Separation and characterization of highly purified forms of liver microsomal cytochrome P-450 from rats treated with polychlorinated biphenyls, phenobarbital, and 3-methylcholanthrene.

Abstract

Highly purified cytochromes P-450, designated P450,, P-450b, and P-450,, have been isolated from hepatic microsomes of rats treated with the polychlorinated biphenyl mixture Aroclor 1254. Each form shows a single protein-staining band on sodium dodecyl sulfate-polyacrylamide gels with minimum molecular weights of 48,000 (P-450,), 52,000 (P-450& and 56,000 (P-450,). Cytochromes P-450, and P-450,, have been obtained from phenobarbital-treated rats, and cytochromes P-450, and P-450, were isolated from 3-methylcholanthrene-treated rats. Cytochrome P-450,, is the major hemeprotein inducible by phenobarbital, and cytochrome P-450, is the predominant hemeprotein induced by 3-methylcholanthrene. The CO-reduced difference spectral peak of cytochrome P-450, is at 452 nm, P-45Ob is at 450 nm, and P-450, is at 447 nm. Cytochrome P-450, preferentially hydroxylates testosterone at the 7a: position but has low catalytic activity for the metabolism of benzphetamine, benzo[a]pyrene, 7-ethoxycoumarin, and zoxazolamine. Cytochrome P-450,, catalyzes the hydroxylation of testosterone at the 16a: position and the demethylation of benzphetamine most efficiently. Cytochrome P-450, has very high catalytic activity for benzo[a]pyrene and zoxazolamine hydroxylation and 7-ethoxycoumarin O-dealkylation. Aniline is hydroxylated by the three forms of cytochrome P-450 at comparable rates. Antisera prepared against cytochrome P-450,, P-450,,, or P-450, do not cross-react with the heterologous antigens in Ouchterlony double diffusion plates. When cytochromes P-450,, P-450b, and P-450,, were subjected to limited proteolysis in the presence of sodium dodecyl sulfate, the resulting peptide maps provided additional evidence that these hemeproteins are structurally distinct. The forms of cytochrome P-450 obtained from phenobarbitaland 3-methylcholanthrene-treated rats are indistinguishable from the corresponding forms from Aroclor 1254~treated rats by all criteria examined.