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)

1. The role of cytochrome P-450 in the one-electron reduction of mitomycin c was studied in rat hepatic microsomal systems and in reconstituted systems of purified cytochrome P-450. Formation of H2O2 from redox cycling of the reduced mitomycin c in the presence of O2 and the alkylation of p-nitrobenzylpyridine (NBP) in the absence of O2 were taken as parameters. 2. With liver microsomes from both 3-methylcholanthrene (MC)- and phenobarbital (PB)-pretreated rats, reverse type I difference spectra were observed, indicative of a weak interaction between mitomycin c and the substrate binding site of cytochrome P-450. Mitomycin c inhibited the oxidative dealkylation of aminopyrine and ethoxyresorufin in both microsomal systems. 3. Under aerobic conditions the H2O2 production in the microsomal systems was dependent on NADPH, O2 and mitomycin c, and was inhibited by the cytochrome P-450 inhibitors, metyrapone and SKF-525A. 4. Although purified NADPH-cytochrome P-450 reductase was also effective in reduction of mitomycin c and the concomitant reduction of O2, complete microsomal systems and fully reconstituted systems of cytochrome P-450b or P-450c and the reductase were much more efficient. 5. Under anaerobic conditions in the microsomal systems both reduction of mitomycin c (measured as the rate of substrate disappearance) and the reductive alkylation of NBP were dependent on cytochrome P-450. 6. The relative rate of reduction of mitomycin c by purified NADPH-cytochrome P-450 reductase was lower than that by a complete microsomal system containing both cytochrome P-450 and a similar amount of NADPH-cytochrome P-450 reductase. 7. It is concluded that although NADPH-cytochrome P-450 reductase is active in the one-electron reduction of mitomycin c, the actual metabolic locus for the reduction of this compound in liver microsomes under a relatively low O2 tension is more likely the haem site of cytochrome P-450.
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PMID:One-electron reduction of mitomycin c by rat liver: role of cytochrome P-450 and NADPH-cytochrome P-450 reductase. 212 7

Azo dyes are reduced to primary amines by the microsomal enzymes NADPH-cytochrome P450 reductase and cytochrome P450. Amaranth, a highly polar dye, is reduced almost exclusively by rat liver microsomal cytochrome P450 and the reaction is inhibited almost totally by oxygen or CO. Activity is induced by pretreatment with phenobarbital or 3-methylcholanthrene. In contrast, microsomal reduction of the hepatocarcinogen dimethylaminoazobenzene (DAB), a lipid soluble, weakly polar compound, is insensitive to both oxygen and CO. However, reconstitution of activity with purified NADPH-cytochrome P450 reductase and a partially purified cytochrome P450 preparation indicates that activity is catalyzed almost exclusively by cytochrome P450. Activity is induced by clofibrate but not phenobarbital, beta-naphthoflavone, 3-methylcholanthrene, isosafrol, or pregnenolone-16 alpha-carbonitrile. These observations suggest the existence of at least two classes of azoreductase activity catalyzed by cytochrome P450. To investigate this possibility, the reduction of a number of azo dyes was investigated using microsomal and partially purified systems and the characteristics of the reactions were observed. Microsomal reduction of azo dyes structurally related to DAB required a polar electron-donating substituent on one ring. Activity was insensitive to oxygen and CO if the substrates had no additional substituents on either ring or contained only electron-donating substituents. Introduction of an electron-withdrawing group into the prime ring conferred oxygen and CO sensitivity on the reaction. Substrates in the former group are referred to as insensitive and substrates in the latter group as sensitive. Inhibitors of cytochrome P450 activity depressed reduction of both insensitive and sensitive substrates. In a fully reconstituted system containing lipid, highly purified NADPH-cytochrome P450 reductase and a partially purified cytochrome P450 preparation, rates of reduction of various insensitive substrates varied several-fold, whereas rates of reduction of sensitive substrates varied by three orders of magnitude. Using purified enzymes, each of the insensitive substrates was shown to be reduced by reductase alone, but only at a fraction of the rate seen in the fully reconstituted system, implying that reducing electrons were transferred to the dyes mainly from cytochrome P450. Conversely, there was substantial, in some cases almost exclusive, reduction of sensitive substrates by purified reductase alone and almost no inhibition by CO. Their reduction, however, was inhibited by CO in microsomal systems.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Characteristics of two classes of azo dye reductase activity associated with rat liver microsomal cytochrome P450. 212 21

