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)

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

Incubation of glyceraldehyde-3-phosphate dehydrogenase (GAPD) with sodium nitroprusside (SNP) decreased its activity in concentration- and time-dependent fashion in the presence of a thiol compound, with DTT being more effective than GSH. Both forward and backward reactions were effected. Coinciding with this, HgCl2-sensitive labelling of the protein by [32P]NAD+ also increased, indicating the stimulation of ADP-ribosylation. Treatment with SNP of GAPD samples from rabbit muscle, sheep brain and yeast inactivated the dehydrogenase activity of the three, but only the mammalian proteins showed ADP-ribosylation activity. The SNP-modified protein of rabbit muscle GAPD, freed from the reagent by Sephadex filtration showed a concentration-dependent restoration of the dehydrogenase activity on preincubation with DTT and GSH. Such thiol-treated preparations also gave increased ADP-ribosylation activity with DTT, and to a lesser extent with GSH. The SNP-modified protein was unable to catalyze this activity with the native yeast enzyme and native and heat-inactivated muscle enzyme. It was possible to generate the ADP-ribosylation activity in muscle GAPD, by an NO-independent mechanism, on dialysis in Tris buffer under aerobic conditions, and on incubating with NADPH, but not NADH, in muscle and brain, but not yeast, enzymes. The results suggest that the inverse relationship of the dehydrogenase and ADP-ribosylation activities is coincidental but not correlated.
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PMID:Inverse relationship of the dehydrogenase and ADP-ribosylation activities in sodium-nitroprusside-treated glyceraldehyde-3-phosphate dehydrogenase is coincidental. 821 90

Brefeldin A (BFA) is a fungal metabolite that exerts profound and generally inhibitory actions on membrane transport. At least some of the BFA effects are due to inhibition of the GDP-GTP exchange on the ADP-ribosylation factor (ARF) catalyzed by membrane protein(s). ARF activation is likely to be a key event in the association of non-clathrin coat components, including ARF itself, onto transport organelles. ARF, in addition to participating in membrane transport, is known to function as a cofactor in the enzymatic activity of cholera toxin, a bacterial ADP-ribosyltransferase. In this study we have examined whether BFA, in addition to inhibiting membrane transport, might affect endogenous ADP-ribosylation in eukaryotic cells. Two cytosolic proteins of 38 and 50 kDa were enzymatically ADP-ribosylated in the presence of BFA in cellular extracts. The 38-kDa substrate was tentatively identified as the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase. The BFA-binding components mediating inhibition of membrane traffic and stimulation of ADP-ribosylation appear to have the same ligand specificity. These data demonstrate the existence of a BFA-sensitive mono(ADP-ribosyl)transferase that may play a role in membrane movements.
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PMID:Stimulation of endogenous ADP-ribosylation by brefeldin A. 830 39


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