Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:Q8NEX9 (reductase)
 26,410 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

NADPH-cytochrome c (cytochrome P-450) reductase (EC 1.6.2.4) has been purified to homogeneity, as judged by sodium dodecyl sulfate disc gel electrophoresis, from detergent-solubilized rat and pig liver microsomes using an affinity chromatography procedure. Treatment of microsomes with a polyethoxynonylphenyl ether plus either cholate or deoxycholate and subsequent batch-wise DEAE-cellulose chromatography followed by biospecific affinity chromatography on Sepharose 4B-bound N6-(6-aminohexyl)-adenosine 2',5'-bisphosphate (2'5'-ADP-Sepharose 4B) result in a greater than 30% yield of purified reductase from microsomes. The enzyme contains 1 mol each of FAD and FMN and exhibits a molecular weight of 78,000 g mol-1 estimated by comparison with protein standards on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The turnover numbers calculated on the basis of flavin are 1360 min-1 and 1490 min-1 at 25 degrees for the pig and rat liver enzymes, respectively. Titration of these purified preparations aerobically with both NADPH and potassium ferricyanide demonstrated unequivocally that the air-stable, reduced form of NADPH-cytochrome c (P-450) reductase contains 2 electron equivalents, confirming recent results obtained by Masters et al. (Masters, B. S. S., Prough, R. A., and Kamin, H. (1975) Biochemistry 14, 607-613) for the proteolytically solubilized enzyme. In addition, these preparations are capable of reconstituting benzphetamine N-demethylation activity in the presence of partially purified cytochrome P-450 and dilauroylphosphatidylcholine, as measured by formaldehyde formation from benzphetamine.
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PMID:Some properties of a detergent-solubilized NADPH-cytochrome c(cytochrome P-450) reductase purified by biospecific affinity chromatography. 82 51

The liver microsomal drug-metabolizing enzyme system consists of two protein components, cytochrome P-450 and NADPH-cytochrome c reductase, and a lipid, phosphatidylcholine. Cytochrome P-450 serves as the binding site for oxygen and substrate while the reductase acts as an electron carrier shuttling electrons from NADPH to cytochrome P-450. The phospholipid facilitates the transfer of electrons from NADPH-cytochrome c reductase to cytochrome P-450 but itself is not an electron carrier. Different cytochromes P-450 and P-448 have been purified; the spectral, catalytic, and immunological properties as well as the molecular weight (determined by SDS-gel electrophoresis) of all these hemeproteins differ from one another. The presence of multiple cytochrome P-450s may explain the species, strain, age, tissue, and sex differences as well as the effect of inducers and nutritional status in mammlian drug metabolism.
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PMID:Liver microsomal drug-metabolizing enzyme system: functional components and their properties. 82 57

In order to examine effects of oral contraceptives (OC) on the microsomal drug matabolizing enzymes in a state of protein malnutrition, weanling Sprague-Dwayley female rats were fed diets containing 3.5% (low protein, LP), 26% (normal protein, NP) or 42% (high protein, HP) casein for 224 days and norethynodrel plus mestranol for the last 182 days. LP rats were smaller in body weight than NP and HP rats and the latter two groups showed depressed weight gain and hypertrophy of liver and kidney due to OC. Concentrations of microsomal proteins and cytochrome P-450 were lowered by LP diet and OC did not induce cytochrome P-450. Activity of biphenyl-4-hydroxylase was lowered in LP and HP rats compared to NP group in which activity of this enzyme was significantly decreased due to OC. The diets alone had no effect on the activity of p-nitrobenzoate reductase, however, its activity was enhanced by OC only in NP rats. LP diet caused reduction of 4-methylumbelliferone glucuronly transferase activity which was lowered by OC in NP as well as in LP rats. It is concluded that NP diet more than either LP or HP diet exposed rats to the modification of microsomal drug metabolizing enzymes by OC.
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PMID:Induction of microsomal drug metabolizing enzymes by oral contraceptives in protein-malnourished rats. 82 20

Three hr after i.p. administration of a single dose of 30 mg/kg of morphine to male mice, an increase in specific activity of NADPH-cytochrome c reductase by about 10% and the content of cytochrome P-450 by about 14% of their liver microsomes was observed.Administration of 30 mg/kg of morphine, once daily,during 5 days, caused about 16% and 9% increases in specific activity of c reductase and the content of P-450 respectively. Administration of a single dose of morphine to male and female mice caused no sex-dependent differences in the specific activity of c reductase and the content of P-450. Repeated administration of morphine up to 100 mg/kg to male mice increased the specific activity of microsomal c reductase by about 70%. Repeated administration of morphine up to 55 mg/kg also increased the microsomal content of P-450 by about 22%, but with higher doses of morphine, the content of P-450 declined and finally dropped below control levels. The levels of c-reductase activity and P-450 content returned to normal levels about 2 weeks after termination of morphine administration.
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PMID:Effects of morphine sulfate on NADPH-cytochrome c reductase and cytochrome P-450 of mouse liver microsomes. 82 27

