Abstract
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.
| Original language | English |
|---|---|
| Pages (from-to) | 67-77 |
| Number of pages | 11 |
| Journal | Gerontology |
| Volume | 42 |
| Issue number | SUPPL. 1 |
| Publication status | Published - 1996 Jun |
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Keywords
- 25-Hydroxyvitamin D 1α-hydroxylase
- Chicken
- Cytochrome P450
- Induction
- Kidney
- Vitamin D
ASJC Scopus subject areas
- Ageing
Cite this
Evidence for 54-kD protein in chicken kidney as a cytochrome P450 with a high molecular activity of 25-hydroxyvitamin D3 1a-hydroxylase. / Wakino, Shu; Meguro, M.; Suzuki, H.; Saruta, T.; Ogishima, T.; Shimada, H.; Ishimura, Y.; Shinki, T.; Suda, T.
In: Gerontology, Vol. 42, No. SUPPL. 1, 06.1996, p. 67-77.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Evidence for 54-kD protein in chicken kidney as a cytochrome P450 with a high molecular activity of 25-hydroxyvitamin D3 1a-hydroxylase
AU - Wakino, Shu
AU - Meguro, M.
AU - Suzuki, H.
AU - Saruta, T.
AU - Ogishima, T.
AU - Shimada, H.
AU - Ishimura, Y.
AU - Shinki, T.
AU - Suda, T.
PY - 1996/6
Y1 - 1996/6
N2 - 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.
AB - 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.
KW - 25-Hydroxyvitamin D 1α-hydroxylase
KW - Chicken
KW - Cytochrome P450
KW - Induction
KW - Kidney
KW - Vitamin D
UR - http://www.scopus.com/inward/record.url?scp=0029974612&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0029974612&partnerID=8YFLogxK
M3 - Article
C2 - 8964524
AN - SCOPUS:0029974612
VL - 42
SP - 67
EP - 77
JO - Gerontology
JF - Gerontology
SN - 0304-324X
IS - SUPPL. 1
ER -