Characterization of pig kidney microsomal cytochrome P-450 catalysing 25-hydroxylation of vitamin D3 and C27 steroids.
The cytochrome P-450 enzyme which catalyses 25-hydroxylation of vitamin D3 (cytochrome P-450(25] from pig kidney microsomes [Postlind & Wikvall (1988) Biochem. J. 253, 549-552] has been further purified. The specific content of cytochrome P-450 was 15.0 nmol.mg of protein-1, and the protein showed a single spot with an apparent isoelectric point of 7.4 and an Mr of 50,500 upon two-dimensional isoelectric-focusing/SDS/PAGE. The 25-hydroxylase activity towards vitamin D3 was 124 pmol.min-1.nmol of cytochrome P-450-1 and towards 1 alpha-hydroxyvitamin D3 it was 1375 pmol.min-1.nmol-1. The preparation also catalysed the 25-hydroxylation of 5 beta-cholestane-3 alpha,7 alpha-diol at a rate of 1000 pmol.min-1.nmol of cytochrome P-450-1 and omega-1 hydroxylation of lauric acid at a rate of 200 pmol.min-1.nmol of cytochrome P-450-1. A monoclonal antibody raised against the 25-hydroxylating cytochrome P-450, designated mAb 25E5, was prepared. After coupling to Sepharose, the antibody was able to bind to cytochrome P-450(25) from kidney as well as from pig liver microsomes, and to immunoprecipitate the activity for 25-hydroxylation of vitamin D3 and 5 beta-cholestane-3 alpha,7 alpha-diol when assayed in a reconstituted system. The hydroxylase activity towards lauric acid was not inhibited by the antibody. By SDS/PAGE and immunoblotting with mAb 25E5, cytochrome P-450(25) was detected in both pig kidney and pig liver microsomes. These results indicate a similar or the same species of cytochrome P-450 in pig kidney and liver microsomes catalysing 25-hydroxylation of vitamin D3 and C27 steroids. The N-terminal amino acid sequence of the purified cytochrome P-450(25) from pig kidney microsomes differed from those of hitherto isolated mammalian cytochromes P-450.
Bergman
T
T
Department of Chemistry I, Karolinska Institutet, Stockholm, Sweden.
Postlind
H
H
D000595:Amino Acid Sequence; D000818:Animals; D002762:Cholecalciferol / Q000378:metabolism*; D053493:Cholestanetriol 26-Monooxygenase; D002777:Cholestanols / Q000378:metabolism*; D002784:Cholesterol / Q000378:metabolism; D003577:Cytochrome P-450 Enzyme System / Q000302:isolation & purification / Q000378:metabolism*; D005260:Female; D006900:Hydroxylation; D015151:Immunoblotting; D007163:Immunosorbent Techniques; D007668:Kidney / Q000201:enzymology* / Q000648:ultrastructure; D007700:Kinetics; D007850:Lauric Acids / Q000378:metabolism; D008297:Male; D008861:Microsomes / Q000201:enzymology; D008862:Microsomes, Liver / Q000201:enzymology; D008969:Molecular Sequence Data; D009249:NADP / Q000494:pharmacology; D009251:NADPH-Ferrihemoprotein Reductase / Q000378:metabolism; D013250:Steroid Hydroxylases / Q000378:metabolism*; D013552:Swine
D002777:Cholestanols; D007850:Lauric Acids; C030358:lauric acid; D002762:Cholecalciferol; C007426:dihydroxycoprostane; D009249:NADP; D003577:Cytochrome P-450 Enzyme System; D002784:Cholesterol; D013250:Steroid Hydroxylases; D053493:Cholestanetriol 26-Monooxygenase; D009251:NADPH-Ferrihemoprotein Reductase