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)

To investigate whether the energy derived from glycolysis is functionally coupled to Ca2+ active transport in sarcoplasmic reticulum (SR), we determined whether glycolytic enzymes were associated with SR membranes and whether metabolism through these enzymes was capable of supporting 45Ca transport. Sealed right-side-out SR vesicles were isolated by step sucrose gradient from rabbit skeletal and cardiac muscle. Intravesicular 45Ca transport was measured after the addition of glycolytic substrates and cofactors specific for each of the glycolytic reactions being studied or after the addition of exogenous ATP and was expressed as transport sensitive to the specific Ca(2+)-ATPase inhibitor thapsigargin. We found that the entire chain of glycolytic enzymes from aldolase onward, including aldolase, GAPDH, phosphoglycerate kinase (PGK), phosphoglyceromutase, enolase, and pyruvate kinase (PK), was associated with SR vesicles from both cardiac and skeletal muscle. Iodoacetic acid, an inhibitor of GAPDH, eliminated 45Ca transport supported by fructose-1,6-diphosphate, the substrate for aldolase, but transport was completely restored by phosphoenolpyruvate (the substrate for PK), indicating that both of the ATP-producing glycolytic enzymes, GAPDH/PGK and PK, were associated with the SR and functionally capable of providing ATP for the Ca2+ pump. Addition of a soluble hexokinase ATP trap eliminated 45Ca transport fueled by exogenous ATP but had markedly less effect on 45Ca transport supported by endogenously produced ATP (via glycolysis). Similarly, at very low concentrations of ATP and ADP (10 to 50 nmol/L), ATP that was produced endogenously from ADP and phosphoenolpyruvate supported 15-fold more 45Ca transport than ATP that was supplied exogenously at the same concentration. These results are consistent with functional coupling of glycolytic ATP to Ca2+ transport and support the hypothesis that ATP generated by SR-associated glycolytic enzymes may play an important role in cellular Ca2+ homeostasis by driving the SR Ca2+ pump.
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PMID:Functional coupling between glycolysis and sarcoplasmic reticulum Ca2+ transport. 778 86

The structure of the key glycolytic enzyme 3-phosphoglycerate kinase (PGK) is known in detail, but there is little information on its reaction pathway. We have studied its equilibrium and transient kinetics in the direction of 1,3-bisphosphoglycerate (1,3-bis-P-glycerate) production: ATP + 3-P-glycerate<==>ADP + 1,3-bis-P-glycerate. We devised a sensitive method for following this production. PGK is mixed with 3-P-glycerate and [gamma-32P]ATP in a rapid flow quench apparatus. The reaction mixtures are aged for 4 ms or more and then quenched in acid in which any [1-32P]-1,3-P-glycerate decomposes to 3-P-glycerate and 32Pi, which is determined specifically. The Pi reflects accurately the 1,3-bis-P-glycerate in the original reaction mixture, and the kcat obtained is identical to that obtained by the conventional linked assay method with glyceraldehyde-3-phosphate dehydrogenase. This does not support the postulate of a rapid direct transfer of the 1,3-bis-P-glycerate between the kinase and the dehydrogenase [Srivastava, D. K., & Bernhard, S. A. (1986) Science 234, 1081-1086]. We fitted our data to a simple scheme with the formation of binary complexes, the interconversion of substrates to products via ternary complexes, and the release of products. Because of the high turnover of PGK, the work was carried out under cryoenzymic conditions with 40% ethylene glycol in the buffer. The glycol decreased kcat from 80 to 8.5 s-1 (pH 7.5, 4 degrees C), but the Km for 3-P-glycerate and ATP and the equilibrium constants in the scheme were little affected. We carried out two types of experiment.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Transient and equilibrium kinetic studies on yeast 3-phosphoglycerate kinase. Evidence that an intermediate containing 1,3-bisphosphoglycerate accumulates in the steady state. 782 41

