Studies on enzyme reactions related to steroid biosynthesis: II. submicrosomal distribution of the enzymes related to androgen production from pregnenolone and of the cytochrome P-450 in testicular gland of rat

Abstract A testicular microsomal fraction containing the enzyme systems necessary for the production of testosterone from pregnenolone was divided in two subfractions by sucrose density gradient centrifugation in the presence of CsCl. When the subfractions were examined by electron microscope, one consisted mainly of smooth-surfaced particles, the other of rough-surfaced particles with ribosomes on their outer surface. Enzymic and spectrometric investigation of these subfractions revealed that most of the enzyme activities related to the androgen synthesis, which remained after the gradient centrifugation, and also of the cytochrome P-450, which would be a direct site of activation of molecular oxygen for the 17α-hydroxylation and the side chain cleavage, were localized in the smooth-surfaced submicrosomal fraction. The enzyme activities of the submicrosomal fraction which were diminished by the gradient centrifugation were largely restored by addition of the heated 105,000 × g supernatant fluid. Enzyme inhibitors such as Amphenone B (3,3-bis ( p -aminophenyl)-2-butanone dihydrochloride) and SKF-525A (2-diethylaminoethyl 2.2-diphenylvalerate hydrochloride) inhibited the testicular microsomal 17α-hydroxylase and the C 17 –C 20 lyase competitively. The apparent inhibition constants of Amphenone B were 9.17 × 10 −5 M against 17 α-hydroxylase and 2.45 × 10 −4 M against C 17 -C 20 lyase, and those of SKF-525 A were 1.61 × 10 14 M against 17α-hydroxylase and 1.28 × 10 −5 M against the C 17 –C 20 lyase.