Hepatic microsomes from phenobarbital-pretreated rats were treated with sodium cholate in the presence of glycerol and the solubilized proteins were absorbed on a column of 8-aminooctyl derivative of Sepharose 4B. Washing the column stepwise with buffers containing suitable detergents in addition to cholate resulted in the elution of cytochrome P-450, NADH-cytochrome b5 reductase [EC 1.6.2.2], NADPH-cytochrome c reductase [EC 1.6.2.4], and cytochrome b5; the former two enzymes were eluted with Emulgen 913 (a polyoxyethylene nonylphenyl ether), and then the latter two were obtained separately by increasing the concentration of added deoxycholate. Thus, cytochrome P-450, NADPH-cytochrome c reductase, and cytochrome b5 were purified to specific contents (or activities) of 9--10 nmoles per mg of protein, 15-16 units per mg of protein, and nearly 35 nmoles per mg of protein, respectively (about 40--50% pure), in 40--50% yields. Practically no mutual contamination of the three enzymes was detected. Benzphetamine N-demethylation activity could be reconstituted on mixing the cytochrome P-450 and NADPH-cytochrome c reductase preparations in the presence of cholate. NADH-cytochrome C reductase activity could be observed at high efficiency under appropriate conditions in a system containing the cytochrome b5 and NADH-cytochrome b5 reductase preparations. Various derivatives of Sepharose 4B were compared for effectiveness in the separation of the microsomal election transfer enzymes.
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PMID:The use of 8-aminooctyl sepharose for the separation of some components of the hepatic microsomal electron transfer system. 82 21

The report by Schacter et al. (J Biol Chem 247: 3601, 1972) that an antibody to NADPH-cytochrome c oxidoreductase inhibited NADPH-cytochrome c reductase and heme oxygenase activities in rat and pig liver and spleen microsomes demonstrated the role of this flavoprotein in microsomal heme oxygenation. Recent studies from other laboratories (Yoshida et al., J Biochem 75, 1187: 1974 and Bissell et al., Fed Proc 33: 1246, 1974) have strongly suggested that cytochrome P-450 is not involved in heme oxygenation. The availability of a homogeneous preparation of NADPH-cytochrome c reductase prompted us to test heme oxygenase activity in a system devoid of hemoprotein contamination. NADPH-cytochrome c reductase catalyzed biliverdin formation at a rate of 8.26 +/- 0.5 SEM nmole min-1mg-1 in the absence of biliverdin reductase. The rate of bilirubin formation in the presence of biliverdin reductase was less than 10% of the rate of biliverdin formation, suggesting that mixture of biliverdin isomers may be produced. Biliverdin production was potently (70--80%) inhibited by catalase, but was unaffected by superoxide dismutase. Epinephrine also inhibited heme oxygenation, presumably by utilizing O2. required for the formation of H2O2 by the reductase. By extrapolation, the NADPH oxidase activity due to NADPH-cytochrome c reductase can account for heme degradation occurring in microsomes. However, the specificity of ring scission at the IXalpha position must be due to another microsomal protein, perhaps the heme oxygenase of Yoshida et al., and not cytochrome P-450.
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PMID:The catalysis of heme degradation by purified NADPH-cytochrome C reductase in the absence of other microsomal proteins. 82 31

Solubilized cytochromes P-450 and P-448 have been prepared from liver microsomes of phenobarbital- and 3-methylcholanthrene-pretreated rats, respectively. These hemoproteins can bind to microsomes and increase the microsomal monoxygenase activities. The binding of cytochrome P-450 enhances the microsomal benzphetamine demethylase activity, whereas cytochrome P-448 enhances the ethoxycoumarin dealkylase and benzo[a]pyrene hydroxylase activities. The added cytochrome P-450 is believed to be incorporated into the microsomal membrane, and the enriched microsomes can be separated from the free hemoprotein by gel filtration. A correlation between the increased cytochrome P-450 content and the enhanced catalytic activity of the microsomes is shown. Several lines of evidence suggest that the exogenous cytochrome P-450 molecules become catalytically active only when they are incorporated into the membrane. By measuring the enhanced ethoxycoumarin dealkylase activity, the rate of the proposed incorporation of cytochrome P-448 into microsomes can be measured, and the temperature dependence of the rate is reported. The addition of cytochromes P-448 and P-450 causes a great increase in the monoxygenase activities of microsomes which have been treated with linoleic acid hydroperoxide. The hydroperoxide treatment denatures almost all the cytochrome P-450 molecules in the microsomes but retains most of the NADPH-cytochrome P-450 reductase activity. Experiments with such microsomes indicate that the added cytochrome P-450 molecules, after incorporation into the membrane, have a direct access to the reductase molecules and are able to receive electrons directly from the latter. The present results are consistent with a nonrigid model for the organization of cytochrome P-450 and NADPH-cytochrome P-450 reductase in the microsomal membrane.
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PMID:Interactions between solubilized cytochrome P-450 and hepatic microsomes. 83 22

