Sexual differentiation of cytochrome P-450 in rat liver. Evidence for a constitutive isozyme as the male-specific 16 alpha-hydroxylase.
Cytochrome P-450 isozyme RLM5 [Cheng, K-C., and J.B. Schenkman, J. Biol. Chem. 257:2378-2385 (1982)], possessing a high steroid 16 alpha-hydroxylating activity, was isolated from male rat livers, and the hypothesis that this protein is the sexually differentiated 16 alpha-hydroxylase was tested. Isozyme RLM5 was not detected when the same purification procedure was applied to female rat liver microsomes. However, a female isozyme named DEa and possessing a similar Mr was obtained from similar column fractions. The DEa isozyme had insignificant steroid-hydroxylating activity and a substrate specificity different from isozyme RLM5. The two proteins could also be distinguished in their primary structures and immunochemical properties. Rabbit antibodies were raised to isozyme RLM5 and were made specific by immunoabsorption with a crude female cytochrome P-450 fraction coupled to Affi-Gel 10. The antibodies were used in a Western blot immunoassay to demonstrate that isozyme RLM5 can be detected in liver microsomes of male rats at levels at least 20 times higher than those in the female, and that its sexual differentiation is neonatally imprinted by androgen. The antibodies were able to specifically inhibit 70% of the testosterone 16 alpha-hydroxylase activity in male rat liver microsomes but had no effect on the activity in females. It was concluded that isozyme RLM5 is the major sexually differentiated microsomal 16 alpha-hydroxylase.
D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases; D002852:Chromatography, Ion Exchange; D002918:Chymotrypsin / Q000378:metabolism; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D007527:Isoenzymes / Q000378:metabolism*; D008099:Liver / Q000196:embryology / Q000201:enzymology*; D008297:Male; D008862:Microsomes, Liver / Q000201:enzymology; D010206:Papain / Q000378:metabolism; D010446:Peptide Fragments / Q000032:analysis; D051381:Rats; D012733:Sex Differentiation; D039181:Steroid 16-alpha-Hydroxylase; D013250:Steroid Hydroxylases / Q000378:metabolism*; D013739:Testosterone / Q000378:metabolism
D007527:Isoenzymes; D010446:Peptide Fragments; D013739:Testosterone; D003577:Cytochrome P-450 Enzyme System; D013250:Steroid Hydroxylases; D001189:Aryl Hydrocarbon Hydroxylases; D039181:Steroid 16-alpha-Hydroxylase; D002918:Chymotrypsin; D010206:Papain