title: Requirements for cytochrome b5 in the oxidation of 7-ethoxycoumarin, chlorzoxazone, aniline, and N-nitrosodimethylamine by recombinant cytochrome P450 2E1 and by human liver microsomes.
abstract: NADH-dependent 7-ethoxycoumarin O-deethylation activities could be reconstituted in systems containing cytochrome b5 (b5), NADH-b5 reductase, and bacterial recombinant P450 2E1 in 100 mM potassium phosphate buffer (pH 7.4) containing a synthetic phospholipid mixture and cholate. Replacement of NADH-b5 reductase with NADPH-P450 reductase yielded a 4-fold increase in 7-ethoxycoumarin O-deethylation activity, and further stimulation (approximately 1.5-fold) could be obtained when NADPH was used as an electron donor. Removal of b5 from the NADH- and NADPH-supported systems caused a 90% loss of 7-ethoxycoumarin O-deethylation activities in the presence of NADPH-P450 reductase, but resulted in complete loss of the activities in the absence of NADPH-P450 reductase. Km values were increased and Vmax values were decreased for 7-ethoxycoumarin O-deethylation when b5 was omitted from the NADPH-supported P450 2E1-reconstituted systems. Requirements for b5 in P450 2E1 systems were also observed in chlorzoxazone 6-hydroxylation, aniline p-hydroxylation, and N-nitrosodimethylamine N-demethylation. In human liver microsomes, NADH-dependent 7-ethoxycoumarin O-deethylation, chlorzoxazone 6-hydroxylation, aniline p-hydroxylation, and N-nitrosodimethylamine N-demethylation activities were found to be about 55, 41, 33, and 50%, respectively, of those catalyzed by NADPH-supported systems. Anti-rat NADPH-P450 reductase immunoglobulin G inhibited 7-ethoxycoumarin O-deethylation activity catalyzed by human liver microsomes more strongly in NADPH- than NADH-supported reactions, while anti-human b5 immunoglobulin G inhibited microsomal activities in both NADH- and NADPH-supported systems to similar extents. These results suggest that b5 is an essential component in P450 2E1-catalyzed oxidations of several substrates used, that about 10% of the activities occur via P450 2E1 reduction by NADPH-P450 reductase in the absence of b5, and that the NADH-supported system contributes, in part, to some reactions catalyzed by P450 2E1 in human liver microsomes.
authors.affiliation: Osaka Prefectural Institute of Public Health, Japan.
authors.forename: H
authors.initials: H
authors.lastname: Yamazaki
authors.forename: M
authors.initials: M
authors.lastname: Nakano
authors.forename: E M
authors.initials: EM
authors.lastname: Gillam
authors.forename: L C
authors.initials: LC
authors.lastname: Bell
authors.forename: F P
authors.initials: FP
authors.lastname: Guengerich
authors.forename: T
authors.initials: T
authors.lastname: Shimada
mesh_terms: D000814:Aniline Compounds / Q000378:metabolism*; D002753:Chlorzoxazone / Q000378:metabolism*; D003374:Coumarins / Q000378:metabolism*; D019388:Cytochrome P-450 CYP1A2; D019392:Cytochrome P-450 CYP2E1; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D015786:Cytochromes b5 / Q000037:antagonists & inhibitors / Q000378:metabolism*; D004128:Dimethylnitrosamine / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008862:Microsomes, Liver / Q000378:metabolism*; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism; D009251:NADPH-Ferrihemoprotein Reductase; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000378:metabolism; D010089:Oxidoreductases, N-Demethylating / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism
chemical_list: D000814:Aniline Compounds; D003374:Coumarins; D065607:Cytochrome P-450 Enzyme Inhibitors; D011994:Recombinant Proteins; C017299:7-ethoxycoumarin; D015786:Cytochromes b5; D003577:Cytochrome P-450 Enzyme System; D010088:Oxidoreductases; D019392:Cytochrome P-450 CYP2E1; D019388:Cytochrome P-450 CYP1A2; D010089:Oxidoreductases, N-Demethylating; D009247:NADH, NADPH Oxidoreductases; D009251:NADPH-Ferrihemoprotein Reductase; D002753:Chlorzoxazone; D004128:Dimethylnitrosamine; C023650:aniline