Purification and characterization of hepatic microsomal prostaglandin omega-hydroxylase cytochrome P-450 from pregnant rabbits.
Prostaglandin omega-hydroxylase, designated as cytochrome P-450 LPG omega (P-450 LPG omega), 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-450 LPG omega was found to be homogeneous on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and to have an apparent 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 omega-hydroxylation of prostaglandin E1 (PGE1), prostaglandin E2 (PGE2), prostaglandin D2 (PGD2), prostaglandin F2 alpha (PGF 2 alpha), prostaglandin A1 (PGA1), and prostaglandin A2 (PGA2), as well as the omega- and (omega-1)-hydroxylation of myristate and palmitate, in a reconstituted system containing cytochrome P-450, NADPH-cytochrome P-450 reductase, phospholipid, and cytochrome b5. Various monovalent and divalent cations further stimulated these reactions in the presence of cytochrome b5. 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-450 LPG omega was very similar or identical to the pulmonary prostaglandin omega-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-450 LPG omega was more unstable than P-450p-2 on storage. In sharp contrast to P-450p-2, P-450 LPG omega was not induced by progesterone.
Kikuta
Y
Y
Toneyama Institute for Tuberculosis Research, Osaka City University Medical School.
Kusunose
E
E
Matsubara
S
S
Funae
Y
Y
Imaoka
S
S
Kubota
I
I
Kusunose
M
M
D000527:Alprostadil / Q000378:metabolism; D000818:Animals; D003577:Cytochrome P-450 Enzyme System / Q000302:isolation & purification* / Q000378:metabolism; D005260:Female; D008168:Lung / Q000201:enzymology; D008862:Microsomes, Liver / Q000032:analysis* / Q000201:enzymology; D006899:Mixed Function Oxygenases / Q000302:isolation & purification* / Q000378:metabolism; D011247:Pregnancy; D011817:Rabbits
D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; C026961:prostaglandin omega hydroxylases; D000527:Alprostadil