Taniguchi T, Eto T A, Shiotsuki H, Sueta H, Higashi S, Iwamura T, Okuda K I, Setoguchi T
Department of Surgery I, Miyazaki Medical College, Kiyotake, Japan.
J Bone Miner Res. 2001 Jan;16(1):57-62. doi: 10.1359/jbmr.2001.16.1.57.
An accurate assay method of 25-hydroxyvitamin D3 24-hydroxylase (24-hydroxylase) was established. Kidney mitochondria prepared from vitamin D-replete rats were treated with polyoxyethylenesorbitan monolaurate. The solubilized suspension was ultracentrifuged at 100,000g for 60 minutes and an aliquot of the supernatant was incubated under the saturating concentrations of substrate NADPH and the mitochondrial-type electron transferring proteins, adrenodoxin and NADPH-adrenodoxin reductase. Products were analyzed by high-performance liquid chromatography (HPLC) monitoring effluents at a wavelength of 265 nm. The maximal velocity of the enzyme in vitamin D-replete rats was 400 pmol/minute per mg of protein, which was considerably higher than those reported by previous authors who used intact kidney mitochondria as the enzyme source. In applying the new assay method, an interesting property was found; Michaelis constant of 24-hydroxylase for 25-hydroxyvitamin D3 [25(OH)D3] was 0.6 microM, which was 35-fold lower than that for 1alpha,25-dihydroxyvitamin D3 [1alpha,25(OH)2D3] which was 20.9 microM. This fact indicates that affinity of the enzyme to 25(OH)D3 is 35-fold higher than that to 1alpha,25(OH)2D3. These data suggest that 25(OH)D3 is the preferred substrate to 1alpha,25(OH)2D3.
建立了一种准确测定25-羟基维生素D3 24-羟化酶（24-羟化酶）的方法。用聚氧乙烯山梨醇酐单月桂酸酯处理从维生素D充足的大鼠制备的肾线粒体。将溶解的悬浮液在100,000g下超速离心60分钟，取上清液的等分试样在底物NADPH以及线粒体型电子传递蛋白、肾上腺铁氧还蛋白和NADPH-肾上腺铁氧还蛋白还原酶的饱和浓度下孵育。通过高效液相色谱（HPLC）在265nm波长下监测流出物来分析产物。维生素D充足的大鼠中该酶的最大速度为每毫克蛋白质400 pmol/分钟，这大大高于以前使用完整肾线粒体作为酶源的作者报道的速度。在应用新的测定方法时，发现了一个有趣的特性；24-羟化酶对25-羟基维生素D3 [25(OH)D3]的米氏常数为0.6 microM，比其对1α,25-二羟基维生素D3 [1α,25(OH)2D3]（为20.9 microM）低35倍。这一事实表明该酶对25(OH)D3的亲和力比对1α,25(OH)2D3的亲和力高35倍。这些数据表明25(OH)D3是比1α,25(OH)2D3更优选的底物。
