Abstract
Hepatic microsomal cytochrome P-450 and P-448 have been purified from phenobarbital(PB) - and 3-methylcholanthrene (MC) -treated rats, by modifications of Imai and Sato' sprocedures (1974). The purified preparations of cytochrome P-450 and P-448 were homogeneousjudging from their specific contents (17 and 16 nmoles per mg protein, respectively),and the results of SDS-polyacrylamide gel electrophoresis and Ouchterlony immunodiffusionanalyses. These two cytochromes have are different in their physico-chemical and immunologicalproperties, and their substrate specificities.In reconstituted systems containing the purified cytochrome and NADPH-cytochrome P-450reductase, ethoxycoumarin deethylation and benzo (a) pyrene hydroxylation catalyzed by cytochromeP-450 and P-448 were completely inhibited by their homologous antibody, while essentiallyno effect was observed with heterologous combinations of antigen and antibody. In contrast,the benzphetamine demethylation activities of cytochrome P-450 and P-448 are markedlyinhibited by the heterologous antibody as well as by the homologous one. These results suggestthat the two cytochromes are immunologically different but have some antigenic determinantsin common.Drug metabolizing activities of microsomes from PB- and MC-treated rats were inhibited bythe antibodies essentially as expected from the results with the reconstituted systems. Theremaining activities in the presence of excess concentrations of the antibody, however, arehigher in MC microsomes treated with anti P-448 antibody than in PB microsomes treated withanti P-450 antibody. These results suggest that cytochrome P-448 molecules may be so localizedin the microsomal membrane so that the membrane structure may hinder the access of theantibody to the antigenic determinant.
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