CLONING AND EXPRESSION OF RAT 25-HYDROXYVITAMIN D-3-1-ALPHA-HYDROXYLASE CDNA

A full-length cDNA for the rat kidney mitochondrial cytochrome P450 mi xed function oxidase, 25-hydroxyvitamin D-3-1 alpha-hydroxylase (P4501 alpha), was cloned from a vitamin D-deficient rat kidney cDNA library and subcloned into the mammalian expression vector pcDNA 3.1(+). When P4501 alpha cDNA was transfected into COS-7 transformed monkey kidney cells, they expressed 25-hydroxyvitamin D-3-1 alpha-hydroxylase activ ity. The sequence analysis showed that P4501 alpha was of 2,469 bp lon g and contained an ORF encoding 501 amino acids, The deduced amino aci d sequence showed a 53% similarity and 44% identity to the vitamin D-3 -25-hydroxylase (CYP27), whereas it has 42.6% similarity and 34% ident ity with the 25-hydroxyvitamin D-3-24-hydroxylase (CYP24), Thus, it co mposes a nem subfamily of the CYP27 family. Further, it is more closel y related to the CYP27 than to the CYP24. The expression of P4501 alph a mRNA was greatly increased in the kidney of vitamin D-deficient rats , In rats with the enhanced renal production of 1 alpha,25-dihydroxyvi tamin D-3 (rats fed a low Ca diet), P4501 alpha mRNA was greatly incre ased in the renal proximal convoluted tubules.