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 binding of nicotinamide--adenine dinucleotide (NAD+), nicotinamide--1,N6-ethenoadenine dinucleotide (epsilon NAD+), acetylpyridine--adenine dinucleotide (AcPyAD+), ATP, and adenosine diphosphoribose (ADP-ribose) to rabbit muscle glyceraldehyde-3-phosphate dehydrogenase (the enzyme) was examined. NAD+ and epsilon NAD+ were found to bind to the apoenzyme in a negatively cooperative manner, whereas AcPyAD+, ATP, and ADP-ribose bind non-cooperatively to the NAD+ sites. The strong negative cooperativity in coenzyme binding was found to be abolished in the presence of AcPyAD+ and strongly weakened by ATP, ADP, and AMP, but was not affected by the addition of ADP-ribose. These findings demonstrate that the mechanism of the negative cooperativity in coenzyme binding to the enzyme involves ligand-induced conformational changes between neighboring sites. These findings cannot be accounted for by the pre-existent asymmetry model. The results support our previous hypothesis that the structure of the pyridine moiety of the coenzyme analogues plays a role in orienting the adenine moiety in the adenine subsite, and thus affects the cooperativity observed in the binding of the coenzyme analogue.
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PMID:The sequential nature of the negative cooperativity in rabbit muscle glyceraldehyde-3-phosphate dehydrogenase. 744 64

Nitric oxide-stimulated modification of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) by [adenylate-32P]NAD has been interpreted in recent reports as ADP-ribosylation. Incubations of GAPDH with the NO-releasing agent sodium nitroprusside (SNP) and NAD resulted, however, in essentially equal incorporation of radiolabel from the adenine, phosphate, and nicotinamide moieties to the extent of approximately 0.02 mol of NAD.mol of GAPDH-1. Modification of GAPDH by free adenosine 5'-diphosphoribose (ADP-ribose) was only 10% of that by NAD. Exposure of GAPDH modified by NAD in the presence of SNP to HgCl2, which acts at thiol linkages, released two products. Both contained nicotinamide and adenylate but did not cochromatograph with NAD. GAPDH activity was inhibited by SNP in a dose-dependent manner in the presence of NAD. When inhibition was 80%, with 1 mM SNP and 1 mM dithiothreitol, covalent modification with NAD was < 2%. This result is consistent with the conclusion that inhibition of GAPDH activity by SNP in the presence of NAD is due primarily to active-site nitrosylation, as reported by other workers, and is not due to the minor modification with NAD. These results demonstrate that NO-stimulated modification of GAPDH with NAD is not ADP-ribosylation as previously reported but rather is covalent binding of NAD through a NO-dependent thiol intermediate, possibly providing an example of an unexpected, altered reactivity of a nitrosylated protein.
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PMID:Stimulation by nitric oxide of an NAD linkage to glyceraldehyde-3-phosphate dehydrogenase. 832 4

A transfer RNA (tRNA) binding protein present in HeLa cell nuclear extracts was purified and identified as the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Studies with mutant tRNAs indicated that GAPDH recognizes both sequence and structural features in the RNA. GAPDH discriminated between wild-type tRNA and two tRNA mutants that are defective in nuclear export, which suggests that the protein may participate in RNA export. The cofactor nicotinamide adenine dinucleotide disrupted complex formation between tRNA and GAPDH and thus may share a common binding site with the RNA. Indirect immunofluorescence experiments showed that GAPDH is present in the nucleus as well as in the cytoplasm.
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PMID:Sequence-specific binding of transfer RNA by glyceraldehyde-3-phosphate dehydrogenase. 842 4

To evaluate the relationship of inositol 1,4,5-trisphosphate (IP3) receptor-mediated signal transduction and cellular energy dynamics, we have characterized effects of nucleotides on IP3 receptor (IP3R)-mediated calcium (Ca2+) flux in purified IP3 receptors reconstituted in lipid vesicles (IP3RV) and examined hypoxia-induced augmentation of intracellular Ca2+ in intact cells. Reduced nicotinamide adenine dinucleotide (NADH) increases IP3-mediated Ca2+ flux in IP3RV. This effect is highly specific for NADH. Hypoxia elicited by brief exposure of nerve growth factor-differentiated PC12 cells or cerebellar Purkinje cells to cyanide elicits rapid increased in internal [Ca2+], which derives from IP3-sensitive stores. Blockade of this effect by 2-deoxyglucose and inhibition of glyceraldehyde-3-phosphate dehydrogenase implicates enhanced glycolytic production of NADH in the Ca2+ stimulation. Internal [Ca2+] is markedly and specifically increased by direct intracellular injection of NADH, and this effect is blocked by heparin, further implicating IP3R stores. These findings indicate that direct regulation of IP3R by NADH is responsible for elevated cytoplasmic [Ca2+] occurring in the earliest phase of hypoxia. This link of IP3R activity with cellular energy dynamics may be relevant to both hypoxic damage and metabolic regulation of IP3 signaling processes.
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PMID:Reduced nicotinamide adenine dinucleotide-selective stimulation of inositol 1,4,5-trisphosphate receptors mediates hypoxic mobilization of calcium. 860 44

