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.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001647:Bile Acids and Salts / Q000096:biosynthesis*; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D006900:Hydroxylation; D007527:Isoenzymes / Q000302:isolation & purification / Q000378:metabolism; D007700:Kinetics; D008297:Male; D009251:NADPH-Ferrihemoprotein Reductase / Q000378:metabolism; D011817:Rabbits; D013053:Spectrophotometry; D013379:Substrate Specificity
D000596:Amino Acids; D001647:Bile Acids and Salts; D007527:Isoenzymes; D003577:Cytochrome P-450 Enzyme System; D009251:NADPH-Ferrihemoprotein Reductase