Purification and characterization of two forms of cytochrome P-450 with omega-hydroxylase activities toward prostaglandin A and fatty acids from rabbit liver microsomes.
Two forms of cytochrome P-450 (P-450), designated as P-450LPGA omega 1 and P-450LPGA omega 2, have been purified to specific contents of 17.9 and 11.1 nmol P-450/mg protein, respectively, from liver microsomes of rabbits treated with di(2-ethylhexyl)phthalate (DEHP), a peroxisomal proliferator. The purified P-450LPGA omega 1 and P-450LPGA omega 2 were found to have apparent molecular weights of 52,500 and 53,000, respectively. They showed absorption maxima at 451 and 450 nm in the carbon monoxide-difference spectra for their reduced forms, respectively. The two P-450s both efficiently catalyzed the omega-hydroxylation of prostaglandins A1 (PGA1) and A2 (PGA2), as well as the omega- and (omega-1)-hydroxylation of fatty acids such as laurate, myristate, and palmitate. In a reconstituted system, various metal ions such as Na+ and Mg2+ stimulated these reactions. The P-450s exhibited no detectable activity toward several xenobiotics tested. The two P-450s showed different peptide map patterns following limited proteolysis with Staphylococcus aureus V8 protease or papain. The NH2-terminal amino acid sequences (ALNPTRLPGSLSGLLQVAGL and ALSLTRLPGSFSGFLQAxGLLGLLL) of P-450LPGA omega 1 and P-450LPGA omega 2 were identical at 18/20 and 19/24 positions with that of the lung prostaglandin omega-hydroxylase from pregnant rabbits, respectively. An antibody against P-450LPGA omega 2 recognized a 52,000-53,000 molecular weight protein(s) in rabbit liver microsomes. The intensity of the immunoblot was significantly increased in liver microsomes from rabbits treated with DEHP, but not with phenobarbital or 3-methylcholanthrene.(ABSTRACT TRUNCATED AT 250 WORDS)
Kikuta
Y
Y
Toneyama Institute for Tuberculosis Research, Osaka City University Medical School.
Kusunose
E
E
Okumoto
T
T
Kubota
I
I
Kusunose
M
M
D000595:Amino Acid Sequence; D000818:Animals; D042926:Cytochrome P-450 CYP4A; D003577:Cytochrome P-450 Enzyme System / Q000302:isolation & purification* / Q000378:metabolism*; D004051:Diethylhexyl Phthalate / Q000494:pharmacology; D005227:Fatty Acids / Q000378:metabolism*; D015151:Immunoblotting; D007527:Isoenzymes / Q000302:isolation & purification*; D008297:Male; D008862:Microsomes, Liver / Q000201:enzymology*; D006899:Mixed Function Oxygenases / Q000378:metabolism*; D008969:Molecular Sequence Data; D011454:Prostaglandins A / Q000378:metabolism*; D011817:Rabbits
D005227:Fatty Acids; D007527:Isoenzymes; D011454:Prostaglandins A; D003577:Cytochrome P-450 Enzyme System; D004051:Diethylhexyl Phthalate; D006899:Mixed Function Oxygenases; D042926:Cytochrome P-450 CYP4A