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:1.6.99.3 (diaphorase)
5,903 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A simple procedure for preparation of highly purified soluble succinate-ubiquinone reductase from bovine heart mitochondrial particles is described. The enzyme exhibits four major bands on sodium dodecyl sulfate gel electrophoresis and contains (nmol per mg protein): covalently bound flavin, 6; non-heme iron, 53; acid-labile sulfur, 50; cytochrome b-560 heme, 1.2. The enzyme catalyzes thenoyltrifluoroacetone, or carboxin-sensitive (pure non-competitive with Q2) reduction of Q2 by succinate with a turnover number close to that in parent submitochondrial particles. The succinate reduced enzyme exhibits ferredoxin-type iron-sulfur center EPR-signal (g = 1.94 species) and a semiquinone signal (g = 2.00). An oxidized preparation shows a symmetric signal centered around g = 2.01. An unusual dissociation of the enzyme in the absence of a detergent is described. When added to the assay mixture from a concentrated protein-detergent solution, the enzyme does not reduce Q2 being highly reactive towards ferricyanide ('low Km ferricyanide reactive site'; Vinogradov, A.D., Gavrikova, E.V. and Goloveshkina, V.G. (1975) Biochem. Biophys. Res. Commun. 65, 1264-1269). The ubiquinone reductase, not the ferricyanide reductase was observed when the enzyme was added to the assay mixture from the diluted protein-detergent solutions. Thus the dissociation of succinate dehydrogenase from the complex occurs in the absence of a detergent dependent on the concentration of the protein-detergent complex in the stock preparation where the samples for the assay are taken from. An active antimycin-sensitive succinate-cytochrome c reductase was reconstituted by admixing of the soluble succinate-ubiquinone reductase and the cytochrome b-c1 complex, i.e., from the complexes which both contain the ubiquinone reactivity conferring protein (QPs). Cytochrome c reductase was also reconstituted from the succinate-ubiquinone reductase and succinate-cytochrome c reductase containing inactivated succinate dehydrogenase. The reconstitution experiments suggest that there exists a specific protein-protein (or lipid) interaction between QPs and a certain component(s) of the b-c1 complex.
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PMID:Studies on the succinate dehydrogenating system. Isolation and properties of the mitochondrial succinate-ubiquinone reductase. 299 19

A simple, rapid procedure suitable for large scale preparation of a lipid deficient cytochrome b6-f complex from spinach chloroplasts has been developed. The procedure involves solubilization with a mixture of sodium cholate and octylglucoside, ammonium sulfate fractionation and calcium phosphate column chromatography. The purified complex contains, in nanomoles per milligram protein, 20.6 cytochrome b, 10.8 cytochrome f and 54 phospholipids. The purified complex has little plastoquinol-cytochrome c reductase activity in the absence of added lipid. Full reductase activity was reconstituted by the addition of plastoquinone prior to the addition of lipid.
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PMID:Preparation and reconstitution of a phospholipid deficient cytochrome b6-f complex from spinach chloroplasts. 299 23

A membrane-associated O2-.-generating oxidase has been purified from activated bovine polymorphonuclear neutrophils (PMN). The oxidase was extracted with Triton X-100 from a PMN membrane fraction largely devoid of lysosomal granules. The Triton extract was purified by a series of steps, including ion-exchange chromatography on DE-52 cellulose, gel filtration on Sephadex G-200, and isoelectric focusing. The O2-.-generating oxidase activity was assayed as a superoxide dismutase inhibitable cytochrome c reductase. The activity of the purified enzyme was strictly dependent on NADPH as electron donor. The purification factor with respect to the phorbol myristate acetate activated PMN was 75, and the recovery was about 6%. The reactivity of the purified oxidase was increased by 3-4-fold after incubation with asolectin. The minimum molecular weight of the oxidase, deduced from migration in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was 65 000 +/- 3000. The optimum pH of the oxidase was 7.5, its KM,NADPH was congruent to 30 microM, and its isoelectric point was at pH 5.0. The enzyme was inhibited by low concentrations of mersalyl (half-inhibition congruent to 10 microM) and Cibacron Blue (half-inhibition less than 10 microM). It was insensitive to 1 mM cyanide. Rapid loss of activity occurred at 0-2 degrees C, concomitantly with a decrease in sensitivity to superoxide dismutase: both activity and sensitivity to superoxide dismutase could be restored by addition of asolectin. The purified oxidase contained no spectrophotometrically detectable cytochrome b, and enzymatic assay failed to detect FAD in oxidase preparations subjected to heat treatment or trypsin digestion.
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PMID:Purification and properties of an O2-.-generating oxidase from bovine polymorphonuclear neutrophils. 300 51

