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)

A versatile fluorimetric assay based on the reduction of resazurin to resorufin demonstrated high specific activities for a number of important pyridine nucleotide-linked dehydrogenases in tobacco leaves. The Michaelis constant for the important photosynthetic enzyme, D-glyceraldehyde-3-phosphate:NADP+ oxidoreductase (EC 1.2.1.13), determined by the fluorimetric method, was considerably lower than constants determined by conventional extraction and assay methods reported for the enzyme from other plants. The sensitivity of the fluorimetric method enabled the use of dilute enzyme preparations with resultant low background and high substrate specificity. Inclusion of the anti-oxidant diethyldithiocarbamate in the extraction medium preserved the enzymes during extraction. Primary amines inhibited competitively, and phenazine methosulfate non-competitively each of the eight dehydrogenases tested with the fluorimetric assay. The Mn2+ dependence of NADP-linked dehydrogenases specific for isocitrate and malate was confirmed. The method is rapid, requires a simple combination of ingredients and should be useful for surveying dehydrogenase activity in leaves.
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PMID:Fluorimetric assay of tobacco leaf dehydrogenases with resazurin. 2 Sep 57

We have previously described a gene named tkl (tyrosine kinase related to lck). It belongs to the src family of protein-tyrosine kinases and among these it has significant homology to the lck gene (lymphoide cell kinase). The tkl gene product may represent the avian homolog of Lck, which is believed to participate in a lymphocyte-specific signal transduction pathway by association with a membrane receptor. To study the biochemical properties of the protein, a nearly complete tkl gene (isolated from a cDNA library from chicken spleen cells) was expressed in a baculovirus system. Approximately 10% of the extracted protein consisted of the soluble 51-kDa Tkl protein (p51tkl) at 40 h post-infection. This protein was found to be phosphorylated on tyrosine and serine residues at a ratio of 5:1. As expected, glycosylation or myristoylation could not be detected. Immunocomplex kinase assays indicated strong autophosphorylation of p51tkl at tyrosine residues and phosphorylation of exogenous substrates such as D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH), histones H2b and H4, and casein. This protein-tyrosine kinase activity also exhibited a marked preference for Mn2+ compared to Mg2+. The high level expression of enzymatically active Tkl should provide an excellent tool to further study the biological functions of this class of enzymes.
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PMID:A strong protein-tyrosine kinase activity is associated with a baculovirus-expressed chicken tkl gene. 151 92

Yeast vectors suitable for high-level expression of heterologous proteins should combine a high copy number with a high mitotic stability under non-selective conditions. Since high stability can best be assured by integration of the vector into chromosomal DNA we have set out to design a vector that is able to integrate into the yeast genome in a large number of copies. The rDNA locus appeared to be an attractive target for such multiple integration since it encompasses 100-200 tandemly repeated units. Plasmids containing several kb of rDNA for targeted homologous recombination, as well as the deficient LEU2-d selection marker were constructed and, after transformation into yeast, tested for both copy number and stability. One of these plasmids, designated pMIRY2 (for multiple integration into ribosomal DNA in yeast), was found to be present in 100-200 copies per cell by restriction analysis. The pMIRY2 transformants retained 80-100% of the plasmid copies over a period of 70 generations of growth in batch culture under non-selective conditions. To explore the potential of pMIRY2 as an expression vector we have inserted the homologous genes for phosphoglycerate kinase (PGK) and Mn2+-dependent superoxide dismutase (SOD) as well as the heterologous genes for thaumatin from Thaumatococcus danielli (under the GAPDH promoter), into this plasmid and analyzed the yield of the various proteins. Under optimized conditions the level of PGK in cells transformed with pMIRY2-PGK was about 50% of total soluble protein. The yield of thaumatin in the pMIRY2-thaumatin transformants exceeded by about a factor of 100 the level of thaumatin observed in transformants carrying only a single thaumatin gene integrated at the TRP1 locus in chromosome IV.
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PMID:High-copy-number integration into the ribosomal DNA of Saccharomyces cerevisiae: a new vector for high-level expression. 267 25

The toxicological effects of manganese chloride on the redox state of thiols and on the lipid peroxidation in cultures of the neuroblastoma clone N1E 115 were studied. The cell cultures were exposed, after a stationary growth phase was attained, to manganese chloride (25-100 microM) for up to 9 days. The non-protein thiols decreased at the most 27% as compared to the controls. Significant effects were obtained at all manganese concentrations tested. The total thiol content was maximally reduced by 40%. This reduced thiol content was also reflected in a lowered activity of the thiolenzyme, glyceraldehyde-3-phosphate dehydrogenase in manganese exposed cells. In addition the lipid peroxide level in the cells was decreased during the manganese treatment.
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PMID:Changes in the redox state of neuroblastoma cells after manganese exposure. 663 53

