MICROSOMAL 25-HYDROXYLATION OF VITAMIN-D-2 AND VITAMIN-D-3 IN PIG-LIVER

A microsomal cytochrome P-450 catalysing 25-hydroxylation of vitamin D-2, was purified from both male and female pigs to apparent homogeneity and a specific cytochrome P-450 content of 13 and 15.4 nmol x mg of protein(-1), respectively. The enzyme also catalysed 25-hydroxylation of vitamin D-3. The ratio between the 25-hydroxylase activities towards vitamin D-2 and D-3 was essentially the same in the different purification steps as well as in the apparently homogeneous enzyme preparation. The two enzyme activities showed the same pH optimum and decreased in parallel upon partial denaturation of the enzyme. Cholecalciferol competitively inhibited 25-hydroxylation of vitamin D-2 and vice versa. The non-steroidal cytochrome P-450 inhibitor ketoconazole inhibited both enzyme activities and the K-i values were the same. The cytochrome P-450 showed the same apparent M(r), substrate specificity and N-terminal amino acid sequence as the previously purified vitamin D-2 25-hydroxylase from pig liver microsomes. A monoclonal antibody raised against the vitamin D, 25-hydroxylase also recognized the vitamin D-2 25-hydroxylase. The antibody immunoprecipitated the 25-hydroxylase activity towards both vitamin D-2 and D-3 in the purified enzyme. Taken together, the results show that the 25-hydroxylation of vitamin D-2 and D-3 is catalysed by the same microsomal cytochrome P-450 in pig liver microsomes. The properties of this 25-hydroxylase are discussed in relation to present knowledge concerning previously well-characterized vitamin D-3 25-hydroxylases that are not able to catalyse 25-hydroxylation of vitamin D-2.