Recombinant plasmids for expression of bovine cytochrome P450c21 (pA gamma 2), both P450c21 and yeast NADPH-cytochrome P450 reductase (pAR gamma 1), P450c21/yeast reductase fused enzymes (pAF gamma R1, pAF gamma R2, and pAF gamma R20), and yeast reductase/P450c21 fused enzymes (pAFR gamma 1 and pAFR gamma 2) were constructed by using expression vector pAAH5. The plasmids were each introduced into the yeast Saccharomyces cerevisiae AH22 cells. The recombinant yeast strains AH22/pA gamma 2 (Y21) and AH22/pAR gamma 1 (Y21R) produced 2-3 X 10(3) molecules of P450c21 per cell. The cultures of both strains converted progesterone and 17 alpha-hydroxyprogesterone into 11-deoxycorticosterone and 11-deoxycortisol, respectively. The 21-hydroxylase activity per cell of the strain Y21R was about three times higher than that of the strain Y21, probably due to overproduction of yeast reductase. The recombinant yeast strains AH22/pAF gamma R1 (Y21RF1), AH22/pAF gamma R2 (Y21RF2), and AH22/pAF gamma R20 (Y21RF20) produced about 1.1-2.0 X 10(4) molecules per cell of the corresponding P450c21/yeast reductase fused enzymes. The specific 21-hydroxylase activity toward 17 alpha-hydroxyprogesterone per cell of the strains Y21RF1, Y21RF2, and Y21RF20 was about 21, 28, and 49 times higher than that of the strain Y21, respectively. Thus, the fused enzymes were superior to P450c21 in the specific activity and in the expression level in the yeast. The Km values for 17 alpha-hydroxyprogesterone of P450c21 in the strains Y21 and Y21R, and of the fused enzymes in the strains Y21RF1 and Y21RF2 were 0.29, 0.30, 0.67, and 0.65 microM, respectively. The Vmax values of P450c21 in the strains Y21 and Y21R, and of the fused enzymes in the strains Y21RF1 and Y21RF2 were 28, 124, 151, and 222 moles/min.mole P450c21 or fused enzyme, respectively. These results indicated that the fused enzymes showed lower affinity for the substrate, probably due to structural modification and higher reaction rates through efficient intramolecular electron transfer as compared with those of P450c21. While the strain AH22/pAFR gamma 2 (YR21F2) produced about 3 X 10(4) molecules per cell of the reductase/P450c21 fused enzyme, the specific 21-hydroxylase activity of the fused enzyme toward 17 alpha-hydroxyprogesterone was extremely low, suggesting that the structure of the fused enzyme might not be suited for electron transfer in yeast microsomes.
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PMID:Expression of bovine cytochrome P450c21 and its fused enzymes with yeast NADPH-cytochrome P450 reductase in Saccharomyces cerevisiae. 212 25

The NADPH-cytochrome P450 reductase gene isolated from the yeast Saccharomyces cerevisiae [Yabusaki et al., J. Biochem. 103, 1004-1010 (1988)] was expressed on a multi-copy plasmid in the yeast. The transformed yeast cells with the recombinant plasmid carrying the reductase gene with a length of 3 kb produced the corresponding mRNA read from the original transcription initiation site under the control of its own promoter with a maximum length of 300 bp. The reductase content in the transformed cells was 25 times higher than that of the endogenous reductase. When the coding region for the reductase was placed between the alcohol dehydrogenase I gene promoter and the terminator of the expression vector pAAH5, the expression level was 32 times higher than at the endogenous level. These recombinant yeast strains showed enhanced cytochrome c reductase activity with increased cellular reductase levels. A simultaneous expression of yeast P450 reductase with rat P450c or bovine P450(17 alpha) resulted in 25 times or a 5 times increase in the corresponding P450-dependent monooxygenase activity of the recombinant yeast strains, respectively, as compared with that of the yeast cells expressing the corresponding P450 species. These results suggested that the overproduction of yeast P450 reductase with a simultaneous expression of the mammalian P450 species enhanced the P450c- and P450(17 alpha)-dependent monooxygenase activities in the recombinant yeast strains, probably due to the increased frequency of the interaction between yeast P450 reductase and P450c or P450(17 alpha) in the yeast microsomes.
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PMID:Expression of cloned yeast NADPH-cytochrome P450 reductase gene in Saccharomyces cerevisiae. 212 33

