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:1.8.1.4 (diaphorase)
2,754 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The distribution of simian virus 40 large tumor antigen in subcellular fractions from simian virus 40-transformed hamster (H-50) and mouse (VLM) cells and from simian virus 40-infected monkey cells was determined. Solubilized [(35)S]-methionine- or (32)P(i)-labeled surface membrane and nuclear fractions were prepared, immunoprecipitated with hamster anti-T serum, and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Tumor antigen with an apparent molecular weight of approximately 96,000 was detected in both subcellular fractions. Minor components of approximately 68,000 and approximately 56,000 with anti-T reactivity which labeled with [(35)S]methionine were also detected in both fractions from H-50 cells, as were components of approximately 140,000 and approximately 56,000 from VLM cells. The 56,000 component appeared to be greatly reduced in (32)P(i)-labeled surface membrane fractions. Normal cells or cells transformed with a heterologous agent, such as polyoma virus or a chemical carcinogen, lacked immunoprecipitable tumor antigen. Cell fractionation was monitored by [(3)H]thymidine labeling, NADH-diaphorase activity, and Na(+)-K(+)-dependent ATPase activity. These analyses revealed only trace contamination of surface membranes by nuclei, extremely low levels of nuclear rupture during homogenization, and an approximate 10-fold enrichment of surface membrane. Reconstruction experiments demonstrated that soluble tumor antigen failed to associate or copurify with surface membranes during fractionation procedures. These results indicate the presence of a protein in the plasma membrane of cells transformed or infected by simian virus 40 that is immunologically indistinguishable from nuclear tumor antigen.
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PMID:Subcellular Localization of simian virus 40 large tumor antigen. 22 15

Specific, polyclonal antisera have been raised to the native branched-chain 2-oxoacid dehydrogenase complex (BCOADC) from bovine kidney and each of its three constituent enzymes: E1, the substrate-specific 2-oxoacid dehydrogenase; E2, the multimeric dihydrolipoamide acyltransferase 'core' enzyme and E3, dihydrolipoamide dehydrogenase. Purified BCOADC, isolated by selective poly(ethyleneglycol) precipitation and hydroxyapatite chromatography, contains only traces of endogenous E3 as detected by a requirement for this enzyme in assaying overall complex activity and by immunoblotting criteria. A weak antibody response was elicited by the E1 beta subunit relative to the E2 and E1 alpha polypeptides employing either purified E1 or BCOADC as antigens. Anti-BCOADC serum showed no cross-reaction with high levels of pig heart E3 indicating the absence of antibody directed against this component. However, immunoprecipitates of mature BCOADC from detergent extracts of NBL-1 (bovine kidney) or PK-15 (porcine kidney) cell lines incubated for 3-4 h in the presence of [35S]methionine contained an additional 55,000-Mr species which was identified as E3 on the basis of immunocompetition studies. Accumulation of newly synthesised [35S]methionine-labelled precursors for E2, E1 alpha and E3 was achieved by incubation of PK-15 cells for 4 h in the presence of uncouplers of oxidative phosphorylation. Pre-E2 exhibited an apparent Mr value of 56,500, pre-E1 alpha, 49,000 and pre-E3, 57,000 compared to subunit Mr values of 50,000, 46,000 and 55,000, respectively, for the mature polypeptides. Thus, like the equivalent lipoate acyltransferases of the mammalian pyruvate dehydrogenase (PDC) and 2-oxoglutarate dehydrogenase (OGDC) complexes, pre-E2 of BCOADC characteristically contains an extended presequence. In NBL-1 cells, pre-E2 was found to be unstable since no cytoplasmic pool of this precursor could be detected; moreover, processed E1 alpha was not assembled into intact BCOADC as evidenced by the absence of E2 or E3 in immunoprecipitates with anti-(BCOADC) serum after a 45-min 'chase' period in the absence of uncoupler. Dihydrolipoamide dehydrogenase (E3), in its precursor state, was not present in immune complexes with anti-(BCOADC) serum, indicating that its co-precipitation with mature complex is by virtue of its high affinity for assembled complex in vivo whereas no equivalent interaction of pre-E3 with its companion precursors occurs prior to mitochondrial import.
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PMID:Immunology, biosynthesis and in vivo assembly of the branched-chain 2-oxoacid dehydrogenase complex from bovine kidney. 200 11

