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)

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

Reference two-dimensional (2-D) gels are presented for human breast ductal carcinoma and histologically normal tissue. Whole biopsy fragments were analyzed, including epithelial and nonepithelial components. Thirty-five spots have been assigned by gel matching to the human liver SWISS-2DPAGE reference map and/or to the human primary keratinocyte IPG map from the Danish Center for Human Genome. N-terminal microsequencing was applied to confirm randomly chosen matching assignments and to identify six new spots. Protein expression profiles in ductal carcinoma and in normal breast tissue appeared to be similar, except for a pattern consisting of 32 spots, which were highly expressed in all carcinoma specimens, and less intense and occasionally undetectable in normal tissue. This difference was statistically significant. Assignment has been obtained for several spots, namely GRP94, GRP78, GRP75, mitochondrial HSP60, calreticulin, protein disulfide isomerase, peptidyl-prolyl cis-trans isomerase, collagen-binding protein 2, fructose bisphosphate aldolase, glyceraldehyde-3-phosphate dehydrogenase, thioredoxin, cytochrome c oxidase VA subunit, tubulin beta isoform and macrophage migration inhibitory factor (MIF). The cancer- and tissue-specificity of the described pattern was assessed by matching to the Swiss-2DPAGE human liver, hepatoma, lymphoma, erythroleukemia reference maps. The pattern of 32 spots was found to be indicative of epithelial neoplasia.
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PMID:Protein expression profiles in human breast ductal carcinoma and histologically normal tissue. 950 17

Leaf metabolites, adenylates, and Rubisco activation were studied in two transgenic tobacco (Nicotiana tabacum L. cv W38) types. Plants with reduced amounts of cytochrome b/f complex (anti-b/f) have impaired electron transport and a low transthylakoid pH gradient that restrict ATP and NADPH synthesis. Plants with reduced glyceraldehyde 3-phosphate dehydrogenase (anti-GAPDH) have a decreased capacity to use ATP and NADPH in carbon assimilation. The activation of the chloroplast NADP-malate dehydrogenase decreased in anti-b/f plants, indicating a low NADPH/NADP(+) ratio. The whole-leaf ATP/ADP in anti-b/f plants was similar to wild type, while it increased in anti-GAPDH plants. In both plant types, the CO(2) assimilation rates decreased with decreasing ribulose 1, 5-bisphosphate concentrations. In anti-b/f plants, CO(2) assimilation was further compromised by reduced carbamylation of Rubisco, whereas in anti-GAPDH plants the carbamylation remained high even at subsaturating ribulose 1,5-bisphosphate concentrations. We propose that the low carbamylation in anti-b/f plants is due to reduced activity of Rubisco activase. The results suggest that light modulation of activase is not directly mediated via the electron transport rate or stromal ATP/ADP, but some other manifestation of the balance between electron transport and the consumption of its products. Possibilities include the transthylakoid pH gradient and the reduction state of the acceptor side of photosystem I and/or the degree of reduction of the thioredoxin pathway.
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PMID:The role of chloroplast electron transport and metabolites in modulating Rubisco activity in tobacco. Insights from transgenic plants with reduced amounts of cytochrome b/f complex or glyceraldehyde 3-phosphate dehydrogenase. 1067 42

Evidence is accumulating that the adverse tumor microenvironment both modifies the malignant progression of tumor cells and contributes to chemotherapy and radiation resistance. We hypothesized that some of the effects on malignant progression are mediated through the transcriptional regulation of genes responsive to the stresses of the microenvironment, such as low oxygen or low glucose conditions. To determine epigenetic changes in gene expression that were consistent with that hypothesis, we used an in vitro subtractive hybridization method, representational difference analysis, to identify hypoxia-induced cDNAs from cultured human cervical epithelial cells. We identified 12 induced genes: two novel genes (HIG1 and HIG2), three genes known to be hypoxia-inducible (tissue factor, GAPDH, thioredoxin), and seven genes not previously identified as hypoxia-inducible [HNRNP(a1), ribosomal L7, annexin V, lipocortin 2, Ku(70), PRPP synthase, and acetoacetyl-CoA thiolase]. In cultured cells, HIG1 and HIG2 expression is induced by hypoxia and by glucose deprivation, but their expression is not induced by serum deprivation, UV, or ionizing radiation. The putative HIG1 and HIG2 open reading frames are expressed in cells, as confirmed by epitope tagging. In addition, tumor xenografts derived from human cervical cancer cells display increased expression of HIG1 and HIG2 when they are deprived of oxygen. Taken together, these data suggest a coordinated transcriptional response of eukaryotic cells to microenvironmental stresses found in the solid tumor.
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PMID:Epigenetic regulation of gene expression in cervical cancer cells by the tumor microenvironment. 1069 May 27

