Hydroxylations in biosynthesis of bile acids. Isolation of subfractions with different substrate specificity from cytochrome P-450LM4.

Chromatography of electrophoretically homogeneous cytochrome P-450LM4 from cholestyramine-treated rabbits on octylamine-Sepharose resulted in the isolation of two subfractions, cytochrome P-450LM4 I and cytochrome P-450LM4 II, with different catalytic properties. The original cytochrome P-450LM4 fraction catalyzed 7 alpha-hydroxylation of cholesterol, 12 alpha-hydroxylation of 5 beta-cholestane-3 alpha, 7 alpha-diol, 25-hydroxylation of 5 beta-cholestane-3 alpha, 7 alpha-diol and 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha-triol, 6 beta-hydroxylation of testosterone, and demethylation of ethylmorphine. Cytochrome P-450LM4 I was inactive in cholesterol 7 alpha-hydroxylation, but catalyzed the other hydroxylations. Cytochrome P-450LM4 II catalyzed efficient cholesterol 7 alpha-hydroxylation. It also catalyzed the other hydroxylations, although at lower rates than cytochrome P-450LM4 I. Emulgen inhibited all steroid hydroxylase activities in cytochrome P-450LM4 II except the cholesterol 7 alpha-hydroxylase activity. Cytochrome P-450LM4 I and cytochrome P-450LM4 II showed the same apparent molecular weight and spectral properties as the original cytochrome P-450LM4 fraction. The two subfractions differed in amino acid composition. They produced similar but not identical one-dimensional peptide maps upon limited proteolysis with papain, chymotrypsin, and trypsin. The results show that cytochrome P-450LM4 from cholestyramine-treated rabbits contains at least two species with different amino acid compositions and different substrate specificities toward C27-steroids involved in biosynthesis of bile acids.