Reconstitution of Steroid 17,20-Lyase Activity after Separation and Purification of Cytochrome P-450 and Its Reductase from Rat Testis Microsomes*

Abstract

The testicular enzyme, 17,20-lyase, catalyzes the removal of the C-17 side chain from steroids in the synthesis of androgens. This activity employs cytochrome P-450 as an oxygen donor. Attempts to purify the cytochrome and its reductase from testis microsomes have previously been unsuccessful due to the low concentrations of these components (2–5% that of liver).The cytochrome and reductase were solubilized from rat testis microsomes using a mixture of sodium cholate and Emulgen 913. The components were then separated by DEAE chromatography. The cytochrome was further purified by chromatographyusing hydroxylapatite for an 8.5-fold enrichment. The reductase was further purified by hydroxylapatite and affinity chromatography. An 84-fold enrichment was achieved. 17,20-Lyase activitycould be partially restored by mixing the cytochrome and reductase in the presence of phospholipid.