Immunochemical studies on two electrophoretically homogeneous forms of rabbit liver microsomal cytochrome P-450: P-450LM2 and P-450LM4.

Two electrophoretically homogeneous forms of liver microsomal cytochrome P-450 (P-~~OLM), designated P-450~~~ and LM~ by their relative mobilities, were previously isolated from rabbits induced with phenobarbital and P-naphthoflavone, respectively. In the present immunochemical study, rabbits and goats were inoculated with these purified proteins. Rabbits produced antibodies against P-450~~~ but not against P-450~M,, whereas goats produced antibodies against both cytochromes. Antibodies against P-450~~~ were purified from antkw, serum by preparation of the P450Lm,-antibody precipitate. The ability of Fab’ fragments prepared from the purified antibodies to bind to P-450~~~ was studied by fluorescence quenching, and it was found that about three Fab’ fragments from rabbit and ten from goat were bound per molecule of cytochrome. In Ouchterlony double diffusion studies, all antisera yielded only one precipitin band with both purified and crude preparations of the corresponding antigen, thereby providing additional evidence for the homogeneity of the cytochromes. No cross-reactions observable by precipitin band formation were detected between anti-LM, sera and P~~OLM*, or between anti+& serum and P-450~~~. Competitive binding studies with radiolabeled cytochromes confirmed that rabbit anti-Lm, does not cross-react with P450~~~; however, slight but significant cross-reactions were detected by this technique between goat anti-Lm, and P~~OLM~, and between goat anti-LMI and P-450~~~. These results indicate that the two cytochromes have significant structural differences. Benzphetamine binding, P-450~~ reduction by NADPH in the presence of the reductase, and benzphetamine hydroxylation were all strongly inhibited by goat anti-LM3 Fab’ fragments in a reconstituted enzyme system containing P~~OLM*, whereas in intact microsomes benzphetamine binding was inhibited much less than the other two activities.