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

When rabbit muscle phosphoglycerate kinase (PGK; a 48-kDa monomeric protein) and glyceraldehyde-3-phosphate dehydrogenase (GraPDH; a 145-kDa homotetrameric protein) are present together in solution in the proportion of 1 mol PGK/1 mol GraPDH monomer (total protein 0.2-1.0 mg/ml), an 80--82-kDa protein species is observed by gel-penetration (dilution factor) method and by the conventional procedure of elution from a gel column. Individually, PGK and GraPDH do not exhibit any self association or dissociation in the concentration range employed. Electrophoresis of the 80-82-kDa peak eluted from the gel column shows a single protein band with mobility intermediate between those of GraPDH and PGK. In titration experiments by the gel-penetration method, plots of dilution factor of PGK (or GraPDH) activity versus GraPDH (or PGK) concentration shows two linear portions intersecting at approximately 1 mol GraPDH monomer/1 mol PGK. From the molecular-mass values and the titration experiments, it has been suggested that, in solution, these enzymes form a complex consisting of 1 molecule of PGK and one monomeric subunit of GraPDH (expected molecular mass 84 kDa). Its dissociation constant has been estimated to be equal to or less than 13 nM. The complex is dissociated in the presence of KCl or NADH, with approximately half dissociation at 0.1 M salt or 0.25 mM NADH. At 0.1 M KCl, the complex is completely dissociated by adding ATP, NADH or 3-phosphoglycerate. AMP, ADP, NAD+, glyceraldehyde-3-phosphate, phosphate ions and fructose-1,6-bisphosphate reverse the effect of KCl.
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PMID:Phosphoglycerate-kinase-glyceraldehyde-3-phosphate-dehydrogenase interaction. Molecular mass studies. 785 37

Nitric oxide (NO) has been suggested to act as a regulator of endogenous intracellular ADP-ribosylation, based on radiolabelling of proteins in tissue homogenates incubated with [32P]NAD and NO. After the NO-stimulated modification was replicated in a defined system containing only the purified acceptor protein, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), the hypothesis of NO-stimulation of an endogenous ADP-ribosyltransferase became moot. The NO-stimulated, NAD-dependent modification of GAPDH was recently characterized as covalent binding of the whole NAD molecule to the enzyme, not ADP-ribosylation. With this result, along with the knowledge that GAPDH is stoichiometrically S-nitrosylated, the role of NO in protein modification with NAD may be viewed as the conferring of an unexpected chemical reactivity upon GAPDH, possibly due to nitrosylation of a cysteine in the enzyme active site.
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PMID:Nitric oxide and NAD-dependent protein modification. 789 64

A 41,000 M(r) cytosolic protein (p41) in Dictyostelium discoideum was shown to be modified by ADP-ribosylation that was not regulated by nitric oxide (NO). This endogenous ADP-ribosylation was optimal at conditions distinct from those optimal for the NO-stimulated ADP-ribosylation of p41. These two activities were also differentially sensitive to reducing agents and modified different amino acids. The addition of haemoglobin, which sequesters NO, and of NO synthase inhibitors failed to block the endogenous ADP-ribosylation. P41 was purified to homogeneity. The N-terminal sequence of the purified protein was shown to be highly homologous to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Both endogenous and NO-stimulated activities ADP-ribosylated three isoforms of the protein, with pI values of 6.6, 6.8 and 7.0. In each case, the isoform with pI 6.8 was preferentially modified. Experiments using purified GAPDH indicate that both the endogenous and NO-stimulated ADP-ribosylation are self-catalysed modifications.
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PMID:Endogenous ADP-ribosylation of glyceraldehyde-3-phosphate dehydrogenase that is not regulated by nitric oxide in Dictyostelium discoideum. 790 97

The ability of compounds releasing nitric oxide (NO) to regulate glyceraldehyde-3-phosphate dehydrogenase (GraPDH) activity was analysed both in cell homogenates and in intact Dictyostelium discoideum. The time course of GraPDH inactivation in cell lysates by NO-releasing compounds suggests that two processes may be involved, one of which accounts for the majority of the inactivation and shows a close correlation with GraPDH ADP-ribosylation. Maximal ADP-ribosylation under these conditions exhibited a stoichiometry of about 0.4 mol ADP-ribose/mol enzyme tetramer. NO-mediated inhibition of GraPDH activity was attenuated if specific substrates, cofactors, or cysteine were added to cytosol preparations. Under such conditions, ADP-ribosylation of the enzyme was correspondingly reduced or negligible. Intact cells treated with NO-releasing compounds were shown to respond by rapidly decreasing their GraPDH activity. This inhibition was transient and, after a 10-min incubation, enzyme activity returned to the level seen in control cells. The time course of these in vivo changes correlated well with those of the NO-stimulated ADP-ribosylation of GraPDH also seen in intact cells. The basis underlying the NO-stimulated inhibition of GraPDH activity was investigated and found to reflect a decreased Vmax. No changes in either the Km of the enzyme for its substrates or its state of polymerization were observed.
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PMID:Nitric oxide regulation of glyceraldehyde-3-phosphate dehydrogenase activity in Dictyostelium discoideum cells and lysates. 792 59

