LIVER MITOCHONDRIAL CYTOCHROME-P450 CYP27 AND RECOMBINANT-EXPRESSED HUMAN CYP27 CATALYZE 1-ALPHA-HYDROXYLATION OF 25-HYDROXYVITAMIN D-3

A cytochrome P450 catalyzing 1 alpha-hydroxylation of 25-hydroxyvitami n D-3 was purified from pig liver mitochondria. It also catalyzed 27-h ydroxylation of 25-hydroxyvitamin D-3 and 25-hydroxylation of vitamin D-3 The ratio between the 1 alpha-, 27-, and 25-hydroxylase activities remained essentially constant during the purification. Substrates for sterol 27-hydroxylase CYP27 inhibited and a monoclonal antibody raise d against CYP27 immunoprecipitated the 1 alpha-, 27-, and 25-hydroxyla se activities. Apparently homogeneous preparations of CYP27 from pig a nd rabbit liver mitochondria catalyzed 1 alpha-hydroxylation. Human li ver mitochondrial CYP27 was expressed from its cDNA in Escherichia col i. The nucleotide sequence encoding the N terminus of CYP27 was modifi ed in the first eight codons to achieve expression in E. coli. The pur ified recombinant-expressed CYP27 reconstituted with the electron-tran sferring system of adrenal mitochondria catalyzed 1 alpha-hydroxylatio n of 25-hydroxyvitamin DJ Expression of unmodified CYP27 cDNA in simia n COS cells confirmed the 1 alpha-hydroxylase activity toward 25-hydro xyvitamin D-3.