Multiple Forms of Cytochrome P ‐450 in Rat‐Liver Microsomes

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 deoxycholate into ten different fractions on a DEAE‐cellulose column eluted with a stepwise gradient of KCI. 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‐ and 16α‐hydroxylases calculated per nmol of cytochrome P‐450 was 9–34% 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 dependency 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