The effects of three calcium antagonists--nifedipin (NF), verapamil (V) and diltiazem (DL)--on the duration of hexobarbital (HB) sleeping time and on monooxygenase activity are studied. The drugs are applied in oral doses of 50, 40 and 30 mg/kg, respectively every day for 3 weeks on male albino rats. NF was found to shorten the duration of HB sleep, to increase the activities of EMD, BPD, AH, ECOD, EROD, NADPH-cytochtome P-450 reductase and the content of cytochrome P-450, and it does not change significantly the content of cytochrome b 5. It increases AD hydroxylation in positions 7-alpha-, 16-beta-, 16-alpha- and 6-beta, which suggests indirectly that NF has probably induced the synthesis of cytochrome P-450a, cytochrome P-450b, cytochrome P-450h and cytochrome P-450p, respectively. Verapamil also shortens the duration of HB sleeping time, increases the activities of EMD, BPD, AH, EROD and NADPH-cytochrome P-450 reductase, not changing the cytochrome P-450 content and the ECOD activity. It increases AD hydroxylation in positions 7-alpha- and 16-alpha-, which suggests probable induction of the synthesis of cytochrome P-450a and cytochrome P-450h. Unlike the other two calcium antagonists, diltiazem slightly shortens the duration of HB sleeping, not changing the enzyme activities studied, the content of cytochrome P-450 and cytochrome b 5, but increases the activities of EROD and NADPH-cytocrome P-450 reductase. It increases AD hydroxylation in positions 7-alpha-, 16-alpha- and 6-beta, which suggests probable induction of cytochrome P-450a, cytochrome P-450h and cytochrome P-450p.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Liver monooxygenase activity after multiple application of nifedipin, verapamil and diltiazem. 213 Jun 25

A flavin-linked NADPH cytochrome c oxido-reductase of molecular mass 77-kDa was extracted from membranes of rabbit peritoneal neutrophils and purified in the presence of Triton X-100. The redox properties of this enzyme were examined. By some criteria including its high sensitivity to mersalyl, and its relatively high specificity for NADPH compared to NADH, the rabbit neutrophil NADPH cytochrome c reductase resembled NADPH-cytochrome P-450 reductase. Limited proteolysis generated water soluble fragments, with molecular masses of 67-kDa and 57-kDa, which were still endowed with a substantial reductase activity. When added to a lysate of neutrophil membranes in octylglucoside, in the presence of an oxidase activation medium consisting of rabbit neutrophil cytosol, GTP-gamma-S, arachidonic acid and Mg2+, the purified reductase enhanced the production of O2-., suggesting that it forms part of the O2-. generating oxidase.
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PMID:Properties of the NADPH dehydrogenase component of the oxidase complex from rabbit peritoneal neutrophils: reconstitution of an oxidase activity with the dehydrogenase component and a membrane extract. 215 16

The light-induced enhancement of the 7-ethoxycoumarin-O-deethylase activity was measured in a reconstituted system, consisting of the enzyme P-450PB-B and the NADPH-cytochrome P-450 reductase. The relative increase of the activity was about 15%. It is shown that the product release process is accelerated by light. The phases of the catalytic cycle of 2 x 10(12) protein complexes were locked by periodic application of light pulses (0.1 s duration, 1.32 s repetition time, and 390-470 nm, 0.27 joule/nmol P-450). More than 80% of the active reconstituted enzyme complexes (= "molecular machines") worked in phase after a few light pulses. The phase relation continued even after switching off the light pulses. The catalytic cycle time was 1.54 s, giving a turnover number of 39 min-1. The turnover number, as determined from the enzyme activity under optimum conditions, was 39 min-1. Due to the dissociation constant of the P-450PB-B:NADPH-P-450 reductase complexes [3] only 24% of the proteins were in the active (= working) state under the conditions used. The lifetime of this complexes is larger than 6 s since more than 4 cycles of the free running enzyme can be observed. This is the first report, that all catalytic active complexes in the test tube can be synchronized by an external light source, if the right repetition time of the pulses is chosen, so that all these "molecular machines" work in phase.
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PMID:Light-induced activation and synchronization of the cytochrome P-450 dependent monooxygenase system. 216 42

The effect of dilauroylphosphatidylcholine (DLPC) concentration on cytochrome P-450 LM2 (LM2)-dependent reduction and monooxygenase activities was examined as a function of preincubation time. Purified NADPH-cytochrome P-450 reductase (reductase) and LM2 were reconstituted at different DLPC to LM2 ratios by preincubation of the proteins in the presence of DLPC for either 5 min or 2 hr at room temperature. After preincubation was complete, the samples were assayed for either monooxygenase activity or first-electron transfer activity. When preincubated for 5 min, overall monooxygenase activity was dependent on the [DLPC]:[LM2] ratio, beginning at a low level in the absence of phospholipid and increasing to a maximum at a 160:1 ratio. At [DLPC]:[LM2] ratios above 160:1, the rate was decreased to 80% of the maximum rate. When the samples were preincubated for 2 hr, again low monooxygenase activities were obtained in the absence of DLPC, which increased to a maximum at 160:1 [DLPC]:[LM2] ratio. Above this [DLPC]:[LM2]ratio, the rate was decreased to less than 50% of the maximum value. These changes in overall activities appear to be related to changes in the amount of functional reductase-LM2 complex formed. Similar results were found when LM2 reduction was examined. When preincubated for 5 min, LM2 reduction was shown to be diminished as the DLPC to LM2 ratio decreased below 160:1. The DLPC-dependent effect on reduction was primarily characterized by alterations in the fraction of LM2 reduced in the first phase, with the first-phase rate constant and the slow phase parameters being largely unaffected. Below a 16:1 ratio [( DLPC]:[LM2]), no phospholipid stimulation of LM2 reduction was observed. When the [DLPC]:[LM2] ratio was increased above a 160:1 ratio, only a small effect on the kinetic constants was observed, which was characterized by a 20% decrease in the fraction of LM2 reduced in the first phase. LM2 reduction was more sensitive to DLPC concentration after longer preincubations (2 hr), with a 50% decrease in the fraction of reduction in the first phase being observed at [DLPC]:[LM2] ratios above 160:1. The results are consistent with a dual role for phospholipid in the stimulation of LM2-dependent activities. First, DLPC facilitates the association of reductase and LM2 and, second, DLPC provides a matrix for the incorporation of LM2 and reductase. Facilitation of the protein association appears to be a relatively rapid process, occurring after a 5-min preincubation, whereas a 2-hr preincubation altered the protein interactions in a manner consistent with incorporation of the LM2 and reductase into the phospholipid.
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PMID:Dual role of phospholipid in the reconstitution of cytochrome P-450 LM2-dependent activities. 216 29

