Evidence for 54-kD protein in chicken kidney as a cytochrome P450 with a high molecular activity of 25-hydroxyvitamin D₃ 1β-hydroxylase

Conversion of 25-hydroxyvitamin D₃ (25(OH)D₃) to the active vitamin D₃, 1α,25-dihydroxyvitamin D₃ (1α,25(OH)₂D₃) is catalyzed by 25(OH)D₃,1α-hydroxylase (1α-hydroxylase). It has been suggested that this enzyme is cytochrome P450 (P450). We purified 1α-hydroxylase 430-fold from cholate-solubilized kidney mitochondria of vitamin D-deficient chickens by utilizing hydrophobic and ion-exchange column chromatographies. Enzymatic activity was assessed by measuring on HPLC the formation of 1α,25(OH)₂D₃ from 25(OH)D₃ in the assay mixture containing NADPH, adrenodoxin reductase, adrenodoxin as a reducing system. The purified enzyme showed a CO-difference spectrum characteristic of P450. The molecular activity of this preparation was calculated to be 8.7 pmol/min/pmol P450. This value was higher by more than 87-fold than those reported so far. The present preparation was found to contain several proteins on SDS-PAGE. Among them, only the 54-kD protein became undetectable when kidney mitochondria from normal and vitamin D-replete chickens, where 1α-hydroxylase activities were 15 and 0% of that found in vitamin D-deficient chicken, respectively, were used as the starting enzyme sources. Furthermore, the band intensity of the 54-kD protein accounted for the spectrophotometrically determined amount of P450 in the preparation. These results suggest that the 54-kD protein is 1α-hydroxylase.