Isolation and partial characterization of a cytochrome P-450 isoenzyme (cytochrome P-450 tu) from mouse liver tumors

Abstract

Experimental hepatomas induced with 5,9-dimethyldibenzo[ c,g]carbazole in female XVIInc/Z mice display a strong microsomal steroid 15α-hydroxylation activity. A cytochrome P-450 isoenzyme (cytochrome P-450 tu), specific for this activity, has been isolated by an HPLC derived method using various Fractogel TSK and hydroxyapatite supports. On SDS polyacrylamide gel electrophoresis the purified protein appeared as one major band with an apparent M r of 50000. Its specific cytochrome P-450 content was 7.55 nmol/mg protein. As deduced from the visible spectrum, the heme iron of the isolated P-450 tu was to 72% in the high-spin state. The CO-bound reduced form showed an absorption maximum at 450 nm. In addition to the stereospecific 15α-hydroxylation of progesterone (2.3 min −1) and testosterone (2.5 min −1), the enzyme catalyzed also 7-ethoxycoumarin O-deethylation, benzphetamine N-demethylation and aniline 4-hydroxylation. Its N-terminal amino-acid sequence (21 residues) was identical to that of cytochrome P-450 15 α , isolated by Harada and Negishi from liver microsomes of 129/J mice. P-450 tu differed from P-450 15 α by its higher molecular weight, its 40-times lower steroid 15α-hydroxylation and its 4-times higher benzphetamine N-demethylation.