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)

For the quantitation of intrinsic factor (IF) mRNA, an assay based on competitive reverse transcription and subsequent polymerase chain reaction (RT-PCR) combined with temperature gradient gel electrophoresis (TGGE) was established and validated with respect to precision and accuracy. IF-specific mRNA segments ("targets") were coamplified with known amounts of homologous "standard" RNA molecules, which differed from the targets by one base substitution. Following amplification, TGGE heteroduplex analysis proved to be a powerful method facilitating the efficient separation of these nearly identical target and standard DNA products. The measured absolute copy numbers of IF mRNA were put into relation to the constitutively expressed mRNA specific for glyceraldehyde-3-phosphate dehydrogenase (GAPDH), quantified simultaneously by competitive multiplex RT-PCR. The resulting normalized IF mRNA expression rate in terms of n copies of IF mRNA/copy of GAPDH mRNA is independent of the mRNA heterogeneity and the abundance of specific transcripts within the RNA population of interest. Therefore, normalization relative to the housekeeping gene GAPDH provides a widely applicable value for comparative studies of gene expression on the level of mRNA. Here, a normalized IF mRNA expression rate of three copies per GAPDH mRNA molecule was measured in human stomach mucosa.
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PMID:Multiplex reverse transcription polymerase chain reaction combined with temperature gradient gel electrophoresis as a tool for the normalized quantitation of intrinsic factor mRNA. 890 14

Myocardial angiotensin receptors of type 1 (AT1) are downregulated at the protein and mRNA levels in human heart failure. No data are available for the transplanted human heart, which frequently exhibits functional alterations. The aim of the present study was the quantitation of ventricular AT1 mRNA content in endomyocardial biopsies from patients after heart transplantation. For the determination of AT1 mRNA we used a novel quantitative reverse transcription polymerase chain reaction with low variance (6%) based on an internal AT1 cRNA standard, liquid-phase hybridization of polymerase chain reaction products in microtiter plates, and quantitation by enzyme-linked immunosorbent assay. Right ventricular biopsies from 16 patients after heart transplantation (left ventricular ejection fraction 67 +/- 7%) were compared with 12 patients with normal cardiac function (left ventricular ejection fraction 62 +/- 5%). A 46% lower AT1 mRNA content was found in biopsies from the 16 patients after heart transplantation than in the 12 controls (heart transplantation, 200 +/- 25 AT1 mRNA copies/ng RNA; controls, 368 +/- 50; P < 0.01). When AT1 mRNA content was related to the stably expressed GAPDH mRNA, a 49% decrease was detected (AT1/GAPDH: patients, 2.4 +/- 0.25; controls, 4.7 +/- 0.6; P < 0.006). No association between the extent of AT1 downregulation and clinical or hemodynamic variables was detected. In the human heart ventricular AT1 is downregulated after orthotopic heart transplantation. The decrease in AT1 mRNA is not associated with altered systolic function. This may partially reflect a loss of autonomic nerves and thus altered nervous control of the heart.
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PMID:Decreased expression of ventricular angiotensin receptor type 1 mRNA after human heart transplantation. 897 19

Recent studies suggest that angiotensin II (angiotensin) may be involved in the regulation of metabolism of the cardiac extracellular matrix (ECM). Two major components of ECM are collagen types I and III which play an important role in maintaining the structure and function of the heart. Although the cellular metabolism of collagen is very complex (especially at the posttranslational level), we chose to address events that occur relatively early in the synthesis of cardiac collagen molecules. To gain an understanding of the role of angiotensin (12, 24, and 48 micrograms/kg/h) on adult heart and cultured neonatal cardiac fibroblasts. The steady-state mRNA abundance of collagen type I and III was monitored using Northern blot analysis in both left and right ventricular samples at day 3 of angiotensin infusion and in cultured cardiac fibroblasts stimulated with angiotensin. In all mRNA abundance studies, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) signal was used to normalize the data for possible differences in loading and/or transfer of total RNA. Both collagen types I/GAPDH and III/GAPDH mRNA signal ratios were increased significantly in left ventricle in all dose regimens used for angiotensin infusion. Only the collagen type I/GAPDH mRNA signal ratio was increased in right ventricle with angiotensin infusion. Angiotensin (10(-7)-10(-5) M) had no effect on the steady-state mRNA abundance of collagen genes in cultured neonatal cardiac fibroblasts after 24 h treatment in serum-free conditions. Our results confirm that infusion of angiotensin may upregulate steady-state collagen gene mRNA abundance in the heart. Angiotensin had no observable effect on collagen mRNA abundance in neonatal fibroblast culture. An explanation for the current results may be that angiotensin causes the release of undefined factors from cardiac myocytes, and that these secondary factors may be involved in either the activation of collagen gene transcription or in alteration of stability of collagen mRNA transcripts via a paracrine mechanism. Although our results indicate hemodynamic loading may potentiate the action of angiotensin, this scenario is unlikely as collagen type I gene expression was increased in the normotensive right ventricle.
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PMID:Effect of angiotensin II on myocardial collagen gene expression. 897 62

