title: Monoclonal antibody-directed characterization of rat hepatic P450 catalyzing the omega-1 and omega-2 hydroxylation of prostaglandins.
abstract: Previous studies have demonstrated that methylcholanthrene (MC) treatment of rats increases 10-fold the omega-2 hydroxylation of prostaglandin E2 (PGE2) by liver microsomes (K. A. Holm, R. J. Engell, and D. Kupfer (1985) Arch. Biochem. Biophys. 237, 477-489). The current study identifies the cytochrome P450 form, which catalyzes a major portion of the omega-2 hydroxylation of prostaglandins in liver microsomes of MC-treated rats (MC-microsomes) and examines whether the same enzyme catalyzes this reaction in microsomes from untreated rats (control microsomes). Three monoclonal antibodies (MAbs), MC 1-7-1, 1-31-2, and 1-36-1, raised against the major liver P450 from MC-treated rats were used. MAb 1-7-1 binds P450(57K) and P450(56K) (P450c and P450d, respectively); MAb 1-31-2 binds primarily P450(57K); and 1-36-1 binds solely P450(57k). MAb 1-7-1 inhibited omega-2 and omega-1 PGE2 hydroxylations in MC-microsomes by 70 and 45%, respectively. By contrast, MAb 1-31-2 and 1-36-1 were not inhibitory. MAb 1-7-1 did not inhibit PGE2 omega-2 hydroxylation in control or in microsomes from phenobarbital-treated rats (PB-microsomes). Since MAb 1-7-1 binds to both P450c and P450d, and 1-31-2 and 1-36-1 bind to P450c but are not inhibitory, these findings did not permit the determination of whether in MC microsomes a single isozyme (P450c or P450d) or both isozymes catalyze the omega-2 hydroxylation. This question was partially resolved by the observation that immunoaffinity-isolated P450c, supplemented with purified NADPH-P450 reductase, catalyzes effectively the omega-2 hydroxylation and to a lesser extent the omega-1 hydroxylation. There was no activity in the absence of reductase. The P450 antibody complex exhibits characteristics similar to those of the omega-2 hydroxylating activity in intact MC-microsomes supported by H2O2, by demonstrating a much higher activity when H2O2 is used instead of reductase and NADPH. Furthermore, a reconstituted monooxygenase composed of rat liver reductase and P450c, purified by conventional means, hydroxylated PGE2 at the omega-2 and omega-1 sites at a ratio of 2.8, similar to that obtained with the P450-antibody complex. These findings demonstrate that a major portion of the omega-2 hydroxylation of PGs in MC-microsomes is catalyzed by P450c; however, the possibility that some omega-2 hydroxylating activity is due to P450d was not ruled out.(ABSTRACT TRUNCATED AT 400 WORDS)
authors.affiliation: Worcester Foundation for Experimental Biology, Shrewsbury, Massachusetts 01545.
authors.forename: K A
authors.initials: KA
authors.lastname: Holm
authors.forename: S S
authors.initials: SS
authors.lastname: Park
authors.forename: H V
authors.initials: HV
authors.lastname: Gelboin
authors.forename: D
authors.initials: D
authors.lastname: Kupfer
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D002846:Chromatography, Affinity; D003577:Cytochrome P-450 Enzyme System / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D006900:Hydroxylation; D007700:Kinetics; D008297:Male; D008748:Methylcholanthrene / Q000494:pharmacology; D008862:Microsomes, Liver / Q000187:drug effects / Q000201:enzymology*; D006899:Mixed Function Oxygenases / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D010634:Phenobarbital / Q000494:pharmacology; D051381:Rats; D011919:Rats, Inbred Strains
chemical_list: D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D008748:Methylcholanthrene; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; C026961:prostaglandin omega hydroxylases; D010634:Phenobarbital