ASSAY OF 25-HYDROXYVITAMIN D-3 1-ALPHA-HYDROXYLASE IN RAT-KIDNEY MITOCHONDRIAL

An assay method for 25-hydroxyvitamin D-3 1 alpha-hydroxylase [calcidi ol, NADPH: oxygen oxidoreductase (1-hydroxylating), EC 1.14.13.13] in rat kidney is described. The mitochondrial and nuclear fraction was so lubilized effectively with olamidopropyl)-dimethylammonio]-1-propanesu lfonate (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 saturat ed amounts of adrenodoxin and NADPH-adrenodoxin reductase. Products we re analyzed by HPLC, monitoring absorbance at 265 nm. The enzyme activ ity 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 en zyme activity was linear to a protein concentration up to 0.8 mg of pr otein/ml. Under the best assay conditions established, the maximal vel ocity 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 oth er authors with the enzyme of rachitic rat. Michaelis constant was 1.8 mu M. Specific activity with the enzyme of normal rat was 0.25 pmol o f product/min/mg. (C) 1998 Academic Press.