Characterization of mitochondrial cytochromes P-450 from pig kidney and liver catalysing 26-hydroxylation of 25-hydroxyvitamin D3 and C27 steroids.
The properties of cytochrome P-450 from pig kidney mitochondria, catalysing 26-hydroxylation of 25-hydroxyvitamin D3 and C27 steroids [Postlind & Wikvall (1989) Biochem. Biophys. Res. Commun. 159, 1135-1140; Postlind (1990) Biochem. Biophys. Res. Commun. 168, 261-266], were compared with those of a 26-hydroxylating cytochrome P-450 from pig liver mitochondria. The liver enzyme was purified to a cytochrome P-450 content of 7.4 nmol/mg of protein and showed only one protein band with an apparent Mr of 53,000 upon SDS/PAGE. The cytochrome P-450 catalysed 26-hydroxylation of 25-hydroxyvitamin D3, cholesterol and 5 beta-cholestane-3 alpha, 7 alpha-diol at rates of 361, 1090 and 2065 pmol/min per nmol of cytochrome P-450. A monoclonal antibody against the purified liver mitochondrial cytochrome P-450 26-hydroxylase (cytochrome P-450(26] was prepared. After coupling to Sepharose, the antibody was able to bind to cytochrome P-450(26) from liver as well as from kidney mitochondria and to immunoprecipitate the 26-hydroxylase activity towards 25-hydroxyvitamin D3 and cholesterol when assayed in a reconstituted system. After SDS/PAGE and immunoblotting with the antibody, the cytochrome P-450(26) was detected in the purified liver and kidney preparations. These results indicate that similar species of cytochrome P-450 catalyse 26-hydroxylation of 25-hydroxyvitamin D3 and C27 steroids in liver and kidney mitochondria. The results with the monoclonal antibody together with the finding that cholesterol competitively inhibits the 26-hydroxylation of 25-hydroxyvitamin D3 further indicate that 26-hydroxylation of 25-hydroxyvitamin D3 and cholesterol is catalysed by the same species of cytochrome P-450 in each tissue. The N-terminal amino acid sequence of cytochrome P-450(26) in kidney mitochondria resembled that of pig kidney microsomal 25-hydroxylase active in 25-hydroxylation of vitamin D3 and C27 steroids, whereas the sequence of pig liver mitochondrial cytochrome P-450(26) differed from those of rabbit and rat liver mitochondrial 26-hydroxylases as well as from those of other hitherto isolated mammalian cytochromes P-450.
Bergman
T
T
Department of Chemistry I, Karolinska Institutet, Stockholm, Sweden.
Postlind
H
H
D000818:Animals; D000911:Antibodies, Monoclonal; D002112:Calcifediol / Q000378:metabolism*; D003577:Cytochrome P-450 Enzyme System / Q000302:isolation & purification / Q000378:metabolism*; D005260:Female; D006900:Hydroxylation; D007668:Kidney / Q000201:enzymology*; D007700:Kinetics; D051379:Mice; D008807:Mice, Inbred BALB C; D008928:Mitochondria / Q000201:enzymology*; D008930:Mitochondria, Liver / Q000201:enzymology*; D013250:Steroid Hydroxylases / Q000302:isolation & purification / Q000378:metabolism*; D013379:Substrate Specificity
D000911:Antibodies, Monoclonal; D003577:Cytochrome P-450 Enzyme System; D013250:Steroid Hydroxylases; D002112:Calcifediol