An azido-ubiquinone derivative, 3-azido-2-methyl-5-methoxy-6-(3,7-dimethyloctyl)-1,4-benzoquinone, was used to study the ubiquinone-protein interaction and to identify the ubiquinone-binding proteins in yeast mitochondrial ubiquinone-cytochrome c reductase. The phospholipids and Q6 in purified reductase were removed by repeated ammonium sulfate precipitation in the presence of 0.5% sodium cholate. The resulting phospholipid- and ubiquinone-depleted reductase shows no enzymatic activity; activity can be completely restored by the addition of phospholipids and Q6 or Q2. The ubiquinone- and phospholipid-replenished ubiquinonol-cytochrome c reductase is also fully active upon reconstituting with bovine succinate-ubiquinone reductase to form succinate-cytochrome c reductase. When an azido-ubiquinone derivative was added to the ubiquinone and phospholipid-depleted reductase in the dark, followed by the addition of phospholipids, partial reconstitutive activity was restored, while full ubiquinol-cytochrome c reductase activity was observed when Q2H2 was used as substrate in the assay mixture. Apparently, the large amount of Q2H2 present in the assay mixture displaces the azido-ubiquinone in the system. Photolysis of the azido-Q-treated reductase with long-wavelength ultraviolet light abolishes about 70% of both the restored reconstitutive activity and Q2H2-cytochrome c reductase activity. The activity loss is directly proportional to the covalent binding of [3H]azido-ubiquinone to the reductase protein. When the photolyzed, [3H]azido-ubiquinone-treated sample was subjected to SDS-polyacrylamide gel electrophoresis followed by analysis of the distribution of radioactivity among the subunits, the cytochrome b protein and a protein with an apparent molecular weight of 14 000 were heavily labeled. The amount of radioactive labeling in both these proteins was affected by the presence of phospholipids.
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PMID:Identification of ubiquinone-binding proteins in yeast mitochondrial ubiquinol-cytochrome c reductase using an azido-ubiquinone derivative. 300 77

Exposure of antimycin-treated Complex III (ubiquinol-cytochrome c reductase) purified from bovine heart mitochondria to [3H]succinic anhydride plus [35S]p-diazobenzenesulfonate (DABS) resulted in somewhat uniform relative labeling of the eight measured subunits of the complex by [3H]succinic anhydride. In contrast, relative labeling by [35S]DABS was similar to [3H]succinic anhydride for the subunits of high molecular mass, i.e., core proteins, cytochromes, and the iron-sulfur protein, but greatly reduced for the polypeptides of molecular mass below 15 kDa. With Complex II depleted in the iron-sulfur protein the relative labeling of core protein I by exposure of the complex to [3H]succinic anhydride was significantly enhanced, whereas labeling of the polypeptides represented by SDS-PAGE bands 7 and 8 was significantly inhibited. Dual labeling of the subunits of Complex III by 14C- and 3H-labeled succinic anhydride before and after dissociation of the complex by sodium dodecyl sulfate, respectively, was measured with the complex in its oxidized, reduced, and antimycin-inhibited states. Subunits observed to be most accessible or reactive to succinic anhydride were core protein II, the iron-sulfur protein, and polypeptides of SDS-PAGE bands 7,8, and 9. Two additional polypeptides of molecular masses 23 and 12kDa, not normally resolved by gel-electrophoresis, were detected. Reduction of the complex resulted in a significant change of 14C/3H labeling ratio of core protein only, whereas treatment of the complex with antimycin resulted in decreases in 14C/3H labeling ratios of core proteins I and II, cytochrome c1, and a polypeptide of molecular mass 13kDa identified as an antimycin-binding protein.
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PMID:Differential labeling of the subunits of respiratory complex III with [3H]succinic anhydride, [14C]succinic anhydride, and p-diazobenzene-[35S]sulfonate. 300 48

