SUBMICROSOMAL DISTRIBUTION OF ADRENAL ENZYMES AND CYTOCHROME P-450 RELATED TO CORTICOIDOGENESIS

By density-gradient centrifugation in the absence of CsCl, the microsomal fractions obtained from porcine and rat adrenal glands were separated into two subfractions: one fraction consisted of smooth-surfaced membrane structures which bore no ribosomes on their outer surface, and the other consisted of abundant free ribosomes with membrane structures, according to electron microscopic observation. The activities of three microsomal enzymes related to corticoidogenesis, namely Δ5-3β-hydroxysteroid dehydrogenase (EC 1.1.1.51) coupled with the Δ5-Δ4 isomerase (EC 5.3.3.1), 17α-hydroxylase (EC 1.14.1.7) and 21-hydroxylase (EC 1.14.1.8), were predominantly located in the smooth-surfaced subfraction. In accordance with the distribution of the hydroxylases, adrenal microsomal cytochrome P-450 was concentrated exclusively in the smooth-surfaced subfraction. As 21-hydroxylation was significantly inhibited in a CO-containing atmosphere, the cytochrome P-450 in the smooth-surfaced subfraction was participating as the direct activating site of O2 during 21-hydroxylation. The Km values of the submicrosomal Δ5-3β-hydroxysteroid dehydrogenase for pregnenolone and of 17α-hydroxylase for progesterone were estimated as 0.2 and 0.13 mM, respectively. The Km of the adrenal 21-hydroxylase was evaluated as 99 μM for progesterone and as 0.22 mM for 17α-hydroxyprogesterone. Substrate specificities of the adrenal submicrosomal enzymes, dynamic analysis of the corticoidogenesis throughout the incubation time, and the relationship between the amounts of enzyme preparations and the products, were examined. © 1969.