Characterization of three forms of cytochrome P-450 isolated from liver microsomes of rats treated with 3-methylcholanthrene.

Abstract

Three forms of cytochrome P-450, designated as P-450MC-I, P-450MC-II, and P-450MC-III, were isolated from liver microsomes of rats treated with 3-methylcholanthrene (MC) by using a high performance liquid chromatography (HPLC) technique. The major MC-inducible forms, P-450MC-I and P-450MC-II showed a single protein band on SDS-polyacrylamide gel electrophoresis giving a minimum molecular weight of 56,000 daltons. The oxidized absolute spectra of both cytochromes P-450 were of low spin type, having a Soret absorption peak at 417 nm. The CO-reduced difference spectra of these two cytochromes P-450 showed a peak at 447 nm. In a reconstituted system, both cytochromes P-450 exhibited similar high levels of catalytic activity for benzo(a)pyrene hydroxylation and 7-ethoxycoumarin O-deethylation. Anti-P-450MC-I IG and anti-P-450MC-II IG, which were produced against the corresponding cytochromes P-450, each formed a single continuous precipitin line with both P-450MC-I and P-450MC-II in Ouchterlony double diffusion tests. Amino acid sequence analysis revealed that the sequence of the NH2-terminal 18 amino acids of both enzymes was the same. Therefore, the major MC-inducible forms, P-450MC-I and P-450MC-II, were highly homologous, being indistinguishable from each other in terms of apparent molecular weight, spectral properties, substrate specificity and the NH2-terminal 18 amino acid residues, but clearly separable by HPLC. The characteristics of both P-450 forms appear to correspond to those of the previously reported P-450c (1). On the other hand, a minor form, P-450MC-III was different from P-450MC-I and P-450MC-II in chromatographic properties, apparent molecular weight, substrate specificity and immunochemical properties, and did not correspond to any P-450 species previously purified from MC-treated rat liver microsomes.