The functional role and topographical orientation in the inner membrane of subunit VII, the ubiquinone-binding protein, of the cytochrome b-c1 complex of yeast mitochondria has been investigated. The apparent molecular weight of this subunit on sodium dodecyl sulfate-urea gels was calculated to be 15,500, while its amino acid composition was similar to that of the Q-binding proteins present in the cytochrome b-c1 complexes isolated from both beef heart and yeast mitochondria. The specific antibody obtained against subunit VII inhibited 30-47% of the ubiquinol-cytochrome c reductase activity in the isolated cytochrome b-c1 complex and in submitochondrial particles but had no effect on cytochrome c reductase activity in mitoplasts, mitochondria from which the outer membrane has been removed. Furthermore, the antibody against subunit VII strongly inhibited (74%) the reduction of cytochrome b by succinate in the presence of antimycin, an inhibitor of center i, but had no effect on cytochrome b reduction in the presence of myxothiazol, an inhibitor of center o. These results suggest that subunit VII, the Q-binding protein, is involved in electron transport at center o of the cytochrome b-c1 complex of the respiratory chain and that subunit VII is localized facing the matrix side of the inner mitochondrial membrane.
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PMID:Subunit VII, the ubiquinone-binding protein, of the cytochrome b-c1 complex of yeast mitochondria is involved in electron transport at center o and faces the matrix side of the membrane. 303 32

A method for the large-scale isolation of beef heart mitochondrial cytochrome c1 in high purity was developed. This method gave higher yield of "one-band" cytochrome c1 than previously reported [Kim, C. H., & King, T. E. (1981) Biochem. Biophys. Res. Commun. 102, 607-614]. In addition, the present method was effective in the preparation of "two-band" cytochrome c1 which was used to prepare the hinge protein according to the principle of sequential resolution [Kim, C. H., & King, T. E. (1983) J. Biol. Chem. 258, 13543-13551]. The isolation of one-band and two-band cytochrome c1 by this procedure could be completed within 3 or 4 days starting with succinate-cytochrome c reductase. One-band cytochrome c1 showed a molecular weight of 44,000 by sedimentation equilibrium and 29,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The disparities in these data from the actual value of 27,924 by amino acid sequence analysis, as previously reported [Wakabayashi, S., Matsubara, H., Kim, C. H., & King, T. E. (1982) J. Biol. Chem. 257, 9335-9344], are most probably due to the formation of detergent or detergent-phosphate complex. A comparison of some properties of one-band cytochrome c1 with those of two-band cytochrome c1 clearly showed significant differences between the two preparations. These results suggest the hypothesis that one of the possible roles of the hinge protein in the mitochondrial respiratory chain is to stabilize the conformation of cytochrome c1.
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PMID:Preparation and properties of cardiac cytochrome c1. 303 14

