Hydroxylations in biosynthesis of bile acids. Isolation of a cytochrome P-450 from rabbit liver mitochondria catalyzing 26-hydroxylation of C27-steroids.
A cytochrome P-450 catalyzing 26-hydroxylation of C27-steroids was purified from liver mitochondria of untreated rabbits. The enzyme fraction contained 10 nmol of cytochrome P-450/mg of protein and showed only one protein band with a minimum Mr = 53,000 upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified mitochondrial cytochrome P-450 showed apparent molecular weight similar to microsomal cytochromes P-450LM4 but differed in spectral and catalytic properties from these microsomal isozymes. The purified cytochrome P-450 catalyzed 26-hydroxylation of cholesterol, 5-cholestene-3 beta,7 alpha-diol, 7 alpha-hydroxy-4-cholesten-3-one, 5 beta-cholestane-3 alpha,7 alpha-diol, and 5 beta-cholestane-3 alpha,7 alpha,12 alpha-triol up to 1000 times more efficiently than the mitochondria. The cytochrome P-450 required both ferredoxin and ferredoxin reductase for catalytic activity. Microsomal NADPH-cytochrome P-450 reductase could not replace ferredoxin and ferredoxin reductase. The cytochrome P-450 was inactive in 7 alpha-, 12 alpha- and 25-hydroxylations of C27-steroids. The results suggest that mitochondrial 26-hydroxylation of various C27-steroids is catalyzed by the same species of cytochrome P-450.
D000302:Adrenal Cortex / Q000378:metabolism; D000818:Animals; D001647:Bile Acids and Salts / Q000096:biosynthesis*; D003577:Cytochrome P-450 Enzyme System / Q000302:isolation & purification / Q000378:metabolism*; D005287:Ferredoxin-NADP Reductase / Q000378:metabolism; D005288:Ferredoxins / Q000378:metabolism; D006900:Hydroxylation; D007700:Kinetics; D008928:Mitochondria / Q000378:metabolism; D008930:Mitochondria, Liver / Q000378:metabolism*; D009251:NADPH-Ferrihemoprotein Reductase / Q000378:metabolism; D011817:Rabbits; D013053:Spectrophotometry; D013379:Substrate Specificity
D001647:Bile Acids and Salts; D005288:Ferredoxins; D003577:Cytochrome P-450 Enzyme System; D005287:Ferredoxin-NADP Reductase; D009251:NADPH-Ferrihemoprotein Reductase