Synaptic vesicles isolated from electric ray electric organ have been shown previously to contain a 34-kDa protein that binds azido-ATP, azido-AMP, and N-ethylmaleimide. The protein was found to share similarities with the mitochondrial ADP/ATP carrier and assumed to represent the synaptic vesicle nucleotide transporter. Synaptic vesicles were purified by sucrose density gradient centrifugation and subsequent chromatography on Sephacryl S-1000 from both Torpedo electric organ and bovine brain cerebral cortex. They contained ATP-binding proteins of 35 kDa and 34 kDa, respectively. ATP binding was inhibited by AMP. Both proteins were highly enriched after column chromatography of vesicle proteins on AMP-Sepharose. Antibodies were obtained against both proteins. Antibodies against the bovine brain synaptic vesicle protein of 34 kDa bound specifically to the 35-kDa protein of Torpedo vesicles. An N-terminal sequence obtained against the 34-kDa protein of bovine brain synaptic vesicles identified it as glyceraldehyde-3-phosphate dehydrogenase. The previously observed molecular characteristics of the putative vesicular nucleotide transporter in Torpedo fit those of glyceraldehyde-3-phosphate dehydrogenase. We, therefore, suggest that the protein previously identified as putative nucleotide transporter is, in fact, glyceraldehyde-3-phosphate dehydrogenase.
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PMID:Putative synaptic vesicle nucleotide transporter identified as glyceraldehyde-3-phosphate dehydrogenase. 793 48

Previous studies have suggested that glyceraldehyde-3-phosphate dehydrogenase (GAPDH) undergoes covalent modification of an active site thiol by a NO.-induced [32P]NAD(+)-dependent mechanism. However, the efficacy of GAPDH modification induced by various NO donors was found to be independent of spontaneous rates of NO. release. To further test the validity of this mechanism, we studied the effects of nitrosonium tertrafluoroborate (BF4NO), a strong NO+ donor. BF4NO potently induces GAPDH labeling by the radioactive nucleotide. In this case, the addition of thiol significantly attenuates enzyme modification by competing for the NO moiety in the formation of RS-NO. Peroxynitrite (ONOO-) also induces GAPDH modification in the presence of thiol, consistent with the notion that this species can transfer NO+ (or NO2+) through the intermediacy of RS-NO. However, the efficiency of this reaction is limited by ONOO- -induced oxidation of protein SH groups at the active site. ONOO- generation appears to account for the modification of GAPDH by SIN-1. Thus, S-nitrosylation of the active site thiol is a prequisite for subsequent post-translational modification with NAD+, and emphasizes the role of NO+ transfer in the initial step of this pathway. Our findings thus provide a uniform mechanism by which nitric oxide and related NO donors initiate non-enzymatic ADP-ribosylation (like) reactions. In biological systems, endogenous RS-NO are likely to support the NO group transfer to thiol-containing proteins.
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PMID:Mechanism of covalent modification of glyceraldehyde-3-phosphate dehydrogenase at its active site thiol by nitric oxide, peroxynitrite and related nitrosating agents. 803 46

We performed experiments to determine whether nitric oxide promoted the formation of intracellular S-nitrosothiol adducts in human neutrophils. At concentrations sufficient to inhibit chemoattractant-induced superoxide anion production, nitric oxide caused a depletion of measurable intracellular glutathione as determined by both the monobromobimane HPLC method and the glutathione reductase recycling assay. The depletion of glutathione could be shown to be due to the formation of intracellular S-nitrosoglutathione as indicated by the ability of sodium borohydride treatment of cytosol to result in the complete recovery of measurable glutathione. The formation of intracellular S-nitrosylated compounds was confirmed by the capacity of cytosol derived from nitric oxide-treated cells to ADP-ribosylate glyceraldehyde-3-phosphate dehydrogenase. Depletion of intracellular glutathione was accompanied by a rapid and concomitant activation of the hexose monophosphate shunt (HMPS) following exposure to nitric oxide. Kinetic studies demonstrated that nitric oxide-dependent activation of the HMPS was reversible and paralleled nitric oxide-induced glutathione depletion. Synthetic preparations of S-nitrosoglutathione shared with nitric oxide the capacity to inhibit superoxide anion production and activate the HMPS. These data suggest that nitric oxide may regulate cellular functions via the formation of intracellular S-nitrosothiol adducts and the activation of the HMPS.
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PMID:Nitric oxide reacts with intracellular glutathione and activates the hexose monophosphate shunt in human neutrophils: evidence for S-nitrosoglutathione as a bioactive intermediary. 817 Sep 69


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