Chloroplast glyceraldehyde-3-phosphate dehydrogenase (phosphorylating, E.C. 1.2.1.13) (GAPDH) of higher plants exists as an A2B2 heterotetramer that catalyses the reductive step of the Calvin cycle. In dark chloroplasts the enzyme exhibits a molecular mass of 600 kDa, whereas in illuminated chloroplasts the molecular mass is altered in favor of the more active 150 kDa form. We have expressed in Escherichia coli proteins corresponding to the mature A and B subunits of spinach chloroplast GAPDH (GapA and GapB, respectively) in addition to a derivative of the B subunit lacking the GapB-specific C-terminal extension (CTE). One mg of each of the three proteins so expressed was purified to electrophoretic homogeneity with conventional methods. Spinach GapA purified from E. coli is shown to be a highly active homotetramer (50-70 U/mg) which does not associate under aggregating conditions in vitro to high-molecular-mass (HMM) forms of ca. 600 kDa. Since B4 forms of the enzyme have not been described from any source, we were surprised to find that spinach GapB purified from E. coli was active (15-35 U/mg). Spinach GapB lacking the CTE purified from E. coli is more highly active (130 U/mg) than GapB with the CTE. Under aggregating conditions, GapB lacking the CTE is a tetramer that does not associate to HMM forms whereas GapB with the CTE occurs exclusively as an aggregated HMM form. The data indicate that intertetramer association of chloroplast GAPDH in vitro occurs through GapB-mediated protein-protein interaction.
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PMID:Functional studies of chloroplast glyceraldehyde-3-phosphate dehydrogenase subunits A and B expressed in Escherichia coli: formation of highly active A4 and B4 homotetramers and evidence that aggregation of the B4 complex is mediated by the B subunit carboxy terminus. 898 Apr 99

Chronic elevation in glucose has pleiotropic effects on the pancreatic beta-cell including a high rate of insulin secretion at low glucose, beta-cell hypertrophy, and hyperplasia. These actions of glucose are expected to be associated with the modulation of the expression of a number of glucose-regulated genes that need to be identified. To further investigate the molecular mechanisms implicated in these adaptation processes to hyperglycemia, we have studied the regulation of genes encoding key glycolytic enzymes in the glucose-responsive beta-cell line INS-1. Glucose (from 5 to 25 mM) induced phosphofructokinase-1 (PFK-1) isoform C, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (4-fold), and L-pyruvate kinase (L-PK) (7-fold) mRNAs. In contrast the expression level of the glucokinase (Gk) and 6-phosphofructo-2-kinase transcripts remained unchanged. Following a 3-day exposure to elevated glucose, a similar induction was observed at the protein level for PFK-1 (isoforms C, M, and L), GAPDH, and L-PK, whereas M-PK expression only increased slightly. The study of the mechanism of GAPDH induction indicated that glucose increased the transcriptional rate of the GAPDH gene but that both transcriptional and post transcriptional effects contributed to GAPDH mRNA accumulation. 2-Deoxyglucose did not mimic the inductive effect of glucose, suggesting that increased glucose metabolism is involved in GAPDH gene induction. These changes in glycolytic enzyme expression were associated with a 2-3-fold increase in insulin secretion at low (2-5 mM) glucose. The metabolic activity of the cells was also elevated, as indicated by the reduction of the artificial electron acceptor 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium. A marked deposition of glycogen, which was readily mobilized upon lowering of the ambient glucose, and increased DNA replication were also observed in cells exposed to elevated glucose. The results suggest that a coordinated induction of key glycolytic enzymes as well as massive glycogen deposition are implicated in the adaptation process of the beta-cell to hyperglycemia to allow for chronically elevated glucose metabolism, which, in this particular fuel-sensitive cell, is linked to metabolic coupling factor production and cell activation.
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PMID:Induction by glucose of genes coding for glycolytic enzymes in a pancreatic beta-cell line (INS-1). 900 60

