25-Hydroxyvitamin D3-24-hydroxylase in rat kidney mitochondria.
25-Hydroxyvitamin D3-24-hydroxylase in rat kidney mitochondria.
复制标题
大鼠肾线粒体中的 25-羟基维生素 D3-24-羟化酶。
DOI：
10.1016/s0021-9258(18)33110-7
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发表时间：
1983
期刊：
影响因子：
--
通讯作者：
Ingemar Bjorkhemt
中科院分区：
文献类型：
--
作者：
Jan I. Pedersen;Hamdan H. Shobaki;Inger Holmberg;Steinar Bergseth;Ingemar Bjorkhemt
确定了测定大鼠肾线粒体25-羟维生素D_3 -24-羟化酶活性的条件。产物24，25-二羟维生素D3通过高压液相色谱法或同位素稀释-质谱法定量。通过这些程序，可以在饱和浓度（大于2.5 × 10（-6）M）的底物下测量酶活性。用氨茶碱预处理动物（Kulkowski，J.A.，周，T.，马丁内斯，J.，和Ghazarian，J. G.（1979）Biochem. Biophys.通信资源90，50-57）在体外刺激24-羟化酶活性至少2至3倍。通过气相色谱-质谱法验证产物的同一性。反应速率在1.5 - 5 pmol/mg线粒体蛋白·min（25 ℃）之间变化，K ′ m测定为4.2 × 10（-7）M。苹果酸盐、琥珀酸盐和异柠檬酸盐都能够支持反应。低O2张力，CO，KCN，和解偶联剂羰基氰间氯苯腙抑制反应，而呼吸抑制剂鱼藤酮没有效果。在2.5 μ mol/ml的浓度下，甲吡酮以50%的抑制率抑制反应。发现该酶定位于线粒体内膜内。结果表明，在大鼠肾脏线粒体25-羟维生素D3-24-羟化酶是一种细胞色素P-450，还原当量主要由NADPH通过能量依赖性转氢酶提供。
Assay conditions for the measurement of 25-hydroxyvitamin D3-24-hydroxylase activity in rat kidney mitochondria have been worked out. The product, 24,25-dihydroxyvitamin D3 was quantitated either by high pressure liquid chromatography or by isotope dilution-mass spectrometry. By these procedures, the enzyme activity could be measured with saturating concentration (greater than 2.5 X 10(-6) M) of substrate. Pretreatment of the animals by aminophylline (Kulkowski, J. A., Chow, T., Martinez, J., and Ghazarian, J. G. (1979) Biochem. Biophys. Res. Commun. 90, 50-57) stimulated the 24-hydroxylase activity in vitro at least 2 to 3-fold. The identity of the product was verified by gas chromatography-mass spectrometry. The rates of the reaction varied between 1.5 and 5 pmol/mg of mitochondrial protein.min (at 25 degrees C), and the K'm was determined to be 4.2 X 10(-7) M. Malate, succinate, and isocitrate were all able to support the reaction. Low O2 tension, CO, KCN, and the uncoupler carbonyl cyanide m-chlorophenylhydrazone inhibited the reaction, while the respiratory inhibitor rotenone had no effect. Metyrapone inhibited the reaction with 50% inhibition at a concentration of 2.5 mumol/ml. The enzyme was found to be localized inside the inner mitochondrial membrane. The results indicate that in the rat the renal mitochondrial 25-hydroxyvitamin D3-24-hydroxylase is a cytochrome P-450 and that the reducing equivalents are primarily supplied by NADPH via the energy-dependent transhydrogenase.