EVIDENCE FOR 54-KD PROTEIN IN CHICKEN KIDNEY AS A CYTOCHROME-P450 WITH A HIGH-MOLECULAR ACTIVITY OF 25-HYDROXYVITAMIN D-3 1-ALPHA-HYDROXYLASE

Conversion of 25-hydroxyvitamin D-3 (25(OH)D-3) to the active vitamin D-3, 1 alpha,25-dihydroxyvitamin D-3 (1 alpha,25(OH)(2)D-3) is catalyz ed by 25(OH)D-3, 1 alpha-hydroxylase (1 alpha-hydroxylase). It has bee n suggested that this enzyme is cytochrome P450 (P450). We purified 1 alpha-hydroxylase 430-fold from cholate-solubilized kidney mitochondri a of vitamin D-deficient chickens by utilizing hydrophobic and ion-exc hange column chromatographies. Enzymatic activity was assessed by meas uring on HPLC the formation of 1 alpha,25(OH)(2)D-3 from 25(OH)D-3 in the assay mixture containing NADPH, adrenodoxin reductase, adrenodoxin as a reducing system. The purified enzyme showed a GO-difference spec trum characteristic of P450. The molecular activity of this preparatio n was calculated to be 8.7 pmol/min/pmol P450. This value was higher b y more than 87-fold than those reported so far. The present preparatio n was found to contain several proteins on SDS-PAGE. Among them, only the 54-kD protein became undetectable when kidney mitochondria from no rmal and vitamin D-replete chickens, where 1 alpha-hydroxylase activit ies were 15 and 0% of that found in vitamin D-deficient chicken, respe ctively, were used as the starting enzyme sources. Furthermore, the ba nd intensity of the 54-kD protein accounted for the spectrophotometric ally determined amount of P450 in the preparation. These results sugge st that the 54-kD protein is 1 alpha-hydroxylase.