Nitric oxide (NO)-related activity has been associated with an NAD+-dependent modification of the glycolytic enzyme, glyceraldehyde-3-phosphate dehydrogenase (GAPDH). However, the mechanism by which NO effects covalent attachment of nucleotide and its role in regulation of enzyme activity are controversial. Recent studies have shown that S-nitrosylation of GAPDH (Cys149) initiates subsequent modification by the pyridinium cofactor. Here we show that NADH rather than NAD+ is the preferred substrate. Transnitrosation from active site S-nitrosothiol to the reduced nicotinamide ring system appears to facilitate protein thiolate attack on the enzyme-bound cofactor. This results in attachment of the intact NADH molecule. Moreover, we find that S-nitrosylation of GAPDH is responsible for reversible enzyme inhibition, whereas attachment of NADH accounts for irreversible enzyme inactivation. S-Nitrosylation may serve to protect GAPDH from oxidant inactivation in settings of cytokine overproduction and to regulate glycolysis. NADH attachment is more likely to be a pathophysiological event associated with inhibition of gluconeogenesis.
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PMID:Posttranslational modification of glyceraldehyde-3-phosphate dehydrogenase by S-nitrosylation and subsequent NADH attachment. 862 64

The individual fluorescence and phosphorescence properties of W84 and W310 in Bacillus stearothermophilus glyceraldehyde-3-phosphate dehydrogenase were identified through the construction of a single tryptophan mutant (W84F) and by comparison of the emission between mutant and wild-type enzymes. The results show that the luminescence of W310 is red-shifted and substantially quenched relative to that of W84. It displays an average subnanosecond fluorescence lifetime (tau F) and a very short, 50 microseconds, room-temperature phosphorescence (RTP) lifetime (tau P). The perturbation of W310 luminescence is believed to arise from a stacking interaction with Y283. In contrast, W84 exhibits a fluorescence lifetime tau F of several nanoseconds and a long-lived phosphorescence lifetime tau P, typical of buried, unperturbed TrP residues. NAD+ binding to the tetrameric enzyme causes a 55% reduction of W310 fluorescence intensity together with a nearly complete quenching of its low-temperature phosphorescence. W84, which is located far from the nicotinamide moiety of NAD+, is much less affected by the binding of the coenzyme; the reduction in fluorescence intensity is 35%, and its phosphorescence intensity is unchanged. Another consequence of NAD+ binding is a significant decrease of the RTP lifetime tau P of W84, manifesting thereby a conformational change in the region of the coenzyme-binding domain. However, no change is observed in the RTP lifetime tau P of W310 located in the catalytic domain. These findings and those obtained at partial coenzyme saturation support the conclusions derived from high-resolution crystallographic structures [Skarzynski, T., & Wonacott, A. J., (1988) J. Mol. Biol. 203, 1097-1118] that the NAD(+)-induced conformational change is sequential and that subtle rearrangement in the structure of unligated subunits might be responsible for the negative cooperative behavior of NAD+ binding.
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PMID:Effects of NAD+ binding on the luminescence of tryptophans 84 and 310 of glyceraldehyde-3-phosphate dehydrogenase from Bacillus stearothermophilus. 882 92

Previous studies have demonstrated that glyceraldehyde-3-phosphate dehydrogenase (GAPDH) undergoes NAD(H) linkage to an active site thiol when it comes into contact with .NO-related oxidants. We found that a free-radical generator 2,2'-azobis-(2-amidinopropane) hydrochloride (AAPH), which does not release either .NO or .NO-related species, was indeed able to induce the NAD(H) linkage to GAPDH. We performed spin-trapping studies with purified apo-GAPDH to identify a putative thiol intermediate produced by AAPH as well as by .NO-related oxidants. As .NO sources we used .NO gas and two .NO-donors, S-nitroso-N-acetyl-D,L-penicillamine and 3-morpholinosydno-nimine hydrochloride (SIN-1). Because SIN-1 produces .NO and a superoxide radical simultaneously, we also tested the effects of peroxynitrite. All the .NO-related oxidants were able to induce the linkage of NAD(H) to GAPDH and the formation of a protein free-radical identified as a thiyl radical (inhibited by N-ethylmaleimide). .NO gas and the .NO-donors required molecular oxygen to induce the formation of the GAPDH thiyl radical, suggesting the possible involvement of higher nitrogen oxides. Thiyl radical formation was decreased by the reconstitution of GAPDH with NAD+. Apo-GAPDH was a strong scavenger of AAPH radicals, but its scavenging ability was decreased when its cysteine residues were alkylated or when it was reconstituted with NAD+. In addition, after treatment with AAPH, a thiyl radical of GAPDH was trapped at high enzyme concentrations. We suggest that the NAD(H) linkage to GAPDH is mediated by a thiyl radical intermediate not specific to .NO or .NO-related oxidants. The cysteine residue located at the active site of GAPDH (Cys-149) is oxidized by free radicals to a thiyl radical, which reacts with the neighbouring coenzyme to form Cys-NAD(H) linkages. Studies with the NAD+ molecule radio-labelled in the nicotinamide or adenine portion revealed that both portions of the NAD+ molecule are linked to GAPDH.
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PMID:Nitric oxide-dependent NAD linkage to glyceraldehyde-3-phosphate dehydrogenase: possible involvement of a cysteine thiyl radical intermediate. 891 69

