Two forms of
cytochrome P-450 (
hepatoma P-450MCI and P-450MCII) were purified from
hepatoma 5123D microsomes of
tumor-bearing rats treated with
3-methylcholanthrene.
Hepatoma P-450MCI 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-450MCII had a specific content of 7.38 nmol/mg
protein and a minimum molecular weight of 50,000. The
carbon monoxide-reduced difference spectral peak of
hepatoma P-450MCI was at 446.5 nm, whereas the peak of
hepatoma P-450MCII was at 451 nm. In the reconstituted system,
hepatoma P-450MCI 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-450MCII did not catalyze hydroxylation of any of the substrates tested. By Ouchterlony double-diffusion analysis,
hepatoma P-450MCI was immunologically indistinguishable from rat liver
cytochrome P-450c, but
hepatoma P-450MCII was distinct from
hepatoma P-450MCI and rat liver
cytochrome P-450c.
Peptide maps of
hepatoma P-450MCI and rat liver
cytochrome P-450c after proteolysis with Staphylococcus aureus V8
protease demonstrated the similarity of the two
cytochromes P-450.