The cytochrome P-450 enzyme which catalyses 25-hydroxylation of vitamin D3 (cytochrome P-45025) from pig kidney microsomes [Postlind and 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 M(r) of 50500 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 1α-hydroxyvitamin D3 it was 1375 pmol·min-1·nmol-1. The preparation also catalysed the 25-hydroxylation of 5β-cholestane-3α,7α-diol at a rate of 1000 pmol·min-1·nmol of cytochrome P-450 and ω-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-45025 from kidney as well as from pig liver microsomes, and to immunoprecipitate the activity for 25-hydroxylation of vitamin D3 and 5β-cholestane-3α,7α-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-45025 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-45025 from pig kidney microsomes differed from those of hitherto isolated mammalian cytochromes P-450.