One major form of cytochrome P450 has been isolated from the hepatopancreas of untreated spiny lobsters, Panulirus argus. This form, termed here D1, was purified to a specific content of 12.1 +/- 1.8 nmol/mg protein. Two minor forms, termed D2 and D3 were partially purified to 4.6 +/- 1.6 and 2.3 +/- 0.2 nmol P450/mg protein, respectively. No NADPH-cytochrome P450 reductase activity was detected in spiny lobster hepatopancreas microsomes and no purification of spiny lobster reductase was achieved in this study. Very low NADPH-cytochrome c reductase activity was found in hepatopancreas microsomes and also in cytosol. Indirect evidence suggested that proteolysis of spiny lobster P450 reductase during the preparation of hepatopancreas microsomes may in part account for the lack of detectable monooxygenase activity in hepatopancreas microsomes. The catalytic activities of the D1 or D2 forms of spiny lobster P450 were measured by mixing D1 or D2 with NADPH-cytochrome P450 reductase isolated from pig or rat liver microsomes. D2 was very efficient in demethylating benzphetamine, with a turnover number of 122 per minute, and D1 was an efficient catalyst of progesterone 16 alpha-hydroxylation, with a turnover number of 43 per minute. Other good substrates for D1 and D2 forms were aminopyrine, testosterone, benzo(a)pyrene, and 7-ethoxycoumarin. Little activity was found with methyl-, ethyl-, pentyl-, or benzyl-phenoxazone ethers as substrates. The profile of metabolites formed by D1 or D2 with benzo(a)pyrene as substrate were more similar to those formed with uninduced rat liver microsomes than to those formed by liver microsomes from uninduced flatfish species.
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PMID:Isolation of cytochrome P450 from hepatopancreas microsomes of the spiny lobster, Panulirus argus, and determination of catalytic activity with NADPH cytochrome P450 reductase from vertebrate liver. 217 37

The omega-hydroxylation of leukotriene B4 (LTB4) by rat liver microsomes requires NADPH and molecular oxygen, suggesting that the hydroxylation is catalyzed by a cytochrome P-450 (P-450)-linked monooxygenase system. The reaction is inhibited by CO, and the inhibition is reversed by irradiation of light at 450 nm in a light-intensity-dependent manner. The extent of the reversal is strongly dependent on the wavelength of the light used, the 450-nm light is most efficient. The finding provides direct evidence for the identification of the LTB4 omega-hydroxylase as a P-450. The P-450 seems to be also responsible for prostaglandin A1 (PGA1) omega-hydroxylation, but not for lauric acid omega-hydroxylation. The LTB4 omega-hydroxylation is competitively inhibited by PGA1, but not affected by lauric acid. The Ki value for PGA1 of 38 microM agrees with the Km value for PGA1 omega-hydroxylation of 40 microM. LTB4 inhibits the PGA1 omega-hydroxylation by rat liver microsomes in a competitive manner with the Ki of 43 microM, which is consistent with the Km for the LTB4 omega-hydroxylation of 42 microM. An antiserum raised against rabbit pulmonary PG omega-hydroxylase (P-450p-2) inhibits slightly the omega-hydroxylations of LTB4 and PGA1, while it has stronger inhibitory effect on lauric acid omega-hydroxylation. In addition to NADPH-cytochrome P-450 reductase, cytochrome b5 appears to participate in the LTB4 omega-hydroxylating system, since the reaction is inhibited by an antibody raised against the cytochrome b5 as well as one raised against the reductase.
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PMID:Leukotriene B4 omega-hydroxylase in rat liver microsomes: identification as a cytochrome P-450 that catalyzes prostaglandin A1 omega-hydroxylation, and participation of cytochrome b5. 217 23


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