Microsomal monooxygenase system in morris hepatoma: Purification and characterization of cytochromes p-450 from morris hepatoma 5123D of 3-methylcholanthrene-treated rats

Two forms of cytochrome P -450 (hepatoma P -450 MCI and P -450 MCII were purified from hepatoma 5123D microsomes of tumor-bearing rats treated with 3-methylcholanthrene. Hepatoma P -450 MCI had a specific content of 18.4 nmol/mg protein and showed a main protein band with a minimum molecular weight of 56,000 on sodium dodecyl sulfate-polyacrylamide gel. Hepatoma P -450 MCII had a specific content of 7.38 nmol/mg protein and a minimum molecular weight of 50,000. The carbon monoxidereduced difference spectral peak of hepatoma P -450 MCI was at 446.5 nm, whereas the peak of hepatoma P -450 MCII was at 451 nm. In the reconstituted system, hepatoma P -450 MCI catalyzed 3-hydroxylation of benzo[ a ]pyrene and O -deethylation of 7-ethoxycoumarin, but showed low activities for N -demethylation of benzphetamine and aminopyrine, O -demethylation of p -nitroanisole, and p -hydroxylation of aniline. On the other hand, hepatoma P -450 MCII did not catalyze hydroxylation of any of the substrates tested. By Ouchterlony double-diffusion analysis, hepatoma P -450 MCI was immunologically indistinguishable from rat liver cytochrome P -450 c , but hepatoma P -450 MCII was distinct from hepatoma P -450 MCI and rat liver cytochrome P -450 c . Peptide maps of hepatoma P -450 MCI and rat liver cytochrome P -450 c after proteolysis with Staphylococcus aureus V8 protease demonstrated the similarity of the two cytochromes P -450.