Evidence for 54-kD protein in chicken kidney as a cytochrome P450 with a high molecular activity of 25-hydroxyvitamin D3 1 alpha-hydroxylase.
Conversion of 25-hydroxyvitamin D3 (25(OH)D3) to the active vitamin D3, 1 alpha,25-dihydroxyvitamin D3 (1 alpha,25(OH)2D3) is catalyzed by 25(OH)D3, 1 alpha-hydroxylase(1 alpha-hydroxylase). It has been suggested that this enzyme is cytochrome P450 (P450). We purified 1 alpha-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 1 alpha,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 1 alpha-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 1 alpha-hydroxylase.
Wakino
S
S
Department of Internal Medicine, School of Medicine, Keio University, Tokyo, Japan.
Meguro
M
M
Suzuki
H
H
Saruta
T
T
Ogishima
T
T
Shimada
H
H
Ishimura
Y
Y
Shinki
T
T
Suda
T
T
D015090:25-Hydroxyvitamin D3 1-alpha-Hydroxylase / Q000302:isolation & purification / Q000378:metabolism*; D000818:Animals; D002645:Chickens / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D007668:Kidney / Q000378:metabolism*; D008297:Male; D008928:Mitochondria / Q000378:metabolism; D008970:Molecular Weight; D014807:Vitamin D / Q000378:metabolism
D014807:Vitamin D; D003577:Cytochrome P-450 Enzyme System; D015090:25-Hydroxyvitamin D3 1-alpha-Hydroxylase