Examination of the properties of Escherichia coli and rabbit muscle D-glyceraldehyde-3-phosphate dehydrogenase (GPDHs) modified by 2,3-butanedione has shown that both tetrameric enzymes are stabilized, on selective modification of arginine residues (probably Arg 231), in an asymmetric state with only two active centers capable of performing the dehydrogenase reaction. The functionally incompetent active centers can be alkylated by iodoacetate or iodoacetamide in the case of E. coli enzyme, but are inaccessible for these reagents in the case of rabbit muscle D-GPDH. These results are consistent with the idea that the two homologous enzymes share common principles of the protein design, but differ somewhat in their active centers geometries. Modification of the arginine procedures marked changes in the shape of the charge transfer complex spectrum in the region of 300-370 nm, suggestive of the alterations in the microenvironment of the nicotinamide ring of NAD(+), although the coenzyme binding characteristics remain largely unaltered. On arginine modification, the enzyme becomes insensitive to the effect of AMP on the kinetic parameters of p-nitrophenyl acetate hydrolysis reaction.
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PMID:D-glyceraldehyde-3-phosphate dehydrogenase. Properties of the enzyme modified at arginine residues. 910 Mar 44

CP12 is a small nuclear encoded chloroplast protein of higher plants, which was recently shown to interact with NAD(P)H-glyceraldehyde-3-phosphate dehydrogenase (GAPDH; EC 1.2.1. 13), one of the key enzymes of the reductive pentosephosphate cycle (Calvin cycle). Screening of a pea cDNA library in the yeast two-hybrid system for proteins that interact with CP12, led to the identification of a second member of the Calvin cycle, phosphoribulokinase (PRK; EC 2.7.1.19), as a further specific binding partner for CP12. The exchange of cysteines for serines in CP12 demonstrate that interaction with PRK occurs at the N-terminal peptide loop of CP12. Size exclusion chromatography and immunoprecipitation assays reveal the existence of a stable 600-kDa PRK/CP12/GAPDH complex in the stroma of higher plant chloroplasts. Its stoichiometry is proposed to be of two N-terminally dimerized CP12 molecules, each carrying one PRK dimer on its N terminus and one A2B2 complex of GAPDH subunits on the C-terminal peptide loop. Incubation of the complex with NADP or NADPH, in contrast to NAD or NADH, causes its dissociation. Assays with the stromal 600-kDa fractions in the presence of the four different nicotinamide-adenine dinucleotides indicate that PRK activity depends on complex dissociation and might be further regulated by the accessible ratio of NADP/NADPH. From these results, we conclude that light regulation of the Calvin cycle in higher plants is not only via reductive activation of different proteins by the well-established ferredoxin/thioredoxin system, but in addition, by reversible dissociation of the PRK/CP12/GAPDH complex, mediated by NADP(H).
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PMID:CP12 provides a new mode of light regulation of Calvin cycle activity in higher plants. 929 36

Glyceraldehyde-3-phosphate dehydrogenase is a key enzyme in the glycolytic pathway. Since its transcript levels do not vary in most experimental conditions, it has been often used as a control in northern blot or reverse transcriptase-polymerase chain reaction analysis. We have cloned and sequenced a gene encoding glyceraldehyde-3-phosphate dehydrogenase (Tthgapdh) from Tetrahymena thermophila cDNA library and determined whether the Tthgapdh mRNA is a loading control in gene expression studies of T. thermophila cell. The open reading frame encoded a protein of 341 amino acid residues (36.8 kDa) containing a nicotinamide adenine dinucleotide-binding domain and a catalytic domain, which was highly similar to those of other organisms. Its mRNA levels at different growth stages were examined by northern blot analysis. The fragment of the isolated cDNA was hybridized to a 1.3-kb mRNA transcript. There was a marked increase in Tthgapdh mRNA level at the mid-exponential phase, followed by a gradual decrease. Therefore, much caution should be made to use Tthgapdh mRNA as an internal standard for northern blot analysis in Tetrahymena.
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PMID:Cloning and sequencing of a cDNA encoding glyceraldehyde-3-phosphate dehydrogenase from Tetrahymena thermophila: growth-associated changes in its mRNA expression. 930 12


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