Two groups (n = 5) of male weanling Wistar rats were housed individually and fed copper (Cu)-deficient (0.5 mg Cu/kg) diets either with or without methionine supplementation (18 g/kg) for 49 days. Plasma caeruloplasmin (EC 1.16.3.1) and erythrocyte superoxide dismutase (EC 1.15.1.1, CuSOD) activities were measured in blood. Tissue Cu levels and the activities of cytochrome c oxidase (EC 1.9.3.1, CCO) and CuSOD were measured in the heart and liver. Hepatic activities of the sulfhydryl-sensitive enzymes, creatine kinase (EC 2.7.3.2), fumarase (EC 4.2.1.2) glutathione S-transferase (EC 2.5.1.18) and lipoamide dehydrogenase (EC 1.6.4.3) were also measured. Apart from cardiac CCO activity all of the measured indices of Cu status were found to be significantly (p less than 0.05) decreased in the methionine supplemented rats. Although fumarase activity was significantly (p less than 0.05) decreased in the methionine-supplemented animals compared with controls, the activities of the other sulfhydryl-sensitive enzymes were not significantly decreased. These results suggest that some of the toxic effects of excess dietary methionine may be mediated through interference with copper metabolism rather than through the previously postulated inhibition of sulfhydryl-sensitive enzymes by metabolites of methionine.
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PMID:Excess dietary methionine decreases indices of copper status in the rat. 216 46

A cDNA clone for the preprotein of spinach ferredoxin:NADP+ reductase has been modified to allow the expression in Escherichia coli of the mature flavoprotein form the lacks the transit peptide. An expression vector, pFNR1, was constructed by subcloning the fragment into the plasmid pDS12/RBSII, SphI. In the crude extracts of transformed cells after induction, two active holoproteins of 35 kDa and 32 kDa, respectively, were found. The 32-kDa protein, purified by immunoaffinity chromatography, was found to lack the first 28 residues of the spinach protein sequence and to have a methionine as the N-terminal residue instead of Val29. A new expression plasmid, pFNR2, was obtained by in vitro mutagenesis of the codon GTG for Val29 to the synonymous GTT; in this case, only the 35-kDa protein was expressed by transformed cells. Both the 35-kDa and 32-kDa enzymes were purified and characterized. All the properties analyzed of the cloned 35-kDa enzyme were very similar to those of the spinach flavoprotein. The 32-kDa form showed the same catalytic efficiency of the spinach enzyme as a diaphorase but its interaction with oxidized ferredoxin was partially impaired.
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PMID:Expression in Escherichia coli of ferredoxin:NADP+ reductase from spinach. Bacterial synthesis of the holoflavoprotein and of an active enzyme form lacking the first 28 amino acid residues of the sequence. 220 97

Combined deficiency of the pyruvate, alpha-ketoglutarate and branched-chain keto acid dehydrogenase complexes is a rare condition in which activity of lipoamide dehydrogenase is either reduced or grossly deficient. Activities in three cell strains from patients with excretion of branched chain ketoacids and alpha-ketoglutarate and lactic-acidemia showed decreased levels of the three alpha-ketoacid dehydrogenases. Lipoamide dehydrogenase activity was 5% of normal in one cell stain and 50-60% in the other two. Antiserum raised against lipoamide dehydrogenase was used to immunoprecipitate labelled lipoamide dehydrogenase from fibroblasts grown on [35S]methionine. After separation of cell proteins from control fibroblasts by sodium dodecyl sulphate/polyacrylamide gel electrophoresis and fluorography, a prominent 55 kilodalton band was evident in cell extracts treated with the antiserum which corresponded to lipoamide dehydrogenase. In the cell lines from patients with combined alpha-ketoacid dehydrogenase deficiency immunoprecipitation of lipoamide dehydrogenase showed that this protein was present in similar amounts to that seen in control cell lines and was also of the correct molecular weight.
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PMID:Immunoextraction of lipoamide dehydrogenase from cultured skin fibroblasts in patients with combined alpha-ketoacid dehydrogenase deficiency. 241 42

