Monoclonal antibody-directed characterization of rat hepatic P450 catalyzing the ω-1 and ω-2 hydroxylation of prostaglandins

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Abstract

Previous studies have demonstrated that methylcholanthrene (MC) treatment of rats increases 10-fold the ω-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 ω-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 P45057K and P45056K (P450c and P450d, respectively); MAb 1-31-2 binds primarily P45057K; and 1-36-1 binds solely P45057K. MAb 1-7-1 inhibited ω-2 and ω-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 ω-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 ω-2 hydroxylation. This question was partially resolved by the observation that immunoaffinity-isolated P450c, supplemented with purified NADPH-P450 reductase, catalyzes effectively the ω-2 hydroxylation and to a lesser extent the ω-1 hydroxylation. There was no activity in the absence of reductase. The P450 antibody complex exhibits characteristics similar to those of the ω-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 ω-2 and ω-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 ω-2 hydroxylation of PGs in MC-microsomes is catalyzed by P450c; however, the possibility that some ω-2 hydroxylating activity is due to P450d was not ruled out. Certain purified P450s (RLM 5 and 5a) from untreated rat liver catalyze the ω-2 hydroxylation of PGE2 (D. Kupfer et al. (1988) Arch. Biochem. Biophys.261, 186–195). The current findings demonstrate that all the ω-2 hydroxylation in control microsomes and about 25% of the activity in MC-microsomes is by P450s which are not inhibited by MAb 1-7-1. It is not known whether these resistant P450s are RLM 5, 5a, or different constitutive P450s.

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    This study was supported in part by Grant ES 00834 from the National Institute of Environmental Health Sciences.

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