Hepatic microsomal cytochrome P-450 from rats treated with isosafrole. Purification and characterization of four enzymic forms.

Abstract

A unique form of liver microsomal cytochrome P-450 induced in rats treated with the methylenedioxyphenyl compound, isosafrole, has been purified to apparent homogeneity.The highly purified hemoprotein, designated cytochrome P-450d, shows a single major proteinstaining band on sodium dodecyl sulfate-polyacrylamide gels, and this cytochrome has a minimum molecular weight of 52,000.Cytochrome P-450d is purified as an isosafrole metabolite -cytochrome P-450 complex; addition of 7-ethoxycoumarin to the oxidized hemoprotein displaces some, but not all, of the bound metabolite.Another microsomal enzyme, epoxide hydrolase, can be obtained and purified during the isolation of cyto- chrome P-450d.Three other hemoproteins, cytochromes P-450,, P-450b, and P-450,, have also been isolated from rats treated with isosafrole; these enzymes appear to be free of the methylenedioxyphenyl metabolite.Cytochrome P-450d has different chromatographic properties than the other hemoproteins on DEW-cellulose.If cytochromes P-450b and P-450d are mixed and electrophoresed in the sodium dodecyl sulfate-polyacrylamide gel system of Laemmli ((1970) Nature 227, 680-685), * The costs of publication of this article were defrayed in part by the payment of page charges.This article must therefore be hereby marked "nduertisernmt" in accordance with 18 U.S.C.Section 1734 solely to indicate this fact.' The term cytochrome P-450 is used to designate any or all forms of microsomal cytochrome P-450.In rats, cytochrome P-450h is the major hemoprotein induced by phenobarbital; cytochrome P-450, is the predominant hemoprotein inducible by 3-methylcholanthrene; and cytochrome P-450, is a minor form induced by both compounds (18).Cytochrome P-450, is also known as cvtochrome P-448 (19) and cytochrome Pi450 (20).'The abbreviations used are: PB, phenobarbital; SDS, sodium dodecyl sulfate; 3-MC, 3-methylcholanthrene.