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

S. Wakino, M. Meguro, H. Suzuki, T. Saruta, T. Ogishima, H. Shimada, Y. Ishimura, T. Shinki, T. Suda

Research output: Contribution to journalArticle

11 Citations (Scopus)

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 languageEnglish
Pages (from-to)67-77
Number of pages11
JournalGerontology
Volume42
Issue number1
DOIs
Publication statusPublished - Jan 1996

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Calcifediol
Mixed Function Oxygenases
Cytochrome P-450 Enzyme System
Chickens
Kidney
Vitamin D
Proteins
Mitochondria
Enzymes
Adrenodoxin
Ferredoxin-NADP Reductase
Cholates
Calcitriol
Cholecalciferol
Ion Exchange Chromatography
Carbon Monoxide
NADP
Polyacrylamide Gel Electrophoresis
High Pressure Liquid Chromatography

All Science Journal Classification (ASJC) codes

  • Ageing
  • Geriatrics and Gerontology

Cite this

Evidence for 54-kD protein in chicken kidney as a cytochrome P450 with a high molecular activity of 25-hydroxyvitamin D3 1β-hydroxylase. / Wakino, S.; Meguro, M.; Suzuki, H.; Saruta, T.; Ogishima, T.; Shimada, H.; Ishimura, Y.; Shinki, T.; Suda, T.

In: Gerontology, Vol. 42, No. 1, 01.1996, p. 67-77.

Research output: Contribution to journalArticle

Wakino, S. ; Meguro, M. ; Suzuki, H. ; Saruta, T. ; Ogishima, T. ; Shimada, H. ; Ishimura, Y. ; Shinki, T. ; Suda, T. / Evidence for 54-kD protein in chicken kidney as a cytochrome P450 with a high molecular activity of 25-hydroxyvitamin D3 1β-hydroxylase. In: Gerontology. 1996 ; Vol. 42, No. 1. pp. 67-77.
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