Studies on cytochrome P-450-dependent microsomal enzymes of testicular androgen and estrogen biosynthesis in a urodele amphibian, Necturus.
The microsomal fraction isolated from the testis of the urodele amphibian, Necturus maculosus, is very rich in cytochrome P-450 and three cytochrome P-450-dependent steroidogenic enzyme activities, 17 alpha-hydroxylase, C-17, 20-lyase, and aromatase. In this study, we investigated aspects of these reactions using both spectral and enzyme techniques. In animals obtained at different points in the annual cycle, Necturus testis microsomal P-450 concentrations ranged from 0.6-1.8 nmol/mg protein. Substrates for the three enzymes generated type I difference spectra; progesterone and 17 alpha-hydroxyprogesterone appeared to bind to one P-450 species while the aromatase substrates, androstenedione, 19-hydroxyandrostenedione, and testosterone, all bound to another P-450 species. Spectral binding constants (Ks) for these interactions were determined. Michaelis constants (Km) and maximum velocities were determined for progesterone 17 alpha-hydroxylation, 17 alpha-hydroxyprogesterone side-chain cleavage, and for the aromatization of androstenedione, 19-hydroxyandrostenedione, and testosterone. Measured either by spectral or kinetic methods, progesterone, androstenedione, and 19-hydroxyandrostenedione were high affinity substrates (Ks or Km less than 0.3 microM), while 17 alpha-hydroxyprogesterone and testosterone were low affinity substrates (Ks or Km = 0.6-4.8 microM). As evidence for the participation of cytochrome P-450 in these reactions, carbon monoxide was found to inhibit each of the enzyme activities studied. The activity of NADPH-cytochrome c reductase, a component of cytochrome P-450-dependent reactions, was also high in Necturus testis microsomes.
D000728:Androgens / Q000096:biosynthesis; D000735:Androstenedione / Q000378:metabolism; D000818:Animals; D001141:Aromatase / Q000378:metabolism; D002248:Carbon Monoxide / Q000494:pharmacology; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D004967:Estrogens / Q000096:biosynthesis; D006801:Humans; D007700:Kinetics; D008297:Male; D008861:Microsomes / Q000201:enzymology; D009251:NADPH-Ferrihemoprotein Reductase / Q000378:metabolism; D009337:Necturus / Q000378:metabolism*; D011374:Progesterone / Q000378:metabolism; D013254:Steroid 17-alpha-Hydroxylase / Q000378:metabolism; D013256:Steroids / Q000378:metabolism; D013737:Testis / Q000201:enzymology*; D013739:Testosterone / Q000378:metabolism
D000728:Androgens; D004967:Estrogens; D013256:Steroids; D013739:Testosterone; D000735:Androstenedione; D011374:Progesterone; D002248:Carbon Monoxide; D003577:Cytochrome P-450 Enzyme System; D001141:Aromatase; D013254:Steroid 17-alpha-Hydroxylase; D009251:NADPH-Ferrihemoprotein Reductase