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.
Previous studies demonstrated that liver microsomes from untreated rats catalyze the omega, omega-1, and omega-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 PGE1 and PGE2 by purified forms of P-450 from untreated male and female rat liver microsomes. PGE1 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 b5. 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 PGE1, RLM 5 catalyzed solely the omega-1 hydroxylation and 5a catalyzed primarily the omega-1 and little omega and omega-2 hydroxylation. By contrast, f4 effectively hydroxylated PGE1 and PGE2 at the omega-1 and at a novel site. Based on retention on HPLC and on limited mass fragmentation, we speculate that this site is omega-3 (i.e., 17-hydroxylation). Kinetic analysis of PGE1 hydroxylation demonstrated that the affinity of f4 for PGE1 is approximately 100-fold higher than that of RLM 5; the Km values for f4, monitoring 19- and 17-hydroxylation of PGE1, were about 10 microM. Surprisingly, cytochrome b5 stimulated the activity of RLM 5a and f4, but not that of RLM 5. Hydroxylation of PGE2 by RLM 5 was at the omega, omega-1, and omega-2 sites, demonstrating a lesser regioselectivity than with PGE1. 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 b5 requirement.
Kupfer
D
D
Worcester Foundation for Experimental Biology, Shrewsbury, Massachusetts 01545.
Jansson
I
I
Favreau
L V
LV
Theoharides
A D
AD
Schenkman
J B
JB
D000818:Animals; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D003573:Cytochrome b Group / Q000378:metabolism; D015786:Cytochromes b5; D015232:Dinoprostone; D005260:Female; D006900:Hydroxylation; D007700:Kinetics; D008297:Male; D008862:Microsomes, Liver / Q000378:metabolism*; D011453:Prostaglandins / Q000378:metabolism*; D011458:Prostaglandins E / Q000378:metabolism; D051381:Rats; D011919:Rats, Inbred Strains; D012737:Sex Factors
D003573:Cytochrome b Group; D011453:Prostaglandins; D011458:Prostaglandins E; D015786:Cytochromes b5; D003577:Cytochrome P-450 Enzyme System; D015232:Dinoprostone