Abstract
Abstract The liver microsomal hydroxylation system which metabolizes drugs and steroids was separated into fractions containing cytochrome, cytochrome reductase, and lipid, all of which are needed for maximal catalytic activity. The enzyme system isolated from rats treated with either phenobarbital or 3-methylcholanthrene has been fractionated, and the role of the hemoprotein, lipid, and reductase fractions in influencing substrate specificity has been studied. The results obtained indicate that the specificity for hydroxylation resides primarily in the cytochrome fraction, rather than in the reductase or lipid fraction. For the hydroxylation of 3,4-benzpyrene, the cytochrome P-448 fraction prepared from rats treated with 3-methylcholanthrene was much more active than the cytochrome P-450 fraction from phenobarbital-treated rats. In contrast, the cytochrome P-450 fraction was more active than the cytochrome P-448 fraction for benzphetamine N-demethylation. For chlorcyclizine N-demethylation, the cytochrome P-448 fraction was about 60 to 70% as effective as the cytochrome P-450 fraction. Studies on the hydroxylation of testosterone in the 6β,7α, and 16α positions revealed that the cytochrome P-450 and P-448 fractions were about equally active for either testosterone 6β- or 7α-hydroxylation, whereas the cytochrome P-448 fraction was only 10 to 20% as active as the cytochrome P-450 fraction for the 16α-hydroxylation reaction. These results indicate that the cytochrome P-450 and P-448 fractions have different substrate specificities and are consistent with the view that cytochromes P-450 and P-448 are different hemoproteins. Depending on the susbtrate used, the reductase fraction may also play a role in determining substrate specificity. The reductase fraction prepared from 3-methyl-cholanthrene-treated animals was only 50 to 60% as active as the reductase fraction from phenobarbital-treated rats in supporting benzphetamine N-demethylation, while the lipid fractions from both sources were equally active. No differences between the two reductase fractions or the two lipid fractions were observed for the hydroxylation of 3,4-benzpyrene.
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