Purification and characterization of cytochrome P-450 specific for prostaglandin and fatty acid hydroxylase activities from the microsomes of rabbit small intestinal mucosa.
Earlier studies (Kusunose, E., Kaku, M., Ichihara, K., Yamamoto, S., Yano, I., & Kusunose, M. (1984) J. Biochem. 95, 1733-1739) showed that a form of cytochrome P-450 isolated from microsomes of rabbit small intestinal mucosa had the highest prostaglandin A1 (PGA1) hydroxylase activity so far reported among cytochrome P-450s. The present paper describes the procedure for the purification and further characterization of this cytochrome (designated as cytochrome P-450ia). Cytochrome P-450ia had a monomeric molecular weight of 53,000. The CO-difference spectra of its reduced form showed a maximal absorption at 451 nm, and the absolute spectra of its oxidized form indicated that cytochrome P-450ia was present largely in the low-spin state, and partially in the high-spin state. The cytochrome efficiently catalyzed the hydroxylation of fatty acids as well as prostaglandins in a reconstituted system containing cytochrome P-450, NADPH-cytochrome P-450 reductase, phospholipid, and cytochrome b5. PGA1 was the most efficient substrate, followed by myristate, laurate, palmitate, caprate, and PGE1 or PGE2. Among phospholipids, didecanoyl- and dilauroylphosphatidylcholines had the most stimulatory effect for both activities. 20-Hydroxy PGA1 was identified as the hydroxylation product of PGA1 by gas chromatography-mass spectrometry and mass fragmentography; the possibility of 19-hydroxy PGA1 being the product was excluded. In contrast, both omega- and (omega-1)-hydroxy fatty acids were identified as hydroxylation products of fatty acids. Cytochrome P-450ia had no detectable activity toward aminopyrine, benzphetamine, p-nitroanisole, 7-ethoxycoumarin, benzo(a)pyrene, or hexadecane.
D000818:Animals; D042926:Cytochrome P-450 CYP4A; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D006900:Hydroxylation; D066298:In Vitro Techniques; D007413:Intestinal Mucosa / Q000201:enzymology*; D008297:Male; D008861:Microsomes / Q000201:enzymology*; D006899:Mixed Function Oxygenases / Q000302:isolation & purification* / Q000378:metabolism; D019814:Myristic Acid; D009227:Myristic Acids / Q000378:metabolism; D011454:Prostaglandins A / Q000378:metabolism; D011817:Rabbits
D009227:Myristic Acids; D011454:Prostaglandins A; D019814:Myristic Acid; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; C026961:prostaglandin omega hydroxylases; D042926:Cytochrome P-450 CYP4A; C100573:prostaglandin A1