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)

Glycerol can be oxidized by rat liver microsomes to formaldehyde in a reaction that requires the production of reactive oxygen intermediates. Studies with inhibitors, antibodies, and reconstituted systems with purified cytochrome P4502E1 were carried out to evaluate whether P450 was required for glycerol oxidation. A purified system containing phospholipid, NADPH-cytochrome P450 reductase, P4502E1, and NADPH oxidized glycerol to formaldehyde. Formaldehyde production was dependent on NADPH, reductase, and P450, but not phospholipid. Formaldehyde production was inhibited by substrates and ligands for P4502E1, as well as by anti-pyrazole P4502E1 IgG. The oxidation of glycerol by the reconstituted system was sensitive to catalase, desferrioxamine, and EDTA but not to superoxide dismutase or mannitol, indicating a role for H2O2 plus non-heme iron, but not superoxide or hydroxyl radical in the overall glycerol oxidation pathway. The requirement for reactive oxygen intermediates for glycerol oxidation is in contrast to the oxidation of typical substrates for P450. In microsomes from pyrazole-treated, but not phenobarbital-treated rats, glycerol oxidation was inhibited by anti-pyrazole P450 IgG, anti-hamster ethanol-induced P450 IgG, and monoclonal antibody to ethanol-induced P450, although to a lesser extent than inhibition of dimethylnitrosamine oxidation. Anti-rabbit P4503a IgG did not inhibit glycerol oxidation at concentrations that inhibited oxidation of dimethylnitrosamine. Inhibition of glycerol oxidation by antibodies and by aminotriazole and miconazole was closely associated with inhibition of H2O2 production. These results indicate that P450 is required for glycerol oxidation to formaldehyde; however, glycerol is not a direct substrate for oxidation to formaldehyde by P450 but is a substrate for an oxidant derived from interaction of iron with H2O2 generated by cytochrome P450.
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PMID:Role of cytochrome P450 in the oxidation of glycerol by reconstituted systems and microsomes. 153 67

We present a novel strategy for increasing the level of functional mammalian cytochrome P450 (Cyt.P450) and NADPH:cytochrome P450 reductase enzymes produced in yeast. A cDNA encoding the rat P450 reductase was modified by the addition of a sequence coding for the N-terminal region of P450 reductase from Saccharomyces cerevisiae. The addition of this hydrophobic tail greatly increased the apparent stability of the reductase protein produced in S. cerevisiae, as compared to the unmodified rat P450 reductase. When the rat hybrid reductase was produced simultaneously with one of two mammalian Cyt.P450s, the rat CYP2B1 or the human CYP2A6, there was a significant increase in the specific activity of each of the Cyt.P450s. The optimization of this approach and its extrapolation to other organisms should lead to a marked improvement in our ability to study and exploit the P450 system.
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PMID:Production of cytochrome P450 reductase yeast-rat hybrid proteins in Saccharomyces cerevisiae. 154 75

Cytochrome P450IA1 (purified from hepatic microsomes of beta-naphthoflavone-treated rats) has been covalently modified with the lysine-modifying reagent acetic anhydride. Different levels of lysine residue modification in cytochrome P450IA1 can be achieved by varying the concentration of acetic anhydride. Modification of lysine residues in P450IA1 greatly inhibits the interaction of P450IA1 with NADPH-cytochrome P450 reductase. Modification of 1.0 and 3.3 mol lysine residues per mole P450IA1 resulted in 30 and 95% decreases, respectively, in 7-ethoxycoumarin hydroxylation by a reconstituted P450IA1/reductase complex. However, modification of 3.3 mol lysine residues per mole P450IA1 decreased only cumene hydroperoxide-supported P450-dependent 7-ethoxycoumarin hydroxylation by 30%. Spectral and fluorescence studies showed no indication of global conformational change of P450IA1 even with up to 8.8 mol lysine residues modified per mole P450IA1. These data suggest that at least three lysine residues in P450IA1 may be involved in the interaction with reductase. Identification of lysine residues in P450IA1 possibly involved in this interaction was carried out by [14C]acetic anhydride modification, trypsin digestion, HPLC separation, and amino acid sequencing. The lysine residue candidates identified in this manner were K97, K271, K279, and K407.
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PMID:The role of cytochrome P450 lysine residues in the interaction between cytochrome P450IA1 and NADPH-cytochrome P450 reductase. 155 Mar 61

