REQUIREMENTS FOR CYTOCHROME B(5) IN THE OXIDATION OF 7-ETHOXYCOUMARIN, CHLORZOXAZONE, ANILINE, AND N-NITROSODIMETHYLAMINE BY RECOMBINANT CYTOCHROME-P450 2E1 AND BY HUMAN LIVER-MICROSOMES

NADH-dependent 7-ethoxycoumarin O-deethylation activities could be rec onstituted in systems containing cytochrome b(5) (b(5)), NADH-b(5) red uctase, and bacterial recombinant P450 2E1 in 100 mM potassium phospha te buffer (pH 7.4) containing a synthetic phospholipid mixture and cho late. Replacement of NADH-b(5) reductase with NADPH-P450 reductase yie lded a 4-fold increase in 7-ethoxycoumarin O-deethylation activity, an d further stimulation (similar to 1.5-fold) could be obtained when NAD PH was used as an electron donor. Removal of b(5) from the NADH- and N ADPH-supported systems caused a 90% loss of 7-ethoxycoumarin O-deethyl ation activities in the presence of NADPH-P450 reductase, but resulted in complete loss of the activities in the absence of NADPH-P450 reduc tase. K-m values were increased and V-max values were decreased for 7- ethoxycoumarin O-deethylation when b(5) was omitted from the NADPH-sup ported P450 2E1-reconstituted systems. Requirements for b(5) in P450 2 E1 systems were also observed in chlorzoxazone 6-hydroxylation, anilin e p-hydroxylation, and N-nitrosodimethylamine N-demethylation. In huma n liver microsomes, NADH-dependent 7-ethoxycoumarin O-deethylation, ch lorzoxazone 6-hydroxylation, aniline P-hydroxylation, and N-nitrosodim ethylamine N-demethylation activities were found to be about 55, 41, 3 3, and 50%, respectively, of those catalyzed by NADPH-supported system s. Anti-rat NADPH-P450 reductase immunoglobulin G inhibited 7-ethoxyco umarin O-deethylation activity catalyzed by human liver microsomes mor e strongly in NADPH- than NADH-supported reactions, while anti-human b (5) immunoglobulin G inhibited microsomal activities in both NADH- and NADPH-supported systems to similar extents. These results suggest tha t b(5) is an essential component in P450 2E1-catalyzed oxidations of s everal substrates used, that about 10% of the activities occur via P45 0 2E1 reduction by NADPH-P450 reductase in the absence of b(5), and th at the NADH-supported system contributes, in part, to some reactions c atalyzed by P450 2E1 in human liver microsomes.