Purification and immunochemical characterization of the two adrenal cortex mitochondrial cytochrome P-450-proteins

Abstract

A procedure for the separation and purification of two distinct P-450 cytochromes, termed P-450 scc and P-450 11 β , solubilized from bovine adrenal cortex mitochondria is reported. Important features of the purification procedure are uses of aniline-substituted Sepharose chromatography and utilization of the markedly different characteristics in stability and solubility of each cytochrome. Polyacrylamide gel electrophoresis of the two purified preparations in sodium dodecyl sulfate reveals single protein bands. The P-450 scc, which catalyzes the formation of pregnenolone from cholesterol, has a turnover number of 16 mol of pregnenolone formed per minute per mole of P-450-heme. The P-450 11 β catalyzes the hydroxylation of deoxycorticosterone at 11β- and 18-positions with turnover numbers of 110 and 18, and of 4-androstene-3,17-dione at 11β- and 19-positions with turnover numbers of 41 and 12, respectively. The recoveries of the P-450 scc and P-450 11 β , in terms of the catalytic activities, are 20% and 15%, respectively, from the crude extract which contains the two activities in a ratio of roughly 2:1. Each rabbit antibody prepared against the two purified P-450-proteins interacts with respective, but not alternative cytochrome P-450, whether in the crude mitochondrial preparation or in the purified preparation. The observed patterns of immunoprecipitation and inhibition of catalytic activity indicated that the two P-450 proteins are immunochemically different from each other. Neither antibody immunoprecipitates with a highly purified bacterial cytochrome P-450, P-450 cam.