Microsomal P450s catalyze the monooxygenation of a large variety of hydrophobic compounds, including drugs, steroids, carcinogens, and fatty acids. The interaction of microsomal P450s with their electron transfer partner, NADPH-P450 reductase, during the transfer of electrons from NADPH to P450, for oxygen activation, may be important in regulating this enzyme system. Highly purified Bacillus megaterium P450BM-3 is catalytically self-sufficient and contains both the reductase and P450 domains on a single polypeptide chain of approximately 120,000 Da. The two domains of P450BM-3 appear to be analogous in their function and homologous in their sequence to the microsomal P450 system components. FAD, FMN, and heme residues are present in equimolar amounts in purified P450BM-3 and, therefore, this protein could potentially accept five electron equivalents per mole of enzyme during a reductive titration. The titration of P450BM-3 with sodium dithionite under a carbon monoxide atmosphere was complete with the addition of the expected five electron equivalents. The intermediate spectra indicate that the heme iron is reduced first, followed by the flavin residues. Titration of the protein with the physiological reductant, NADPH, also required approximately five electron equivalents when the reaction was performed under an atmosphere of carbon monoxide. Under an atmosphere of argon and in the absence of carbon monoxide, one of the flavin groups was reduced prior to the reduction of the heme group. The titration behavior of P450BM-3 with NADPH was surprising because no spectral changes characteristic of flavin semiquinone intermediates were observed. The results of the titration with NADPH can only be explained if (a) there was "rapid" intermolecular electron transfer between P450BM-3 molecules, (b) there is no kinetic barrier to the reduction of P450 by the one-electron-reduced form of the reductase, and (c) the "air-stable semiquinone" form of the reductase does not accumulate in this complex multidomain enzyme.
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PMID:P450BM-3: reduction by NADPH and sodium dithionite. 156 20

A specific form of flavin monooxygenase has been identified in the lungs of a number of species. Distribution of the pulmonary flavin-containing monooxygenase (FMOp) is of interest because it oxidatively metabolizes a wide variety of nitrogen-, sulfur-, and phosphorous-containing xenobiotics, some of which form highly toxic reactive intermediates. We have identified the nonciliated bronchiolar epithelial (Clara) cell as the predominant location for this enzyme in rabbit lung. In addition, protein in ciliated, endothelial, type I, and type II cells and in tracheal lining layer reacted with antibodies to FMOp. In all these cell types antigen was found associated with cytoplasmic organelles, and in the Clara cell antigen was most concentrated in areas rich in smooth endoplasmic reticulum. Staining of ciliated surfaces was also observed at both the light and electron microscopy levels. Extracellular antigen was also apparent in tracheal lining layer smeared onto glass slides. We compared the location of the FMOp with that of two enzymes of the cytochrome P-450 monooxygenase system (studied here and elsewhere), cytochrome P450 IIB (P450 IIB), and NADPH cytochrome P450 reductase (reductase), and concluded that (1) FMOp is detected in all cells where P450 IIB and reductase are both present (Clara, type II, and ciliated); (2) FMOp and P450 IIB, but not reductase, are detected in endothelial cells; (3) P450 IIB alone is detected in the plasma membrane, cilia, and microvillae of ciliated cells and plasma membrane of endothelial cells; and (4) FMOp alone is detected in type I cells.
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PMID:Cellular localization of flavin-containing monooxygenase in rabbit lung. 157 20

