DIFFERENT MECHANISMS OF HYDROXYLATION SITE SELECTION BY LIVER AND KIDNEY CYTOCHROME-P450 SPECIES (CYP27 AND CYP24) INVOLVED IN VITAMIN-D METABOLISM

A series of homologated 1 alpha-hydroxyvitamin D-3 and 1,25-dihydroxyv itamin D-3 molecules with one to three extra carbons in the side chain were used to examine the substrate preferences and hydroxylation site selection mechanisms of the liver vitamin D-3-25-hydroxylase (CYP27) and the target cell 25-hydroxyvitamin D-3-24-hydroxylase (CYP24). Cult ured and transfected cell models, used as sources of these hydroxylase s, gave 23-, 24-, 25-, and 27-hydroxylated metabolites which were iden tified by their high performance liquid chromatography and GC-MS chara cteristics. Lengthening the side chain is tolerated by each cytochrome P450 isoform such that 25-hydroxylation or 24-hydroxylation continues to occur at the same rate as in the native side chain, while the site of hydroxylation remains the same for the liver enzyme in that CYP27 continues to hydroxylate at C-25 and C-27 (minor) despite the two-carb on-atom extension. Somewhat surprising is the finding that C-24 and C- 23 (minor) hydroxylations also do not change as the side chain is exte nded by as much as three carbons. We conclude that CYP24 must be direc ted to its hydroxylation site(s) by the distance of carbon 24 from the vitamin D ring structure and not as in CYP27 by the distance of the h ydroxylation site from the end of the side chain.