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)

The influence of Ebselen, an organoselenium anti-inflammatory agent, on the two electron transport chains present in rat liver microsomes has been studied. At low micromolar concentrations, Ebselen markedly inhibited the flow of reducing equivalents from NADPH-cytochrome P450 reductase to both its natural electron acceptor, cytochrome P450, and its artificial electron acceptor, cytochrome c. Similarly, the microsomal NADH-cytochrome c reductase system consisting of cytochrome b5 and its flavoprotein, NADH-cytochrome b5 reductase, was also significantly inhibited by Ebselen. The inhibition appears to be due to the inability of the reduced pyridine nucleotide to transfer electrons to the flavin (FAD and/or FMN) in the flavoprotein reductase. This was shown with the purified NADPH-cytochrome P450 reductase, which in the presence of Ebselen was not converted to the semiquinone form following the addition of NADPH. The addition of Ebselen to a suspension of hepatic microsomes from either untreated or phenobarbital-treated rats did not result in any spectral change characteristic of type I, type II, or reverse type I.
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PMID:Disruption of rat hepatic microsomal electron transport chains by the selenium-containing anti-inflammatory agent Ebselen. 291 42

A study of the steady-state kinetics of NADH(NADPH)-cytochrome c reductase (FMN-containing) from ale yeast (M. S. Johnson and S. A. Kuby (1985) J. Biol. Chem. 260, 12341-12350) has led to a postulated three-substrate random-ordered hybrid mechanism, where NAD(P)H and FMN add randomly and very likely in a steady-state fashion, followed by an ordered addition of cytochrome c. Kinetic parameters have been derived from this mechanism. Arrhenius plots showed large differences between NADH and NADPH, as the substrate-reductant. Menadione accelerated cytochrome c reduction and also O2 uptake, but vitamin K1 and coenzyme Q10 were ineffective as electron mediators, possibly as a result of their insolubility. With NADPH as the substrate-reductant, the order of the rate of reduction of electron acceptors was ferricyanide greater than DCIP greater than cytochrome c greater than oxygen; with menadione, the specificity sequence was cytochrome c greater than ferricyanide greater than DCIP greater than oxygen. With NADH, the order was ferricyanide greater than cytochrome c greater than oxygen greater than DCIP, which changed to cytochrome c greater than ferricyanide greater than oxygen greater than DCIP on addition of menadione. Cytochrome b5 was also reduced in the absence of oxygen. No transhydrogenase activity was observed, but the reduced thionicotinamide analogs of NADH and NADPH acted as substrates. Superoxide dismutase inhibited cytochrome c reduction in air by 50%, but O2-. was not necessary for cytochrome c reduction, as evidenced by the increase in rate in the absence of O2. The product of the reaction with oxygen appeared to be H2O2.
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PMID:Studies on NADH(NADPH)-cytochrome c reductase (FMN-containing) from yeast: steady-state kinetic properties of the flavoenzyme from top-fermenting ale yeast. 308 Sep 58

A unique cytochrome P-450-dependent fatty acid monooxygenase from Bacillus megaterium ATCC 14581 is strongly induced by phenobarbital (Narhi, L. O., and Fulco, A. J. (1982) J. Biol. Chem. 257, 2147-2150) and many other barbiturates (Kim, B.-H., and Fulco, A. J. (1983) Biochem. Biophys. Res. Commun. 116, 843-850). This monooxygenase has now been purified to homogeneity from pentobarbital-induced bacteria as a single polypeptide with a molecular weight of 119,000 +/- 5,000 daltons. In the presence of NADPH and O2, it can catalyze the oxygenation of long chain fatty acids without the aid of any other protein. The enzyme has a catalytic center activity of 4,600 nmol of fatty acid oxygenated per nmol of P-450 (the highest activity yet reported for a P-450-dependent monooxygenase) and also functions as a highly active cytochrome c reductase in the presence of NADPH. The purified holoenzyme is a soluble protein containing 40 mol % hydrophobic amino acid residues and 1 mol each of FAD and FMN/mol of heme. It is isolated and purified in the low spin form but is converted to the high spin form in the presence of long chain fatty acids. The enzyme, which catalyzes the omega-2 hydroxylation of saturated fatty acids and the hydroxylation and epoxidation of unsaturated fatty acids has its highest affinity (Km = 2 +/- 1 microM) for the C15 and C16 chain lengths.
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PMID:Characterization of a catalytically self-sufficient 119,000-dalton cytochrome P-450 monooxygenase induced by barbiturates in Bacillus megaterium. 308 9

The location of ferrochelatase in bovine heart mitochondria has been studied. When the mitochondria were fractionated into Complexes I, II and III, ferrochelatase activity was only found in Complex I. Complex I also showed heme synthesis from ferric ion in the presence of NADH as an electron donor. Immunoblot experiments confirmed the presence of ferrochelatase in Complex I, but not in Complexes II or III. Some phospholipids, including phosphatidylserine and cardiolipin, stimulated NADH-dependent heme synthesis from ferric ion. When purified ferrochelatase was incubated with the low molecular weight form of NADH dehydrogenase prepared from Complex I, heme synthesis from ferric ion occurred by the addition of NADH. FMN markedly elevated the synthesis. These results indicate that ferrous ion is produced by NADH oxidation in Complex I and is then utilized for heme synthesis by ferrochelatase.
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PMID:Association of ferrochelatase with Complex I in bovine heart mitochondria. 309 Oct 80