A cDNA containing the complete coding nucleotide sequence for rat liver NADPH-cytochrome P-450 oxidoreductase was constructed from two overlapping cDNA clones. This full-length cDNA was inserted into the plasmid expression vector pCQV2, transfected into Escherichia coli, and expressed reductase was identified in cell lysates by electrophoresis followed by electrophoretic transfer to nitrocellulose and immunodetection. Various strains were screened for maximal expression and minimal intracellular degradation of the expressed protein, and strain C-1A was selected for preparation of the expressed enzyme. Induced cells from 12-liter cultures were pelleted, lysed in a French press, and the 50,000g supernate was fractionated by DEAE-cellulose and 2'5'-ADP agarose chromatography. Thirty-five grams of packed cells yielded approximately 2 mg of affinity-purified protein that was essentially free of E. coli proteins. The final preparation exhibited considerable proteolytic degradation and only an estimated 5-10% of the immunoreactive protein was undegraded. Four principal forms could be distinguished upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis, with molecular weights of 65,000, 66,000, 74,000, and 78,000, the latter being equivalent to that of intact reductase. High-performance liquid chromatography with a Spherogel-DEAE column resolved these forms but resulted in the loss of the 78-kDa form; three peaks eluted with molecular weights of 65,000. Several of the HPLC fractions exhibited cytochrome c reductase activity, indicating correct incorporation of both flavin prosthetic groups, with the 66-kDa form showing the highest specific activity (44 mumol of cytochrome c reduced/mg reductase/min at 22 degrees C). HPLC assay of flavin content demonstrated equimolar FMN and FAD concentrations, and spectrophotometric analysis of the 66-kDa form revealed a spectrum essentially identical to that of reductase purified from rat liver. When the affinity-purified preparation was reconstituted with cytochrome P-450c, rates of benzo[a]pyrene metabolism approaching rates observed with liver reductase were obtained, indicating that the undegraded component in the affinity-purified preparation was able to interact with cytochrome P-450 and catalyze electron transfer from NADPH.
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PMID:Expression of a functional 78,000 dalton mammalian flavoprotein, NADPH-cytochrome P-450 oxidoreductase, in Escherichia coli. 310 72

Ferredoxin-NADP reductase from Euglena gracilis Klebs var. Bacillaris Cori purified to apparent homogeneity, yields a typical 36 kDa and an unusual 15 kDa polypeptide on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, exhibits a typical flavoprotein spectrum, contains FAD, and catalyzes NADPH-dependent iodonitrotetrazolium-violet diaphorase, NADPH-specific ferredoxin-dependent cytochrome-c-550 reductase and NADPH-NAD transhydrogenase activities. Rabbit antibody to the purified FNR blocks these activities specifically and also blocks the iodonitrotetrazolium-violet diaphorase activity of Euglena chloroplast completely. The low iodonitrotetrazolium-violet diaphorase activity in the plastidless mutant, W10BSmL, is mitochondrial and is not specifically blocked by the ferredoxin-NADP reductase antibody. Dark-grown non-dividing (resting) wild-type Euglena cells show a 4-fold increase in ferredoxin-NADP reductase activity during greening at 970 lx. Half of the low ferredoxin-NADP reductase activity in dark-grown cells is initially soluble, but by the end of chloroplast development nearly all of the enzyme is membrane-bound. The binding of ferredoxin-NADP reductase on exposure to light correlates with the extent of thylakoid membrane formation. Immunoblots of wild-type extracts during greening indicate that the 15 kDa polypeptide increases in the same manner as the extent of reductase binding to thylakoid membranes.
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PMID:Purification, properties, and cellular localization of Euglena ferredoxin-NADP reductase. 312 Jul 72

NADPH-cytochrome c reductase [NADPH: ferricytochrome oxidoreductase, EC 1.6.2.4] was highly purified from the membrane fraction of porcine polymorphonuclear leukocytes by column chromatographies on DEAE cellulose DE-52, 2',5'-ADP-agarose, Sephacryl S-300, and Bio-gel HTP. Upon sodium dodecyl sulfate polyacrylamide gel electrophoresis, the purified preparation gave a main band with a molecular weight of 80,000. The enzyme contained 0.79 mol of FAD and 0.88 mol of FMN per mol, and was capable of exhibiting a benzphetamine N-demethylation activity in the presence of cytochrome P-450 purified from rabbit liver microsomes and dilauroylphosphatidylcholine, as is the case with liver NADPH-cytochrome P-450 reductase. The cytochrome c reductase activity of the polymorphonuclear leukocytes (PMN) enzyme was precipitated with rabbit anti-guinea pig liver NADPH-cytochrome P-450 reductase IgG followed by addition of guinea pig anti-rabbit IgG antibody. The biochemical and immunological properties of the PMN enzyme so far examined were similar to those of the liver enzyme, although its function in leukocytes has not yet been determined.
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PMID:Purification and characterization of NADPH-cytochrome c reductase from porcine polymorphonuclear leukocytes. 312 59


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