The activation of oxidized phosphoribulokinase either "free" or as part of a bi-enzyme complex by reduced thioredoxins during the enzyme reaction was studied. In the presence of reduced thioredoxin, the product of the reaction catalyzed by phosphoribulokinase within the bi-enzyme complex does not appear in a linear fashion. It follows a mono-exponential pattern that suggests a slow dissociation process of the bi-enzyme complex in the assay cuvette. A plot of the steady state of product appearance against thioredoxin concentration gave a sigmoid curve. On the basis of our experimental results, we propose a minimum model of the activation of phosphoribulokinase by reduced thioredoxin. Reduced thioredoxin may act on the phosphoribulokinase, either within the complex or in the dissociated metastable form. However, the time required to activate the enzyme as part of the complex is shorter (about 20 s) than that required to activate the dissociated form (about 10 min). This might be of physiological relevance, and we discuss the role of the interactions between phosphoribulokinase and glyceraldehyde-3-phosphate dehydrogenase in the regulation of the Calvin cycle.
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PMID:Thioredoxin activation of phosphoribulokinase in a bi-enzyme complex from Chlamydomonas reinhardtii chloroplasts. 1073 91

Protein disulfide isomerase (PDI), a member of the thioredoxin (Trx) superfamily, consists of five consecutive domains, a-b-b'-a'-c. Domain combinations, AB, A'C, B'A'C and AB-C, and hybrids of PDI domains with Trx, Trx-C and Trx-B'A'C, have been constructed and expressed in Escherichia coli to examine the contributions of PDI domains to its enzyme and chaperone activities. All the combination and hybrid products are considerably less active than intact PDI in their enzyme activities. Recombinant products containing C, at low concentrations, inhibit the reactivation of lysozyme in HEPES buffer, while those without C do not. Only the intact PDI molecule and the hybrid molecule, Trx-B'A'C, but to a much lower level, show general chaperone activity in assisting the reactivation of denatured D-glyceraldehyde-3-phosphate dehydrogenase. It is suggested that all domains of PDI contribute to the binding of target protein for its chaperone activity.
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PMID:Contributions of protein disulfide isomerase domains to its chaperone activity. 1100 77

The rat luminal endoplasmic-recticulum calcium-binding proteins 1 and 2 (CaBP1 and CaBP2 respectively) are members of the protein disulphide-isomerase (PDI) family. They contain two and three thioredoxin boxes (Cys-Gly-His-Cys) respectively and, like PDI, may be involved in the folding of nascent proteins. We demonstrate here that CaBP1, similar to PDI and CaBP2, can complement the lethal phenotype of the disrupted Saccharomyces cerevisiae PDI gene, provided that the natural C-terminal Lys-Asp-Glu-Leu sequence is replaced by His-Asp-Glu-Leu. Both the in vitro RNase AIII-re-activation assays and in vivo pro-(carboxypeptidase Y) processing assays using CaBP1 and CaBP2 thioredoxin (trx)-box mutants revealed that, whereas the three trx boxes in CaBP2 seem to be functionally equivalent, the first trx box of CaBP1 is significantly more active than the second trx box. Furthermore, only about 65% re-activation of denatured reduced RNase AIII could be obtained with CaBP1 or CaBP2 compared with PDI, and the yield of PDI-catalysed reactions was significantly reduced in the presence of either CaBP1 or CaBP2. In contrast with PDI, neither CaBP1 nor CaBP2 could catalyse the renaturation of denatured glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which is a redox-independent process, and neither protein had any effect on the PDI-catalysed refolding of GAPDH. Furthermore, although PDI can bind peptides via its b' domain, a property it shares with PDIp, the pancreas-specific PDI homologue, and although PDI can bind malfolded proteins such as 'scrambled' ribonuclease, no such interactions could be detected for CaBP2. We conclude that: (1) both CaBP2 and CaBP1 lack peptide-binding activity for GAPDH attributed to the C-terminal region of the a' domain of PDI; (2) CaBP2 lacks the general peptide-binding activity attributed to the b' domain of PDI; (3) interaction of CaBP2 with substrate (RNase AIII) is different from that of PDI and substrate; and (4) both CaBP2 and CaBP1 may promote oxidative folding by different kinetic pathways.
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PMID:Functional roles and efficiencies of the thioredoxin boxes of calcium-binding proteins 1 and 2 in protein folding. 1141 39

