High affinity binding of substrate and effector ligands to testicular microsomal cytochrome P-450.
The binding characteristics of substrate and effector ligands to testicular microsomal cytochrome P-450 (17 alpha-hydroxylase/C17-20 lyase) have been investigated by difference spectroscopy. Steroid products and their analogs induce oxygen-mediated damage of microsomal P-450 activities of cultured Leydig cells, whereas testosterone acetate protects P-450 from this damage [1]. Progesterone and 17 alpha-hydroxyprogesterone, the enzyme's substrates, bound stoichiometrically to microsomal P-450 with unusually high affinity (Kd = 23-51 nM). These properties of the enzyme may be responsible for the extremely efficient metabolism of these intermediates in androgen biosynthesis. The binding affinities of inhibitory effector ligands were determined in competition experiments with the substrates. Several steroids which have varying effects on damage of P-450 in cultured Leydig cells inhibited substrate binding similarly (KdI = 3.8-12.8 microM). Aminoglutethimide did not inhibit substrate binding. The results suggest that steroid binding to P-450 is necessary but not sufficient to increase oxygen free-radical damage of the testicular microsomal P-450. Testosterone acetate, which diminishes P-450 loss in Leydig cell cultures, was also bound stoichiometrically with high affinity (Kd = 17 nM) and produced a unique spectra, with maxima at 408 nm, an isosbestic point at 418 nm and minima at 428 nm. Therefore, the protection of P-450 in cultured Leydig cells probably results from binding of testosterone acetate at the active site of P-450.
D019326:17-alpha-Hydroxyprogesterone; D000616:Aminoglutethimide / Q000494:pharmacology; D000818:Animals; D001667:Binding, Competitive; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D006908:Hydroxyprogesterones / Q000378:metabolism; D008297:Male; D051379:Mice; D008861:Microsomes / Q000201:enzymology*; D011374:Progesterone / Q000378:metabolism; D013053:Spectrophotometry; D013737:Testis / Q000201:enzymology*; D013739:Testosterone / Q000031:analogs & derivatives / Q000378:metabolism
D006908:Hydroxyprogesterones; D000616:Aminoglutethimide; D013739:Testosterone; D011374:Progesterone; C040594:testosterone acetate; D019326:17-alpha-Hydroxyprogesterone; D003577:Cytochrome P-450 Enzyme System