Rat liver endoplasmic reticulum has been separated into four ribosome-containing subfractions, two from rapidly sedimentation endoplasmic reticulum and two from the microsomes, by differential centrifugation and sucrose density centrifugation. Ribosomes from one of the rapidly sedimenting subfractions were extracted by Trion X-100 as a complex with cytochrome P-450, optimally at a detergent protein ratio of 2/1 (w/w). Upon extraction approximately 50% of the cytochrome P-450 in the membrane appeared complex-bound to ribosomes, and, maximally, 6-7 subunit molecules of the cytochrome were attached per ribosome. The specific concentration of cytochrome P-450 on these ribosomes was 2.5-times higher than in the parent membrane. Cytochrome b5, glucose-6-phosphatase, NADPH-cytochrome c reductase, NADH-ferricyanide reductase, cytochrome oxidase and phospholipids were present in small or trace amounts on the ribosomes in relation to cytochrome P-450. Ribosomes extracted from other subfractions contained much less bound cytochrome P-450. Phenobarbital treatment induced an increase in the cytochrome P-450 content that was different for the various subfractions. This increase could not be correlated with changes in the amounts of cytochrome-ribosome complexes released by detergent. We propose that cytochrome P-450 is part of a specific binding site in the membrane for a fraction of the ribosomes attached to the endoplasmic reticulum. The ribosomes may be anchored to cytochrome P-450 via nascent chain proteins.
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PMID:On the involvement of cytochrome P-450 in the binding of ribosomes to a subfraction of rat-liver rapidly sedimenting endoplasmic reticulum. 83 30

Human hemoglobin was characterized as an enzyme in a reconstituted aniline hydroxylase system containing hemoglobin, NADPH, rat liver cytochrome P-450 reductase, aniline and atmospheric O2. This system catalyzed p-aminophenol formation (turnover number 0.2 mol/min/mol of hemoglobin) with an efficiency similar to that which has been reported for either microsomal cytochrome P-450 or cytochrome P-450 solubilized from rat liver. The rate of the reaction was linearly dependent on hemoglobin concentration up to approximately 1 nmol of hemoglobin/ml. This linear range of hemoenzyme concentration is also similar to cytochrome P-450-catalyzed reactions. Unlike the cytochrome P-450 system, the hemoglobin system did not require a lipid cofactor for maximal activity, and much less reductase was needed for maximal activity. Aniline displayed typical Michaelis-Menten saturation kinetics as substrate, and its Km (8 mM) was the same in the absence of presence of the reductase. Catalase essentially completely inhibited p-aminophenol formation in the absence or presence of reductase. In contrast, superoxide dismutase inhibited the reductase-mediated reaction only to a small extent (if at all). No detectable hydrogen peroxide accumulated during the course of the reaction in the absence of catalase. These findings suggested a hypothetical mechanism for hemoglobin-catalyzed hydroxylation of aniline involving a hemoglobin-bound form of hydrogen peroxide (aniline-Hb3+-OOH-) as an intermediate preceding the rate-determining formation of products.
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PMID:Characterization of Enzyme-like activity of human hemoglobin. Properties of the hemoglobin-P-450 reductase-coupled aniline hydroxylase system. 93 94

25- and 26-Hydroxylation of 5beta-cholestane-3alpha, 7alpha, 12alpha-triol was studied with reconstituted systems from rat liver microsomes consisting of partially purified cytochrome P -450, NADPH-cytochrome P -450 reductase, a phospholipid, and an NADPH -generating system. Cytochrome P -450 was prepared either by sodium cholate treatment and ammonium sulfate fractionation or by subtilisin and sodium deoxycholate treatment followed by DEAE-cellulose chromatography. No side chain hydroxylation was observed when cytochrome P-450 was omitted. With ammonium sulfate-fractionated cytochrome P-450 25- and 26-hydroxylation was stimulated 5- to 8-fold by addition of NADPH-cytochrome P-450 reductase. With subtilisin-treated cytochrome P-450 an almost absolute requirement for NADPH-cytochrome P-450 reductase was observed. Omission of lipid did not reduce the rate of hydroxylation. Centrifugation of the cytochrome P-450 preparation at 100,000 X g for 1 hour just before incubation increased markedly lipid dependency. A significant difference between 25- and 26-hydroxylation was observed with respect to substrate saturation. The stimulatory effect of phenobarbital treatment on 25-hydroxylation and the inhibitory effect of this treatment on 26-hydroxylation were associated with the cytochrome P-450 fraction. The use of increasing amounts of sodium cholate in the solubilization of cytochrome P -450 resulted in a gradual decrease of 25-hydroxylase activity and a gradual increase of 26-hydroxylase activity. 25- and 26-Hydroxylase activities were separated partially by chromatography of subtilisintreated cytochrome P-450 fraction on DEAE-cellulose. The question whether different species of cytochrome P-450 are involved in 25- and 26-hydroxylation is discussed.
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PMID:Side chain hydroxylations in biosynthesis of cholic acid. 25- and 26-Hydroxylation of 5beta-cholestane-3alpha, 7alpha, 12alpha-triol by reconstituted systems from rat liver microsomes. 93 95


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