The subunit structures and conservation of the dihydrolipoyl transacylase (E2) components of bovine and human branched-chain alpha-keto acid dehydrogenase complexes were investigated by Western blotting, peptide sequencing, and cDNA cloning methods. Rabbit antiserum prepared against the sodium dodecyl sulfate (SDS) denaturated bovine E2 subunit recognized the inner E2 core, and the first hinge region of the E2 chain, but failed to react with the lipoyl-bearing domain as determined by Western blot analysis. The lack of antigenicity in the lipoyl-bearing domain was confirmed with antibodies directed against the native E2 component. A human E2 cDNA (1.6 kb) was isolated from a human liver cDNA library in lambda gt11 with a combination of the above anti-native and anti-SDS-denatured E2 immunoglobulin G's as a probe. The fidelity of the human E2 cDNA was established by nucleotide sequencing which showed the determined peptide sequences of the amino terminus and tryptic fragments of bovine E2. A bovine E2 cDNA (0.7 kb) was also isolated from a bovine liver cDNA library in lambda ZAP with the human E2 cDNA as a probe. Northern blot analysis using the human E2 cDNA probe showed that E2 mRNAs in bovine liver and human kidney mesangial cells are 3.3 and 4.6 kb in size, respectively. Primary structures derived from human and bovine E2 cDNAs show leader sequences including the initiator methionine and the homologous mature peptides consisting of complete lipoyl-bearing and dihydrolipoyl dehydrogenase (E3) binding domains and two hinge regions. In addition, the human E2 cDNA contains a portion of the inner E2 core sequence, a 3'-untranslated region, and a poly(A+) tail. Deduced amino acid sequences of the mammalian E2's were compared with those of Escherichia coli transacetylase and transsuccinylase and bovine kidney transacetylase. The results indicate a high degree of conservation in the sequence flanking the lipoyl-attachment site and in the E3-binding domain. Models are presented to discuss implications for the conserved structure-function relationship in the lipoyl-bearing and E3-binding domains of alpha-keto acid dehydrogenase complexes.
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PMID:Conservation of primary structure in the lipoyl-bearing and dihydrolipoyl dehydrogenase binding domains of mammalian branched-chain alpha-keto acid dehydrogenase complex: molecular cloning of human and bovine transacylase (E2) cDNAs. 283 77

The activity and turnover of dihydrolipoamide dehydrogenase (E3), the common component of the three 2-oxoacid dehydrogenase complexes, were measured during the differentiation of 3T3-L1 preadipocytes into 3T3-L1 adipocytes. The specific activity of E3 increased approx. 3-4-fold in 3T3-L1 adipocytes differentiated under a regimen of insulin, dexamethasone and 3-isobutyl-1-methylxanthine for 48 h, followed by insulin alone thereafter. A rabbit antibody to pig heart E3 quantitatively precipitated the enzyme from 3T3-L1 adipocytes. By using immunoprecipitation and gel electrophoresis, a 3.3-fold increase was observed in E3 protein in 3T3-L1 adipocytes as compared with 3T3-L1 preadipocytes, on a DNA basis. Pulse-labelling experiments with L-[35S]methionine revealed a 3.5-fold increase in the rate of synthesis of E3 in 3T3-L1 adipocytes compared with that observed in 3T3-L1 preadipocytes. In contrast, the apparent half-lives of the E3 in 3T3-L1 preadipocytes (43 h) and 3T3-L1 adipocytes (33 h) were not significantly different. Therefore, the 3-4-fold increase in the specific activity of E3 in 3T3-L1 adipocytes resulted from an increased rate of synthesis of the enzyme.
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PMID:Induction of dihydrolipoamide dehydrogenase in 3T3-L1 cells during differentiation. 335 2

