Purification and Characterization of Hepatic Microsomal Prostaglandin ω-Hydroxylase Cytochrome P-450 from Pregnant Rabbits1

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Abstract
Prostaglandin w-hydroxylase, designated as cytochrome P-450LPGω (P-450LPGω) has been purified, to a specific content of 15 nmol of cytochrome P-450/mg of protein, from liver microsomes of pregnant rabbits. The purified P-450LPGω was found to be homogeneous on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and to have an apprent molecular weight of 52, 000. The enzyme showed a maximum at 450 nm in the carbon monoxide (CO)-difference spectrum for its reduced form. This cytochrome P-450 efficiently catalyzed the ω-hydroxylation of prostaglandin E1 (PGE1), prostaglandin E2 (PGE2), prostaglandin D2 (PGD2), prostaglandin F2a (PGF2a), prostaglandin AI (PGA1), and prostaglandin A2 (PGA2), as well as the ω- and (ω-l)-hydroxylation of myristate and palmitate, in a reconstituted system containing cytochrome P-450, NADPH-cytochrome P-450 reductase, phospholipid, and cytochrome β5. Various monovalent and divalent cations further stimulated these reactions in the presence of cytochrome β5. In addition, the reactions were also markedly enhanced by various organic solvents, such as ethanol and acetone. This cytochrome P-450 showed no detectable activity toward several xenobiotics tested. P-450LPGω was very similar or identical to the pulmonary prostaglandin ω-hydroxylase (P-450p-2) (Yamamoto, S., Kusunose, E., Ogita, K., Kaku, M., Ichihara, K., & Kusunose, M. (1984) J. Biochem. 96, 593–603) in its molecular weight, absorption spectra, catalytic activity, peptide mapping pattern, and N-terminal amino acid sequence. However, P-450LPGω was more unstable than P-450p-2 on storage. In sharp contrast to P-450p-2, P-450LPGω. was not induced by progesterone.