We have reported that overexpression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH; EC 1.2.1.12) is involved in age-induced apoptosis of the cultured cerebellar granule cells that grow in a depolarizing concentration (25 mM) of KCI. The present study was undertaken to investigate whether GAPDH overexpression also occurs and participates in apoptosis of the cerebellar granule cells that result from switching the culturing conditions from high (25 mM) to low (5 mM) concentrations of KCl. We found that exposure of granule cells to low potassium (K+) for 24 hr induces not only apoptosis but also necrotic damage. The latter is supported by the morphological observations that a subpopulation of neurons showed cell swelling, extensive cytoplasmic vacuolization, damaged mitochondria, and apparently intact nuclei. Treatments with two antisense but not sense oligodeoxyribonucleotides directed against GAPDH attenuated low K+-induced neuronal death by approximately 50%. Morphological inspection revealed that GAPDH antisense oligonucleotides preferentially blocked low K+-induced apoptosis with little or no effect on necrotic damage. Similar to antisense oligonucleotides, actinomycin-D partially inhibited low K+-induced death of granule cells with a predominant effect on apoptosis. In contrast, cycloheximide almost completely blocked low K+-induced neuronal death and seemed to prevent both apoptotic and necrotic damage. The levels of GAPDH mRNA and protein were markedly increased in a time-dependent manner after low K+ exposure. The overexpression of GAPDH mRNA and protein was completely blocked by cycloheximide, actinomycin-D, and its antisense but not sense oligonucleotides. Taken together, these results lend credence to the view that exposure of cerebellar granule cells to low K+ induces both apoptosis and necrosis and that only the apoptotic component involves overexpression of GAPDH.
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PMID:Overexpression of glyceraldehyde-3-phosphate dehydrogenase is involved in low K+-induced apoptosis but not necrosis of cultured cerebellar granule cells. 910 17

GapB-encoded protein of Escherichia coli and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) share more than 40% amino acid identity. Most of the amino acids involved in the binding of cofactor and substrates to GAPDH are conserved in GapB-encoded protein. This enzyme shows an efficient non-phosphorylating erythrose-4-phosphate dehydrogenase activity (Zhao, G., Pease, A. J., Bharani, N., and Winkler, M. E. (1995) J. Bacteriol. 177, 2804-2812) but a low phosphorylating glyceraldehyde-3-phosphate dehydrogenase activity, whereas GAPDH shows a high efficient phosphorylating glyceraldehyde-3-phosphate dehydrogenase activity and a low phosphorylating erythrose-4-phosphate dehydrogenase activity. To identify the structural factors responsible for these differences, comparative kinetic and binding studies have been carried out on both GapB-encoded protein of Escherichia coli and GAPDH of Bacillus stearothermophilus. The KD constant of GapB-encoded protein for NAD is 800-fold higher than that of GAPDH. The chemical mechanism of erythrose 4-phosphate oxidation by GapB-encoded protein is shown to proceed through a two-step mechanism involving covalent intermediates with Cys-149, with rates associated to the acylation and deacylation processes of 280 s-1 and 20 s-1, respectively. No isotopic solvent effect is observed suggesting that the rate-limiting step is not hydrolysis. The rate of oxidation of glyceraldehyde 3-phosphate is 0.12 s-1 and is hydride transfer limiting, at least 2000-fold less efficient compared with that of erythrose 4-phosphate. Thus, it can be concluded that it is only the structure of the substrates that prevails in forming a ternary complex enzyme-NAD-thiohemiacetal productive (or not) for hydride transfer in the acylation step. This conclusion is reinforced by the fact that the rate of oxidation for erythrose 4-phosphate by GAPDH is 0.1 s-1 and is limited by the acylation step, whereas glyceraldehyde 3-phosphate acylation is efficient and is not rate-determining (>/=800 s-1). Substituting Asn for His-176 on GapB-encoded protein, a residue postulated to facilitate hydride transfer as a base catalyst, decreases 40-fold the kcat of glyceraldehyde 3-phosphate oxidation. This suggests that the non-efficient positioning of the C-1 atom of glyceraldehyde 3-phosphate relative to the pyridinium of the cofactor within the ternary complex is responsible for the low catalytic efficiency. No phosphorylating activity on erythrose 4-phosphate with GapB-encoded protein is observed although the Pi site is operative as proven by the oxidative phosphorylation of glyceraldehyde 3-phosphate. Thus the binding of inorganic phosphate to the Pi site likely is not productive for attacking efficiently the thioacyl intermediate formed with erythrose 4-phosphate, whereas a water molecule is an efficient nucleophile for the hydrolysis of the thioacyl intermediate. Compared with glyceraldehyde-3-phosphate dehydrogenase activity, this corresponds to an activation of the deacylation step by >/=4.5 kcal.mol-1. Altogether these results suggest subtle structural differences between the active sites of GAPDH and GapB-encoded protein that could be revealed and/or modulated by the structure of the substrate bound. This also indicates that a protein engineering approach could be used to convert a phosphorylating aldehyde dehydrogenase into an efficient non-phosphorylating one and vice versa.
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PMID:Comparative enzymatic properties of GapB-encoded erythrose-4-phosphate dehydrogenase of Escherichia coli and phosphorylating glyceraldehyde-3-phosphate dehydrogenase. 918 30

