Regioselective hydroxylation of prostaglandins by constitutive forms of cytochrome P-450 from rat liver: Formation of a novel metabolite by a female-specific P-450

Abstract

Previous studies demonstrated that liver microsomes from untreated rats catalyze the ω, ω-1, and ω-2 hydroxylation of prostaglandins [ K. A. Holm, R. J. Engell, and D. Kupfer (1985) Arch. Biochem. Biophys. 237, 477–489 ]. The current study examined the regioselectivity of hydroxylation of PGE 1 and PGE 2 by purified forms of P-450 from untreated male and female rat liver microsomes. PGE 1 was incubated with a reconstituted system containing cytochrome P-450 RLM 2, 3, 5, 5a, 5b, 6, or f4, NADPH- P-450 reductase, and dilauroylphosphatidylcholine in the presence or absence of cytochrome b 5. Among the P-450 forms examined, only RLM 5 (male specific), 5a (present in both sexes), and f4 (female specific) yielded high levels of PGE hydroxylation. With PGE 1, RLM 5 catalyzed solely the ω-1 hydroxylation and 5a catalyzed primarily the ω-1 and little ω and ω-2 hydroxylation. By contrast, f4 effectively hydroxylated PGE 1 and PGE 2 at the ω-1 and at a novel site. Based on retention on HPLC and on limited mass fragmentation, we speculate that this site is ω-3 (i.e., 17-hydroxylation). Kinetic analysis of PGE 1 hydroxylation demonstrated that the affinity of f4 for PGE 1 is approximately 100-fold higher than that of RLM 5; the K m values for f4, monitoring 19- and 17-hydroxylation of PGE 1, were about 10 μ m. Surprisingly, cytochrome b 5 stimulated the activity of RLM 5a and f4, but not that of RLM 5. Hydroxylation of PGE 2 by RLM 5 was at the ω, ω-1, and ω-2 sites, demonstrating a lesser regioselectivity than with PGE 1. These findings show that the constitutive P-450s differ dramatically in their ability to hydroxylate PGs, in their regioselectivity of hydroxylation, and in their cytochrome b 5 requirement.