glutathione was predominant. These results indicate that during the respiratory burst in monocytes, low molecular weight thiols can bind to specific cytosolic proteins, including GAPDH. It is possible that S-thiolation of cytosolic proteins serves to modulate cellular metabolic events during phagocytosis.
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PMID:S-thiolation of glyceraldehyde-3-phosphate dehydrogenase induced by the phagocytosis-associated respiratory burst in blood monocytes. 792 87

Drawing upon the capacity of pyruvate to detoxify H2O2, we demonstrate that pyruvate (i) protects against H2O2-dependent, hydroxyl radical-mediated degradation of isolated DNA; (ii) reduces the amount of 8-hydroxy-2-deoxyguanosine detected following oxidative injury to isolated DNA and (iii) diminishes the amounts of detectable hydroxyl radical generated by a H2O2-dependent system. Compared to mannitol, pyruvate protects weakly against oxidative degradation of DNA induced by a H2O2-independent, hydroxyl radical-generating system. The protective effects of pyruvate against H2O2-instigated DNA damage were also evinced in cells in culture exposed to H2O2. In contrast to its protective effects against H2O2-dependent injury to DNA, pyruvate failed to offer convincing protection to another intracellular, H2O2-vulnerable target, glyceraldehyde-3-phosphate dehydrogenase. The protection conferred by pyruvate to intracellular H2O2-vulnerable targets is thus influenced by the nature of the target exposed to H2O2. Pyruvate was markedly protective in a model of cytotoxicity induced by the concomitant depletion of cellular glutathione and inhibition of catalase activity; pyruvate can thus function as an intracellular antioxidant and in this latter model, no evidence of DNA damage was observed. Pyruvate, in contrast to catalase, is a potent protector against cytotoxicity induced by organic peroxides, a finding that cannot be explained by the scavenging of organic peroxides, differences in glutathione content or attenuation in oxidative injury to DNA. We conclude that while DNA damage is a key pathogenetic event in oxidative stress induced by H2O2, such nuclear changes may not universally subserve a critical role in models of H2O2-dependent cell death. We also conclude that the antioxidant capabilities of pyruvate extend beyond scavenging of H2O2 to include potent protection against cytotoxicity induced by organic peroxides.
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PMID:Effect of pyruvate on oxidant injury to isolated and cellular DNA. 812 6

Sulfenic acids (R-SOH) result from the stoichiometric oxidations of thiols with mild oxidants such as H2O2; in solution, however, these derivatives accumulate only transiently due to rapid self-condensation reactions, further oxidations to the sulfinic and/or sulfonic acids, and reactions with nucleophiles such as R-SH. In contrast, oxidations of cysteinyl side chains in proteins, where disulfide bond formation can be prevented and where the reactivity of the nascent cysteine-sulfenic acid (Cys-SOH) can be controlled, have previously been shown to yield stable active-site Cys-SOH derivatives of papain and glyceraldehyde-3-phosphate dehydrogenase. More recently, however, functional Cys-SOH residues have been identified in the native oxidized forms of the FAD-containing NADH peroxidase and NADH oxidase from Streptococcus faecalis; these two proteins constitute a new class within the flavoprotein disulfide reductase family. In addition, Cys-SOH derivatives have been suggested to play important roles in redox regulation of the DNA-binding activities of transcription factors such as Fos and Jun, OxyR, and bovine papillomavirus type 1 E2 protein. Structural inferences for the stabilization of protein-sulfenic acids, drawn from the refined 2.16-A structure of the streptococcal NADH peroxidase, provide a molecular basis for understanding the proposed redox functions of these novel cofactors in both enzyme catalysis and transcriptional regulation.
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PMID:Protein-sulfenic acid stabilization and function in enzyme catalysis and gene regulation. 826 33

Hydrogen peroxide (H2O2) may incite cardiac ischemia-reperfusion injury. We evaluate herein the influence of H2O2-induced oxidative stress on heart muscle hexose metabolism in cultured neonatal rat cardiomyocytes, which have a substrate preference for carbohydrate. Cardiomyocyte exposure to 50 microM-1.0 mM bolus H2O2 transiently activated the pentose phosphate cycle and thereafter inhibited cellular glucose oxidation and glycolysis. These metabolic derangements were nonperoxidative in nature (as assessed in alpha-tocopherol-loaded cells) and occurred without acute change in cardiomyocyte hexose transport or glucose/glycogen reserves. Glycolytic inhibition was supported by the rapid, specific inactivation of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The degree of GAPDH inhibition correlated directly with the magnitude of the oxidative insult and was independent of both metal-catalyzed H2O2 reduction to free radicals and lipid peroxidation. Severe GAPDH inhibition was required for a rate-limiting effect on glycolytic flux. Cardiomyocyte pyruvate dehydrogenase was also inhibited by H2O2 overload, but to a lesser degree than GAPDH such that entry of hexose-derived acetyl units into the tricarboxylic acid cycle was not as restrictive as GAPDH inactivation to glycolytic ATP production. An increase in phosphofructokinase activity accompanied GAPDH inactivation, leading to the production and accumulation of glycolytic sugar phosphates at the expense of ATP equivalents. Cardiomyocyte treatment with iodoacetate or 2-deoxyglucose indicated that GAPDH inactivation/glycolytic blockade could account for approximately 50% of the maximal ATP loss following H2O2 overload. Partial restoration of GAPDH activity after a brief H2O2 "pulse" afforded some ATP recovery. These data establish that specific aspects of heart muscle hexose catabolism are H2O2-sensitive injury targets. The biochemical pathology of H2O2 overload on cardiomyocyte carbohydrate metabolism has implications for post-ischemic cardiac bioenergetics and function.
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PMID:Hydroperoxide-induced oxidative stress impairs heart muscle cell carbohydrate metabolism. 830 15


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