A series of site-directed mutants, E35Q, E39Q, and E35Q-D179N, in the gene encoding manganese peroxidase isozyme 1 (mnp1) from Phanerochaete chrysosporium, was created by overlap extension, using the polymerase chain reaction. The mutant genes were expressed in P. chrysosporium during primary metabolic growth under the control of the glyceraldehyde-3-phosphate dehydrogenase promoter. The mutant manganese peroxidases (MnPs) were purified and characterized. The molecular masses of the mutant proteins, as well as UV-vis spectral features of their oxidized states, were very similar to those of the wild-type enzyme. Resonance Raman spectral results indicated that the heme environment of the mutant MnP proteins also was similar to that of the wild-type protein. Steady-state kinetic analyses of the E35Q and E39Q mutant MnPs yielded K(m) values for the substrate MnII that were approximately 50-fold greater than the corresponding K(m) value for the wild-type enzyme. Likewise, the kcat values for MnII oxidation were approximately 300-fold lower than that for wild-type MnP. With the E35Q-D179N double mutant, the K(m) value for MnII was approximately 120-fold greater, and the kcat value was approximately 1000-fold less than that for the wild-type MnP1. Transient-state kinetic analysis of the reduction of MnP compound II by MnII allowed the determination of the equilibrium dissociation constants (KD) and first- order rate constants for the mutant proteins. The KD values were approximately 100-fold higher for the single mutants and approximately 200-fold higher for the double mutant, as compared with the wild-type enzyme. The first-order rate constants for the single and double mutants were approximately 200-fold and approximately 4000-fold less, respectively, than that of the wild-type enzyme. In contrast, the K(m) values for H2O2 and the rates of compound I formation were similar for the mutant and wild-type MnPs. The second-order rate constants for p-cresol and ferrocyanide reduction of the mutant compounds II also were similar to those of the wild-type enzyme.
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PMID:Characterization of manganese(II) binding site mutants of manganese peroxidase. 868 36

The glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is induced by hypoxia in endothelial cells (EC). To define the mechanisms by which GAPDH is regulated by hypoxia, EC were exposed to cobalt, other transition metals, carbon monoxide (CO), deferoxamine, or cycloheximide in the presence or absence of hypoxia for 24 h, and GAPDH protein and mRNA levels were measured. GAPDH was induced in cells by the transition metals cobalt, nickel, and manganese and by deferoxamine, and GAPDH mRNA induction by hypoxia was blocked by cycloheximide. GAPDH induction by hypoxia, unlike that of other hypoxia-regulated genes, was not inhibited by CO or by 4,6-dioxoheptanoic acid, an inhibitor of heme synthesis. GAPDH induction was not altered by mediators of protein phosphorylation, a calcium channel blocker, a calcium ionophore, or alterations in redox state. GAPDH induction by hypoxia or transitional metals was partially blocked by sodium nitroprusside but was not altered by the inhibitor of nitric oxide synthase N omega-nitro-L-arginine. These findings suggest that GAPDH induction by hypoxia in EC occurs via mechanisms other than those involved in other hypoxia-responsive systems.
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PMID:Hypoxic regulation of endothelial glyceraldehyde-3-phosphate dehydrogenase. 948 23

Manganism is a disorder characterized by hyperintensities in basal ganglia structures on magnetic resonance imaging which may be the consequence of manganese deposition in these areas. Since manganese is taken up avidly into astrocytes and is known to interfere with cerebral energy metabolism, we studied the effect of this metal on the expression and activity of the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in primary cultures of astrocytes. Treatment with 100 microM manganese for 7 days increased both the Vmax and Km values for GAPDH which was not reproducible with other divalent metals. Using RT-PCR, increased GAPDH expression was detected in cells exposed to manganese compared with controls. No changes in cytochrome oxidase activity or ATP levels were observed, and lactate production was unaffected, in manganese-treated cells. These findings provide evidence of a possible role for GAPDH in the mediation of the effects of manganese on central nervous system function.
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PMID:Increased expression of glyceraldehyde-3-phosphate dehydrogenase in cultured astrocytes following exposure to manganese. 1040 26