An NADH dehydrogenase complex was isolated from the plasma membranes of aerobically grown Paracoccus denitrificans cells by extraction with NaBr and purification on an NAD-agarose column. The NADH-ubiquinone-1 reductase activity of the isolated NADH dehydrogenase complex was about 10 times higher than that of the NaBr extract. The preparation was composed of 10 (6 major and 4 minor) unlike polypeptides, and lacked identifiable components and activities characteristic of other enzyme complexes of the oxidative phosphorylation system. The purified enzyme contained noncovalently bound FMN, nonheme iron, and acid-labile sulfide. The ratio of FMN to nonheme iron to acid-labile sulfide was 1:13 approximately 14:11 approximately 12, suggestive of the presence of multiple iron-sulfur clusters. The isolated NADH dehydrogenase complex cross-reacted with antisera to beef heart mitochondrial complex I and protein fraction derived therefrom, indicating the presence in the Paracoccus enzyme of antigenic sites similar to those in the intact complex I and its iron-sulfur protein and possibly hydrophobic protein fractions.
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PMID:Purification and characterization of NADH dehydrogenase complex from Paracoccus denitrificans. 309 11

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

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

An enzyme (NADPH-dependent diaphorase) present in rat brain microsomes has been solubilised and shown to utilise both nitrobluetetrazolium and cytochrome c as electron acceptors, when reduced by NADPH. The kinetics of the enzyme have been determined using cytochrome c (Km = 1.3 microM), NADPH (Km = 1.4 microM) and the Vmax (4.7 nmol/min/mg solubilised microsome protein). The subunit Mr is approximately 73,000 D and that of the native enzyme is 170,000-180,000 D, indicating that the enzyme is probably a dimer. Evidence is also provided to show that the enzyme is a flavoprotein, and that it has equimolar amounts of FAD and FMN with respect to the subunit concentration. It seems a possibility that the rat brain diaphorase enzyme may be cytochrome P450 reductase, EC 1.6.2.4.
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PMID:Rat brain NADPH-dependent diaphorase. A possible relationship to cytochrome P450 reductase. 313 10

Two types of the NADH-quinone reductase were isolated from Thermus thermophilus HB-8 membranes, by use of the nonionic detergent, dodecyl beta-maltoside, and NAD-agarose affinity, DEAE-cellulose, hydroxyapatite, and Superose 6 column chromatography. One of these (NADH dehydrogenase 1) is a complex composed of 10 unlike polypeptides, and the other (NADH dehydrogenase 2) exhibits a single band (Mr 53,000) upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The NADH-ubiquinone-1 reductase activity of the isolated NADH dehydrogenase 1 was about 14 times higher than that of the dodecyl beta-maltoside extract and partially rotenone sensitive. The NADH-ubiquinone-1 reductase activity of the isolated NADH dehydrogenase 2 was about 30-fold as high as that of the dodecyl beta-maltoside extract and rotenone insensitive. The purified NADH dehydrogenase 1 contained noncovalently bound FMN, non-heme iron, and acid-labile sulfide. The ratio of FMN to non-heme iron to acid-labile sulfide was 1:11-12:7-9. The high content of iron and labile sulfide is suggestive of the presence of several iron-sulfur clusters. The purified NADH dehydrogenase 2 contained noncovalently bound FAD and no non-heme iron or acid-labile sulfide. The activities of both NADH dehydrogenases were stable at temperatures of greater than or equal to 80 degrees C. The occurrence of two distinct types of NADH dehydrogenase as a common feature in the membranes of various aerobic bacteria is discussed.
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PMID:Purification and characterization of two types of NADH-quinone reductase from Thermus thermophilus HB-8. 337 42

Monospecific antibody to the respiratory NADH dehydrogenase from Paracoccus denitrificans was prepared by using as antigen specific immunoprecipitates containing NADH dehydrogenase which were excised from crossed-immunoelectrophoresis plates. The latter were run with selectively solubilized plasma membranes and antibodies against plasma membranes. The antibody immunoprecipitated NADH dehydrogenase from P. denitrificans membranes biosynthetically labelled with 14C and solubilized with a wide range of detergents. All immunoprecipitates contained the two subunits of Mr 48,000 and 25,000, in an approximate 1:1 stoichiometry, that had previously been assigned to NADH dehydrogenase. A polypeptide of Mr 46,000 in P. denitrificans membranes, previously shown to cross-react with a subunit-specific antibody to mitochondrial NADH dehydrogenase (complex I), was not detected in any immunoprecipitate. Under some conditions a third polypeptide, of Mr 31,000, was also detected, but in variable and non-stoichiometric amounts relative to the two other subunits. It was concluded that this polypeptide was incorporated into the immunoprecipitates as an artefact and that the polypeptides of Mr 48,000 and 25,000 are the sole polypeptides firmly identified in the NADH dehydrogenase. Flavoproteins were specifically radiolabelled by growth of P. denitrificans in the presence of [14C]riboflavin. Crossed immunoelectrophoresis of membranes from such cells showed that succinate dehydrogenase contained flavin, but that there was no detectable flavin in NADH dehydrogenase under these conditions. Analysis of excised immunoprecipitates of succinate dehydrogenase showed that flavin was covalently bound to a polypeptide of Mr 56,000. Flavin was retained by NADH dehydrogenase under mild conditions of detergent solubilization. Subsequent immunoprecipitation, followed by analysis of the acid-extracted flavin, established that FMN is a cofactor, in common with mitochondrial NADH-ubiquinone oxidoreductase (complex I).
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PMID:Immunochemical probing of the structure and cofactor of NADH dehydrogenase from Paracoccus denitrificans. 344 83


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