Multiple forms of cytochrome P-450 in liver microsomes from beta-naphthoflavone-pretreated rats. Separation, purification, and characterization of five forms.

Abstract

Five forms of cytochrome P-450 have been purified from liver microsomes of beta-naphthoflavone-pretreated rats by chromatography on DEAE-Sephadex, DEAE-cellulose, and hydroxylapatite or CM-Sepharose columns. Over 50% of the starting cytochrome P-450 content can be accounted for in these five forms after resolution on the DEAE-cellulose column, and after further purification, the combined total recovery is 30%. The five forms have the following Mr: 47,000, 50,500, 51,500, 53,500, and 56,500. The absorption maxima in reduced carbon monoxide difference spectra are 452.5, 449, 449, 447.5, and 447.5 nm, respectively. Antibody has been prepared in rabbits to each of the five forms; each antibody reacts with the antigen for which it was prepared, but not with the other four heterologous antigens. In addition, each form gives a unique peptide map pattern when partially digested with Staphylococcus aureus V-8 protease and electrophoresed in sodium dodecyl sulfate gels. Each form also shows an individual pattern of catalytic activities when tested with benzphetamine, ethylmorphine, p-nitroanisole, benzo[alpha]pyrene, and 7-ethoxycoumarin as substrates. By all criteria examined, these five forms appear to be distinct forms of cytochrome P-450.