Induction of cytochrome P450 1A1 (P450 1A1) in a variety of tissues is a well established consequence of exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and related compounds. Although localization of the induced protein within the lung has been described, the precise intracellular distribution of the enzyme is not clear. Analysis of tissue sections, microsomal proteins, and mRNA from lungs of treated and untreated rabbits established that P450 1A1 had been induced by treatment with TCDD. Rabbit lungs from animals treated with TCDD were examined with immunocytochemistry and in situ hybridization, to identify the cell types that contain P450 1A1 and those that contain mRNA encoding P450 1A1. Endothelial cells of the entire vascular bed of rabbit lung reacted markedly with anti-P450 1A1. Likewise, cells lining both arteries and veins, as well as capillary endothelial cells, reacted strongly with the cDNA probe for mRNA encoding P450 1A1. Clara cells at all levels of airway labeled prominently for both P-450 1A1 and P450 1A1 mRNA. In addition, type 2 cells, alveolar macrophages, and to a lesser degree, ciliated cells reacted with the cDNA probe. P450 reductase, which is required for P450 activity, has previously been identified in Clara cells, type 2 cells, and alveolar macrophages, but not in endothelium of rabbit lung. We have now obtained similar results for the localization of mRNA encoding P-450 reductase. This finding brings into question the function of P450 1A1 in endothelium.
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PMID:Distribution of cytochrome P450 1A1 and NADPH-cytochrome P450 reductase in lungs of rabbits treated with 2,3,7,8-tetrachlorodibenzo-p-dioxin: ultrastructural immunolocalization and in situ hybridization. 161 8

Cytochrome P-450 coded for by the 3A gene family requires specific conditions in a reconstituted system, if its catalytic activity is to be efficient. We investigated the mechanism of activation of the catalytic activity of cytochrome P450 3A by phospholipids. Rat P450 PB-1 (3A2), human P450NF (3A4), and rabbit P450 3c (3A6) were used. They had low activity in a reconstituted system (system I) with dilauroylphosphatidylcholine (DLPC) but had high activity with a mixture of phospholipids (DLPC, dioleoylphosphatidylcholine, and phosphatidylserine) and sodium cholate (system II). P450 3A forms are cationic (having a high content of lysine residues) and needed the anionic phospholipid phosphatidylserine to have sufficient activity. Double-reciprocal plots of the metabolic rate of cytochrome P-450 versus the concentration of NADPH-cytochrome P-450 reductase showed that cytochrome P-450 and the reductase interacted more in system II than in system I. P450 PB-1 did not absorb at 450 nm in the presence of reductase, CO, DLPC, and NADPH, although other cytochrome P-450s absorbed at around 450 nm in such a mixture. However, P450 PB-1 was reduced in the presence of the phospholipid mixture and sodium cholate instead of DLPC. These results suggested that the stimulation of catalytic activity by phospholipids involved increased interaction between cytochrome P-450 and the reductase. Studies of proteolytic digestion and chemical cross-linking in systems I and II showed that a P450 3A form needed disaggregation of cytochrome P-450 and/or the reductase, not the formation of an aggregated complex necessary for the catalytic activity of other cytochrome P-450s.
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PMID:Role of phospholipids in reconstituted cytochrome P450 3A form and mechanism of their activation of catalytic activity. 162 48

Human liver P450 NF25 (CYP3A4) had been previously expressed in Saccharomyces cerevisiae using the inducible GAL10-CYC1 promoter and the phosphoglycerate kinase gene terminator [Renaud, J. P., Cullin, C., Pompon, D., Beaune, P. and Mansuy, D. (1990) Eur. J. Biochem. 194, 889-896]. The use of an improved expression vector [Urban, P., Cullin, C. and Pompon, D. (1990) Biochimie 72, 463-472] increased the amounts of P450 NF25 produced/culture medium by a factor of five, yielding up to 10 nmol/l. The availability of recently developed host cells that simultaneously overexpress yeast NADPH-P450 reductase and/or express human liver cytochrome b5, obtained through stable integration of the corresponding coding sequences into the yeast genome, led to biotechnological systems with much higher activities of yeast-expressed P450 NF25 and with much better ability to form P450 NF25-iron-metabolite complexes. 9-fold, 8-fold, and 30-fold rate increases were found respectively for nifedipine 1,4-oxidation, lidocaine N-deethylation and testosterone 6 beta-hydroxylation between P450 NF25-containing yeast microsomes from the basic strain and from the strain that both overexpresses yeast NADPH-P450 reductase and expresses human cytochrome b5. Even higher turnovers (15-fold, 20-fold and 50-fold rate increases) were obtained using P450 NF25-containing microsomes from the yeast just overexpressing yeast NADPH-P450 reductase in the presence of externally added, purified rabbit liver cytochrome b5. This is explained by the fact that the latter strain contained the highest level of NADPH-P450 reductase activity. It is noteworthy that for the three tested substrates, the presence of human or rabbit cytochrome b5 always showed a stimulating effect on the catalytic activities and this effect was saturable. Indeed, addition of rabbit cytochrome b5 to microsomes from a strain expressing human cytochrome b5 did not further enhance the catalytic rates. The yeast expression system was also used to study the formation of a P450-NF25-iron-metabolite complex. A P450 Fe(II)-(RNO) complex was obtained upon oxidation of N-hydroxyamphetamine, catalyzed by P450-NF25-containing yeast microsomes. In microsomes from the basic strain expressing P450 NF25, 10% of the starting P450 NF25 was transformed into this metabolite complex, whereas more than 80% of the starting P450 NF25 led to complex formation in microsomes from the strain overexpressing yeast NADPH-P450 reductase.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Optimization of yeast-expressed human liver cytochrome P450 3A4 catalytic activities by coexpressing NADPH-cytochrome P450 reductase and cytochrome b5. 162 42

