Sex differences in cytochrome P-450-dependent 25-hydroxylation of C27-steroids and vitamin D3 in rat liver microsomes.
Monoclonal antibodies directed against the cytochrome P-450 catalyzing 25-hydroxylation of C27-steroids and vitamin D3 (Andersson, S., Holmberg, I., and Wikvall, K. (1983) J. Biol. Chem. 258, 6777-6781) have been prepared by immunization of mice in hind footpads. Lymph node cells from the mice were fused with the Sp 2/0-Ag 14 line of mouse myeloma cells. A monoclonal antibody, designated MAb-25-6, monospecific for cytochrome P-450(25) was, after coupling to Sepharose, able to bind to cytochrome P-450(25) and to immunoprecipitate the activity for 25-hydroxylation of 5 beta-cholestane-3 alpha,7 alpha,12 alpha-triol and vitamin D3 as well as that for 16 alpha-hydroxylation of testosterone when assayed in a reconstituted system. Two-dimensional gel electrophoresis of adult male rat liver microsomes and immunoblotting with MAb-25-6 showed a single spot with an apparent isoelectric point of 7.4 and Mr approximately 51,000. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting with MAb-25-6, cytochrome P-450(25) was shown to be male-specific and not detectable in adult female rat liver microsomes. N-terminal sequence analysis of cytochrome P-450(25) showed a structure identical with that of the male-specific steroid 16 alpha-hydroxylase isolated in several laboratories.
D000818:Animals; D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D001189:Aryl Hydrocarbon Hydroxylases; D002762:Cholecalciferol / Q000378:metabolism*; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D000072467:Cytochrome P450 Family 2; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D006900:Hydroxylation; D007700:Kinetics; D008297:Male; D008862:Microsomes, Liver / Q000187:drug effects / Q000378:metabolism*; D010634:Phenobarbital / Q000494:pharmacology; D051381:Rats; D012737:Sex Factors; D039181:Steroid 16-alpha-Hydroxylase; D013256:Steroids / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D013256:Steroids; D002762:Cholecalciferol; D003577:Cytochrome P-450 Enzyme System; D001189:Aryl Hydrocarbon Hydroxylases; C450279:CYP2C11 protein, rat; D000072467:Cytochrome P450 Family 2; D039181:Steroid 16-alpha-Hydroxylase; D010634:Phenobarbital