Multiple Forms of Cytochrome P‐450 in Rat‐Liver Microsomes: Separation and Some Properties of Different Hydroxylases Active on Free and Sulphoconjugated Steroids

Cytochrome P 450 from male and female rat liver microsomes has been solubilized with sodium deoxycholate, precipitated with ammonium sulphate and separated in the presence of deoxycolate into ten different fractions on a DEAE cellulose column eluted with a stepwise gradient of KCl. Each fraction was characterized with respect to its ability to catalyze different hydroxylation reactions of free and disulphurylated 5α androstane 3α,17β diol, using a reconstituted system. All hydroxylases active on 5α androstane 3α,17β diol (i.e., the 2α, 2β, 7α, 7β, 12β, 15α, 16α and 18 hydroxylases) and 5α androstane 3α,17β diol, 3,17 disulphate (i.e., the 15β hydroxylase) were solubilized with cholate. However, the 2β and 18 hydroxylases were partially inactivated when cholate was added to intact microsomes and these were also the only hydroxylase activities that could not be detected in reconstitution experiments with lipid, NADPH cytochrome P 450 reductase and the cytochrome P 450 fractions recovered after DEAE cellulose chromatography. Using microsomal preparations from male rat liver it was possible to obtain a partial separation of the 2α and 7α hydroxylases from the 12β, 15α and 16α hydroxylases and also from the 7β hydroxylase. With preparations from female rat livers the 15β hydroxylase was well separated from the 2α and 7α hydroxylases. The specific activities of the partially separated 2α, 7α, 7β, 12β, 15α an 16a hydroxylases calculated per nmol of cytochrome P 450 was 9 to 34% of the original activities in sonicated microsomes. There was an absolute requirement of the cytochrome P 450 fractions for all hydroxylase activities except for the 2α hydroxylase activity. The requirement for NADPH cytochrome P 450 reductase was not absolute for any of the hydroxylase activities and no lipid dependence was observed. Based on their behavior during solubilization and purification, elution pattern of the DEAE cellulose column and different modes of regulation, the various hydroxylases studied can be divided into different groups. It is suggested that one form of cytochrome P 450 catalyzes the hydroxylation of 5α androstane 3α,17β diol in the 2β and 18 positions, another form the 12β, 15α and 16α hydroxylations of the same substrate and a third form the 15β hydroxylation of 5α androstane 3α,17β diol 3,17 disulphate. It is concluded that rat liver microsomes contain multiple forms of cytochrome P 450 active on steroid hormones. It is suggested that the specificity of these forms is determined by the nature of the substrate binding site and by the distance between the binding and catalytic sites on the enzyme.