NADPH-cytochrome P450 reductase was purified to apparent homogeneity and cytochrome P450 partially purified from whole rat brain. Purified reductase from brain was identical to liver P450 reductase by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western blot techniques. Kinetic studies using cerebral P450 reductase reveal Km values in close agreement with those determined with enzyme purified from rat liver. Moreover, the brain P450 reductase was able to function successfully in a reconstituted microsomal system with partially purified brain cytochrome P450 and with purified hepatic P450c (P450IA1) as measured by 7-ethoxycoumarin and 7-ethoxyresorufin O-deethylation. Our results indicate that the reductase and P450 components may interact to form a competent drug metabolism system in brain tissue.
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PMID:Reconstitution of the brain mixed function oxidase system: purification of NADPH-cytochrome P450 reductase and partial purification of cytochrome P450 from whole rat brain. 162 29

SR 4233 or WIN 59075 (3-amino-1,2,4-benzotriazine-1,4-dioxide) is a novel and highly selective hypoxic cell cytotoxin requiring reductive bioactivation for its impressive antitumour effects. Expression of appropriate reductases will contribute to therapeutic selectivity. Here we provide more detailed information on the role of cytochrome P450 and cytochrome P450 reductase in SR 4233 reduction by mouse liver microsomes. Reduction of SR 4233 to the mono-N-oxide SR 4317 (3-amino-1,2,4-benzotriazine-1-oxide) is NADPH, enzyme and hypoxia dependent. An inhibitory antibody to cytochrome P450 reductase decreased the microsomal SR 4233 reduction rate by around 20%. Moreover, studies with purified rat cytochrome P450 reductase showed unequivocally that this enzyme was able to catalyse SR 4233 reduction at a rate of 20-30% of that for microsomes with equivalent P450 reductase activity. Exposure to the specific cytochrome P450 inhibitor carbon monoxide (CO) inhibited microsomal reduction by around 70% and CO plus reductase antibody blocked essentially all activity. Additional confirmation of cytochrome P450 involvement was provided by the use of other P450 ligands: beta-diethylaminoethyl diphenylpropylacetate hydrochloride gave a slight stimulation while aminopyrine, n-octylamine and 2,4-dichloro-6-phenylphenoxyethylamine were inhibitory. Induction of SR 4233 reduction was seen with phenobarbitone, pregnenalone-16-alpha-carbonitrile and beta-napthoflavone, suggesting that cytochrome P450 subfamilies IIB, IIC and IIIA may be involved. Since cytochrome P450 and P450 reductase catalyse roughly 70 and 30%, of mouse liver microsomal SR 4233 reduction respectively, we propose that expression of these and other reductases in normal and tumour tissue is likely to be a major factor governing the toxicity and antitumour activity of the drug.
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PMID:The role of cytochrome P450 and cytochrome P450 reductase in the reductive bioactivation of the novel benzotriazine di-N-oxide hypoxic cytotoxin 3-amino-1,2,4-benzotriazine-1,4-dioxide (SR 4233, WIN 59075) by mouse liver. 164 40


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