Human P5 (hP5) was expressed in the Escherichia coli pET system and purified by sequential Ni(2+)-chelating resin column chromatography. Characterization of purified hP5 indicated that it has both isomerase and chaperone activities, but both activities are lower than those of human protein disulfide isomerase (PDI). Moreover, hP5 was observed to have peptide-binding ability, and its chaperone activity was confirmed with rhodanese and citrate synthase as substrates, but not with D-glyceraldehyde-3-phosphate dehydrogenase, showing that hP5 has substrate specificity with respect to chaperone activity. Mutation of two thioredoxin-related motifs in hP5 revealed that the first motif is more important than the second for isomerase activity and that the first cysteine in each motif is necessary for isomerase activity. Since thioredoxin motif mutants lacking isomerase activity retain chaperone activity with the substrate citrate synthase, the isomerase and chaperone activities of hP5 are probably independent, as was shown for PDI.
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PMID:Functional analysis of human P5, a protein disulfide isomerase homologue. 1220 15

The regulatory isoform of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a light-activated enzyme constituted by subunits GapA and GapB. The NADPH-dependent activity of regulatory GAPDH from spinach chloroplasts was affected by the redox potential (E(m,7.9), -353 +/- 11 mV) through the action of thioredoxin f. The redox dependence of recombinant GapB (E(m,7.9), -347 +/- 9 mV) was similar to native GAPDH, whereas GapA was essentially redox-insensitive. GapB mutants having one or two C-terminal cysteines mutated into serines (C358S, C349S, C349S/C358S) were less redox-sensitive than GapB. Different mutants with other cysteines substituted by serines (C18S, C274S, C285S) still showed strong redox regulation. Fully active GapB was a tetramer of B-subunits, and, when incubated with NAD, it associated to a high molecular weight oligomer showing low NADPH-dependent activity. The C-terminal GapB mutants (C358S, C349S, C349S/C358S) were active tetramers unable to aggregate to higher oligomers in the presence of NAD, whereas other mutants (C18S, C274S, C285S) again behaved like GapB. We conclude that a regulatory disulfide, between Cys-349 and Cys-358 of the C-terminal extension of GapB, does form in the presence of oxidized thioredoxin. This covalent modification is required for the NAD-dependent association into higher oligomers and inhibition of the NADPH-activity. By leading to GAPDH autoinhibition, thioredoxin and NAD may thus concur to the dark inactivation of the enzyme in vivo.
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PMID:The C-terminal extension of glyceraldehyde-3-phosphate dehydrogenase subunit B acts as an autoinhibitory domain regulated by thioredoxins and nicotinamide adenine dinucleotide. 1227 Sep 27

Chloroplast glyceraldehyde-3-phosphate dehydrogenase (GAPDH) of higher plants uses both NADP(H) and NAD(H) as coenzyme and consists of one (GapA) or two types of subunits (GapA, GapB). AB-GAPDH is regulated in vivo through the action of thioredoxin and metabolites, showing higher kinetic preference for NADPH in the light than in darkness due to a specific effect on kcat(NADPH). Previous crystallographic studies on spinach chloroplast A4-GAPDH complexed with NADP or NAD showed that residues Thr33 and Ser188 are involved in NADP over NAD selectivity by interacting with the 2'-phosphate group of NADP. This suggested a possible involvement of these residues in the regulatory mechanism. Mutants of recombinant spinach GapA (A4-GAPDH) with Thr33 or Ser188 replaced by Ala (T33A, S188A and double mutant T33A/S188A) were produced, expressed in Escherichia coli, and compared to wild-type recombinant A4-GAPDH, in terms of crystal structures and kinetic properties. Affinity for NADPH was decreased significantly in all mutants, and kcat(NADPH) was lowered in mutants carrying the substitution of Ser188. NADH-dependent activity was unaffected. The decrease of kcat/Km of the NADPH-dependent reaction in Ser188 mutants resembles the behaviour of AB-GAPDH inhibited by oxidized thioredoxin, as confirmed by steady-state kinetic analysis of native enzyme. A significant expansion of size of the A4-tetramer was observed in the S188A mutant compared to wild-type A4. We conclude that in the absence of interactions between Ser188 and the 2'-phosphate group of NADP, the enzyme structure relaxes to a less compact conformation, which negatively affects the complex catalytic cycle of GADPH. A model based on this concept might be developed to explain the in vivo light-regulation of the GAPDH.
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PMID:Coenzyme site-directed mutants of photosynthetic A4-GAPDH show selectively reduced NADPH-dependent catalysis, similar to regulatory AB-GAPDH inhibited by oxidized thioredoxin. 1523 65


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