Radiometric assay for cytochrome P-450-catalyzed progesterone 16 alpha-hydroxylation and determination of an apparent isotope effect.
In the course of studies on the oxygenation of steroids by purified P-450 cytochromes, particularly rabbit liver microsomal cytochrome P-450 form 3b, a rapid and reliable radiometric assay has been devised for progesterone 16 alpha-hydroxylation. In view of the lack of a commercially available, suitably tritiated substrate, [1,2,6,7,16,17-3H]progesterone was treated with alkali to remove the label from potential hydroxylation sites other than the 16 alpha position. The resulting [1,7,16-3H]progesterone was added to a reconstituted enzyme system containing cytochrome P-450 form 3b, NADPH-cytochrome P-450 reductase, and NADPH, and the rate of 16 alpha-hydroxylation was measured by the formation of 3H2O. This reaction was shown to be linear with respect to time and to the cytochrome P-450 concentration. An apparent tritium isotope effect of 2.1 was observed by comparison of the rates of formation of tritium oxide and 16 alpha-hydroxyprogesterone, and the magnitude of the isotope effect was confirmed by an isotope competition assay in which a mixture of [1,7,16-3H]progesterone and [4-14C]progesterone was employed.
Osawa
Y
Y
Department of Biological Chemistry, Medical School, University of Michigan, Ann Arbor 48109.
Coon
M J
MJ
D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases; D002250:Carbon Radioisotopes; D003577:Cytochrome P-450 Enzyme System / Q000032:analysis*; D000072467:Cytochrome P450 Family 2; D006900:Hydroxylation; D006908:Hydroxyprogesterones / Q000096:biosynthesis; D066298:In Vitro Techniques; D008862:Microsomes, Liver / Q000201:enzymology; D011374:Progesterone; D011817:Rabbits; D011874:Radiometry / Q000379:methods*; D039181:Steroid 16-alpha-Hydroxylase; D014316:Tritium
D002250:Carbon Radioisotopes; D006908:Hydroxyprogesterones; D014316:Tritium; C018975:16-hydroxyprogesterone; D011374:Progesterone; D003577:Cytochrome P-450 Enzyme System; D001189:Aryl Hydrocarbon Hydroxylases; C450239:CYP2C3 protein, Oryctolagus cuniculus; D000072467:Cytochrome P450 Family 2; D039181:Steroid 16-alpha-Hydroxylase