Reconstitution of steroid 17,20-lyase activity after separation and purification of cytochrome P-450 and its reductase from rat testis microsomes.
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 chromatography using 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 activity could be partially restored by mixing the cytochrome and reductase in the presence of phospholipid.
D000446:Aldehyde-Lyases / Q000302:isolation & purification / Q000378:metabolism*; D000818:Animals; D003577:Cytochrome P-450 Enzyme System / Q000302:isolation & purification / Q000378:metabolism*; D006908:Hydroxyprogesterones / Q000302:isolation & purification / Q000378:metabolism; D007700:Kinetics; D008297:Male; D008861:Microsomes / Q000201:enzymology*; D009251:NADPH-Ferrihemoprotein Reductase / Q000302:isolation & purification / Q000378:metabolism*; D051381:Rats; D013254:Steroid 17-alpha-Hydroxylase; D013737:Testis / Q000201:enzymology*
D006908:Hydroxyprogesterones; D003577:Cytochrome P-450 Enzyme System; D013254:Steroid 17-alpha-Hydroxylase; D009251:NADPH-Ferrihemoprotein Reductase; D000446:Aldehyde-Lyases