Assay of 25-hydroxyvitamin D3 1 alpha-hydroxylase in rat kidney mitochondria.
An assay method for 25-hydroxyvitamin D3 1alpha-hydroxylase [calcidiol, NADPH: oxygen oxidoreductase (1-hydroxylating), EC 1.14. 13.13] in rat kidney is described. The mitochondrial and nuclear fraction was solubilized effectively with 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate(Chaps). By subsequent ultracentrifugation of the solubilized suspension the effect of inhibitory factor(s) in mammals was removed. The enzyme was then assayed by the reconstitution method using saturated amounts of adrenodoxin and NADPH-adrenodoxin reductase. Products were analyzed by HPLC, monitoring absorbance at 265 nm. The enzyme activity depended on not only pH of the medium but also the kind of buffers. N,N-Bis(2-hydroxyethyl)glycine was the best buffer. At 30 degrees C, the reaction velocity was linear at least up to 10 min, by which time enough amounts of the product needed for analysis were formed. The enzyme activity was linear to a protein concentration up to 0.8 mg of protein/ml. Under the best assay conditions established, the maximal velocity of enzyme in the rachitic rat was 12.9 pmol of product/min/mg of protein, which was 30- to 1000-fold higher than those reported by other authors with the enzyme of rachitic rat. Michaelis constant was 1.8 microM. Specific activity with the enzyme of normal rat was 0.25 pmol of product/min/mg.
Eto
T A
TA
Department of Surgery I, Miyazaki Medical College, Kiyotake, 889-16, Japan.
Nakamura
Y
Y
Taniguchi
T
T
Miyamoto
K
K
Nagatomo
J
J
Maeda
Y
Y
Higashi
S
S
Okuda
K
K
Setoguchi
T
T
D015090:25-Hydroxyvitamin D3 1-alpha-Hydroxylase / Q000032:analysis* / Q000378:metabolism; D000325:Adrenodoxin; D000818:Animals; D002021:Buffers; D002851:Chromatography, High Pressure Liquid; D005287:Ferredoxin-NADP Reductase; D006863:Hydrogen-Ion Concentration; D007668:Kidney / Q000201:enzymology*; D007700:Kinetics; D008928:Mitochondria / Q000201:enzymology; D051381:Rats; D017207:Rats, Sprague-Dawley; D012279:Rickets / Q000201:enzymology; D012995:Solubility; D013056:Spectrophotometry, Ultraviolet; D013696:Temperature
D002021:Buffers; D000325:Adrenodoxin; D015090:25-Hydroxyvitamin D3 1-alpha-Hydroxylase; D005287:Ferredoxin-NADP Reductase