An immunological analysis has been conducted of early events in the biosynthesis, import and assembly of the mammalian pyruvate dehydrogenase complex (PDC). For this purpose, monospecific polyclonal antisera were produced against the intact assembly from ox heart, Mr 8.5 x 10(6), and each of its component polypeptides, E1 alpha, E1 beta, E2, E3 and protein X. Optimal detergent-based incubation mixtures were developed for obtaining clean immunoprecipitation of PDC polypeptides and their precursors from [35S]methionine-labelled extracts of PK-15 (pig kidney), NBL-1 (bovine kidney) and BRL (Buffalo Rat liver) cells. In PK-15 cells, independent higher Mr species, corresponding to precursors of the E2, E1 alpha and E1 beta subunits of PDC, could be detected by immune precipitation and fluorography after incubation of intact cells for 4 h with [35S]methionine and 1-2 mM-2,4-dinitrophenol or 10-15 microM-carbonyl cyanide p-trifluoromethoxyphenylhydrazone. Similar precursor states could be observed in uncoupler-treated BRL or NBL-1 cells. Pre-E1 alpha, pre-E1 beta and also pre-E3, have signal sequences in the Mr range 1500-3000 while pre-E2 contains a long additional segment of Mr 7000-9000. All of these forms exhibit similar kinetics of processing to the mature subunits with a transit time of 10-12 min. In NBL-1 cells, E3 is present in the immune complexes formed with anti-PDC serum whereas this is not the case in PK-15 cells. Thus, there are significant variations in the affinity of lipoamide dehydrogenase (E3) for the E2 core structure in different species. Pre-E1 alpha accumulates only poorly in PK-15 cells and is aberrantly processed on removal of uncoupler. This precursor is markedly more stable in NBL-1 and BRL cells. The lack of detection of a precursor form of component X is also discussed.
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PMID:Biosynthesis, import and processing of precursor polypeptides of mammalian mitochondrial pyruvate dehydrogenase complex. 341 48

We have cloned and sequenced the mouse NMO1 cDNA, which encodes the NAD(P)H:menadione oxidoreductase [also called NAD(P)H:(quinone acceptor) oxidoreductase; quinone reductase; azo dye reductase; DT diaphorase; EC 1.6.99.2]. The cDNA is 1528 bp in length excluding the poly(A+) tail, and has 5' and 3' nontranslated regions of 108 bp and 595 bp, respectively. The deduced protein contains 274 amino acids, including the first methionine (M(r) = 30,959). The mouse NMO1 protein is: 94% similar to the rat NMO1 and 86.5% to the human NMO1 proteins; 49.3% identical to the human NQO2 protein; and < 20% similar to several dozen other proteins in the quinone oxidoreductase superfamily. Southern hybridization analysis of mouse DNA reveals that the Nmo1 gene is likely to span less than a total of 20 kb. The Nmo1 gene is highly inducible by 2,3,7,8,-tetrachlorodibenzo-p-dioxin (dioxin; TCDD) in mouse liver and mouse cell cultures. TCDD inducibility of NMO1 is detectable at 12 and 18 days of gestation, but markedly elevated at 1-3 weeks post partum as compared with the 6- and 12-week-old mouse. NMO1 mRNA levels are strikingly elevated in the untreated mouse hepatoma Hepa-1c1c7 mutant line c37 lacking CYP1A1 (aryl hydrocarbon hydroxylase) activity, and in the untreated 14CoS/14CoS mouse cell line having an 'oxidative stress response' caused by homozygous deletion of about 3800 kb on chromosome 7. Previous work and the data in this report show that the murine Nmo1 gene is regulated by three distinct mechanisms: CYP1A1 metabolism-dependent repression, Ah receptor-mediated induction by TCDD, and activation by the chromosome 7-mediated oxidative stress response.
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PMID:Mouse dioxin-inducible NAD(P)H: menadione oxidoreductase: NMO1 cDNA sequence and genetic differences in mRNA levels. 770 40

Both phenylbutazon and mofebutazon inhibit oxidative fragmentation of the methionine derivative, 2-keto-4-methylthio-butyric acid (KMB) by xanthine oxidase--or diaphorase mediated OH radical production. Differentiation of the two non-steroidal antiinflammatory drugs is possible by means of determining oxygen reduction by xanthine oxidase or diaphorase in the presence of the naphthoquinone, juglone, where only mofebutazon shows an inhibitory effect.
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PMID:Antioxidative properties of phenazone derivatives: differentiation between phenylbutazon and mofebutazon. 821 10


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