Hydroxylations in Biosynthesis of Bile Acids. Cytochrome P-450 LM4 and 12alpha-Hydroxylation of 5beta-Cholestane-3alpha,7alpha-diol

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Abstract
12.alpha.-Hydroxylation of 5.beta.-cholestane-3.alpha.,7.alpha.-diol was studied in reconstituted systems consisting of electrophoretically homogeneous cytochrome P-450 LM4 fractions and NADPH-cytochrome P-450 reductase from rabbit liver microsomes. Cytochrome P-450 LM4 fractions were prepared from untreated, phenobarbital-treated, .beta.-naphthoflavone-treated and starved rabbits. The purified cytochromes catalyzed 12.alpha.-hydroxylation more efficiently than the corresponding microsomes. In the reconstituted systems, CO inhibited 12.alpha.-hydroxylation by 50-80%. The rate of 12.alpha.-hydroxylation was 3-4 times higher with cytochrome P-450 LM4 fractions from starved rabbits than with cytochrome P-450 LM4 fractions from untreated, phenobarbital-treated and .beta.-naphthoflavone-treated animals. Amino acid analyses, peptide mapping experiments, as well as absorption spectral and circular dichroism spectral analyses, revealed physical differences between cytochrome P-450 LM4 fractions starved animals and preparations from phenobarbital-treated animals. The results indicate the presence of a cytochrome P-450 species in the cytochrome P-450 LM4 fraction specific for 12.alpha.-hydroxylation.