Purification and partial characterization of hepatic microsomal cytochrome P-450s from phenobarbital- and 3-methylcholanthrene-treated rats.
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's procedures )1974). The purified preparations of cytochrome P-450 and P-448 were homogeneous judging from their specific contents (17 and 16 nmol per mg protein, respectively) and the results of SDS-polyacrylamide gel electrophoresis and Ouchterlony immunodiffusion analyses. These two cytochromes are different in their physico-chemical and immunological properties, and their substrate specificities. In reconstituted systems containing the purified cytochrome and NADPH-cytochrome P-450 reductase, ethoxycoumarin deethylation and benzo(a)pyrene hydroxylation catalyzed by cytochrome P-450 and P-448 were completely inhibited by the homologous antibody, while essentially no effect was observed with heterologous conbinations of antigen and antibody. In contrast, the benzphetamine demethylation activities of cytochrome P-450 and P-448 were markedly inhibited by the heterologous antibody as well as by the homologous one. These results suggest that the two cytochromes are immunologically different but have some antigenic determinants in common. Drug metabolizing activities of microsomes from PB- and MC-treated rats were inhibited by the antibodies, essentially as expected from the results with the reconstituted systems. The remaining activities in the presence of excess concentrations of the antibody, however, were higher in MC-microsomes treated with anti P-448 antibody than in PB microsomes treated with anti P-450 antibody. These results suggest that cytochrome P-448 molecules may be so localized in the microsomal membrane that the membrane structure may hinder the access of the antibody to the antigenic determinant.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D000906:Antibodies; D003577:Cytochrome P-450 Enzyme System / Q000302:isolation & purification / Q000378:metabolism*; D007118:Immunoassay; D005779:Immunodiffusion; D007700:Kinetics; D008297:Male; D008748:Methylcholanthrene / Q000494:pharmacology*; D008862:Microsomes, Liver / Q000187:drug effects / Q000378:metabolism*; D009251:NADPH-Ferrihemoprotein Reductase / Q000378:metabolism; D010634:Phenobarbital / Q000494:pharmacology*; D051381:Rats; D013053:Spectrophotometry; D013379:Substrate Specificity
D000596:Amino Acids; D000906:Antibodies; D008748:Methylcholanthrene; D003577:Cytochrome P-450 Enzyme System; D009251:NADPH-Ferrihemoprotein Reductase; D010634:Phenobarbital