Recent studies indicated that expression of the housekeeping gene GAPDH is highly regulated during proliferation and differentiation. The objective of this study was to characterize by Northern blot the GAPDH mRNA expression in rat pancreas development and regeneration following acute pancreatitis induction by caerulein. Pancreatic GAPDH mRNA levels were the highest between fetal day 19 and the 11 postnatal day; they decreased to their lowest level after weaning on day 26. In acute pancreatitis, GAPDH mRNA levels were clearly increased 18 h after its initiation, were maximal during the first two days of induction and then decreased to control values after 9 days. These data demonstrate that overexpression of GAPDH may be implicated in pancreatic development, maturation and pancreas regeneration after acute pancreatitis.
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PMID:Pancreatic GAPDH gene expression during ontogeny and acute pancreatitis induced by caerulein. 920 11

Little is known about any alterations in sarcoplasmic reticulum (SR) gene expression associated with cardiac diseases of varying degrees of severity. We assessed, using the reverse transcription-polymerase chain reaction (RT-PCR) technique, SR Ca2+ transport protein gene expression in small tissue samples from failing hearts in patients undergoing cardiac surgery. Total RNA was extracted from 30- to 50-mg samples from the hearts of 13 patients with coronary artery disease, congenital heart disease, or valvular heart disease. We used RT-PCR to synthesize and amplify cDNA encoding cardiac SR Ca(2+)-ATPase, ryanodine receptor (RYR), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The amount of each mRNA in the sample was expressed relative to the amount of GAPDH mRNA. The expression level of each mRNA was correlated with the cardiac functional index. The mRNA levels for Ca(2+)-ATPase and RYR varied between heart samples, but showed a positive correlation with left ventricular ejection fraction. Ca(2+)-ATPase mRNA levels showed in inverse relationship with plasma brain natriuretic peptide. In addition, we isolated partial cDNA encoding a human cardiac RYR. The cDNA consisted of 487 nucleotides, and the nucleotide and deduced amino acid sequences showed 93% and 99% homology, respectively, to those of rabbit cardiac RYR. These results suggest that decreased levels of mRNA for SR Ca2+ transport protein could be related to abnormal cardiac function, regardless of the etiology of the heart disease. RT-PCR provides a rapid and economical way of quantifying the expression of multiple genes in small specimens and may, therefore, aid understanding of the pathophysiology and treatment of heart disease.
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PMID:Differences in sarcoplasmic reticulum gene expression in myocardium from patients undergoing cardiac surgery. Quantification of steady-state levels of messenger RNA using the reverse transcription-polymerase chain reaction. 928 54

Acetaminophen (4'-hydroxyacetanilide, APAP) is a widely used analgesic and antipyretic drug that can cause hepatic necrosis under some circumstances via cytochrome P450-mediated oxidation to a reactive metabolite, N-acetyl-p-benzoquinone imine (NAPQI). Although the mechanism of hepatocellular injury caused by APAP is not fully understood, it is known that NAPQI forms covalent adducts with several hepatocellular proteins. Reported here is the identification of one of these proteins as glyceraldehyde-3-phosphate dehydrogenase [GAPDH, D-glyceraldehyde-3-phosphate: NAD+ oxidoreductase (phosphorylating), EC 1.2.1.12]. Two hours after the administration of hepatotoxic doses of [14C]APAP to mice, at a time prior to overt cell damage, hepatocellular GAPDH activity was significantly decreased concurrent with the formation of a 14C-labeled GAPDH adduct. A nonhepatotoxic regioisomer of APAP, 3'-hydroxyacetanilide (AMAP), was found to decrease GAPDH activity to a lesser extent than APAP, and radiolabel from [14C]AMAP bound to a lesser extent to GAPDH at a time when its overall binding to hepatocellular proteins was almost equivalent to that of APAP. In order to determine the nature of the covalent adduct between GAPDH and APAP, its major reactive and toxic metabolite, NAPQI, was incubated with purified porcine muscle GAPDH. Microsequencing analysis and fast atom bombardment mass spectrometry (FAB-MS) with collision-induced dissociation (CID) were used to characterize one of the adducts as APAP bound to the cysteinyl sulfhydryl group of Cys-149 in the active site peptide of GAPDH.
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PMID:Inactivation of glyceraldehyde-3-phosphate dehydrogenase by a reactive metabolite of acetaminophen and mass spectral characterization of an arylated active site peptide. 934 31


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