Site-directed mutations R177A and R177K in the gene encoding manganese peroxidase isozyme 1 (mnp1) from Phanerochaete chrysosporium were generated. The mutant enzymes were expressed in P. chrysosporium during primary metabolic growth under the control of the glyceraldehyde-3-phosphate dehydrogenase gene promoter, purified to homogeneity, and characterized by spectroscopic and kinetic methods. The UV-vis spectra of the ferric and oxidized states and resonance Raman spectra of the ferric state were similar to those of the wild-type enzyme, indicating that the heme environment was not significantly affected by the mutations at Arg177. Apparent K(m) values for Mn(II) were approximately 20-fold greater for the R177A and R177K MnPs than for wild-type MnP. However, the apparent K(m) values for the substrates, H(2)O(2) and ferrocyanide, and the k(cat) values for Mn(II) and ferrocyanide oxidation were similar to those of the wild-type enzyme. The second-order rate constants for compound I (MnPI) reduction of the mutant MnPs by Mn(II) were approximately 10-fold lower than for wild-type MnP. In addition, the K(D) values calculated from the first-order plots of MnP compound II (MnPII) reduction by Mn(II) for the mutant enzymes were approximately 22-fold greater than for wild-type MnP. In contrast, the first-order rate constants for MnPII reduction by Mn(II) were similar for the mutant and wild-type MnPs. Furthermore, second-order rate constants for the wild-type and mutant enzymes for MnPI formation, for MnPI reduction by bromide, and for MnPI and MnPII reduction by ferrocyanide were not significantly changed. These results indicate that both the R177A and R177K mutations specifically affect the binding of Mn, whereas the rate of electron transfer from Mn(II) to the oxidized heme apparently is not affected.
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PMID:Arginine 177 is involved in Mn(II) binding by manganese peroxidase. 1047

The hexosamine pathway has been implicated in the pathogenesis of diabetic complications. We determined first that hyperglycemia induced a decrease in glyceraldehyde-3-phosphate dehydrogenase activity in bovine aortic endothelial cells via increased production of mitochondrial superoxide and a concomitant 2.4-fold increase in hexosamine pathway activity. Both decreased glyceraldehyde-3-phosphate dehydrogenase activity and increased hexosamine pathway activity were prevented completely by an inhibitor of electron transport complex II (thenoyltrifluoroacetone), an uncoupler of oxidative phosphorylation (carbonyl cyanide m-chlorophenylhydrazone), a superoxide dismutase mimetic [manganese (III) tetrakis(4-benzoic acid) porphyrin], overexpression of either uncoupling protein 1 or manganese superoxide dismutase, and azaserine, an inhibitor of the rate-limiting enzyme in the hexosamine pathway (glutamine:fructose-6-phosphate amidotransferase). Immunoprecipitation of Sp1 followed by Western blotting with antibodies to O-linked GlcNAc, phosphoserine, and phosphothreonine showed that hyperglycemia increased GlcNAc by 1.7-fold, decreased phosphoserine by 80%, and decreased phosphothreonine by 70%. The same inhibitors prevented all these changes. Hyperglycemia increased expression from a transforming growth factor-beta(1) promoter luciferase reporter by 2-fold and increased expression from a (-740 to +44) plasminogen activator inhibitor-1 promoter luciferase reporter gene by nearly 3-fold. Inhibition of mitochondrial superoxide production or the glucosamine pathway prevented all these changes. Hyperglycemia increased expression from an 85-bp truncated plasminogen activator inhibitor-1 (PAI-1) promoter luciferase reporter containing two Sp1 sites in a similar fashion (3.8-fold). In contrast, hyperglycemia had no effect when the two Sp1 sites were mutated. Thus, hyperglycemia-induced mitochondrial superoxide overproduction increases hexosamine synthesis and O-glycosylation of Sp1, which activates expression of genes that contribute to the pathogenesis of diabetic complications.
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PMID:Hyperglycemia-induced mitochondrial superoxide overproduction activates the hexosamine pathway and induces plasminogen activator inhibitor-1 expression by increasing Sp1 glycosylation. 1105 Feb 44

Increasing evidence suggests that astrocytes are the site of early dysfunction and damage in manganese neurotoxicity. Astrocytes accumulate manganese by a high affinity, high capacity, specific transport system. Chronic exposure to manganese leads to increased pallidal signal hyperintensities on T1-weighted magnetic resonance images and selective neuronal loss in basal ganglia structures together with characteristic astrocytic changes known as Alzheimer type II astrocytosis. Manganese is sequestered in mitochondria where it inhibits oxidative phosphorylation. Exposure of astrocytes to manganese results in important changes including (i) decreased uptake of glutamate; (ii) increased densities of binding sites for the "peripheral-type" benzodiazepine receptor (PTBR), a class of receptor localized to mitochondria of astrocytes and involved in oxidative metabolism, mitochondrial proliferation, and neurosteroid synthesis; (iii) increased gene expression and activity of the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH), known to be associated with apoptosis; (iv) increased uptake of L-arginine, a precursor of nitric oxide, together with increased expression of the inducible form of nitric oxide synthase (iNOS). Potential consequences of these alterations in astrocytic gene expression include failure of energy metabolism, production of reactive oxygen species (ROS), increased extracellular glutamate concentration and excitotoxicity which could play a key role in manganese-induced neuronal cell death as a direct result of impaired astrocytic-neuronal interactions.
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PMID:Astrocytes and manganese neurotoxicity. 1210 78


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