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Abstract

Two forms of cytochrome P-450 (P-450), designated as P-450LPGω1 and P-450LPGaω2, 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), aperoxisomal proliferator. The purified P-450Lpgawi and P-450LPGAω2 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 co-hydroxylation of prostaglan-dins A, (PGA1) and A2 (PGA2), as well as the ω- and (ω>-l)-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 ALSLTRLPGSFS-GFLQAxGLLGLLL) of P-450LPCAω1 and P-450LPGAω2 were identical at 18/20 and 19/24 positions with that of the lung prostaglandin ω-hydroxylase from pregnant rabbits, respectively. An antibody against P-450LPgaω2 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. The laurate and PGA <ω-hydroxylase activities of liver microsomes from both untreated and DEHP-treated rabbits were markedly inhibited by pretreatment with the antibody against P-450LPCaωi or P-450LPCAω2. The results suggest that the two P-450s are responsible for a major portion of the fatty acid and PGA eu-hydroxylase activities in rabbit liver microsomes. To our knowledge, this is the first time that P-450s specific for <ω-hydroxylase activities have been isolated from liver microsomes of DEHP-treated rabbits.

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© COPYRIGHT, 1990 BY THE JOURNAL OF BIOCHEMISTRY
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