Mitochondrial Cytochrome P450 A COMPONENT OF CHICK KIDNEY 25-HYDROXYCHOLECALCIFEROL-1α-HYDROXYLASE
Mitochondrial Cytochrome P450 A COMPONENT OF CHICK KIDNEY 25-HYDROXYCHOLECALCIFEROL-1α-HYDROXYLASE
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发表时间：
1974-05
影响因子：
4.8
通讯作者：
J. G. Ghazarian;C. Jefcoate;J. Knutson;W. Orme-Johnson;H. DeLuca
中科院分区：
文献类型：
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作者：
J. G. Ghazarian;C. Jefcoate;J. Knutson;W. Orme-Johnson;H. DeLuca
摘要利用细胞色素氧化酶在抗霉素A存在下直接还原的新方法，提供了用一氧化碳差示光谱揭示细胞色素P450在鸡肾线粒体中存在的方法。这种线粒体色素被发现具有与肾微粒体细胞色素P450明显不同的性质。细胞色素P450的线粒体含量约为每毫克蛋白质0.20nmole。溶解的细胞色素的重组研究表明，这种一氧化碳结合的血红蛋白参与了肾脏25-羟基胆钙素-1α-羟基酶的活性。重组需要添加黄素蛋白和铁硫蛋白，以及NADPH和溶解的血红蛋白。甲孕酮和氨基谷氨酰亚胺竞争性抑制完整线粒体25-羟基胆钙素-1α羟基酶活性的动力学实验表明，这两种药物的抑制常数Ki分别为1.6×10~(-4)M和1.1×10~(-3)M。由差谱测得甲替拉酮和氨基戊二酰亚胺的光谱解离常数Ks分别为1.4×10-4m和4.5×10-4m。甲替拉酮的微粒体光谱解离常数为3.3x10-5m。线粒体Ki和Ks值之间的相似性清楚地表明，所观察到的线粒体1α羟基酶抑制的程度是与细胞色素P450结合的甲替拉酮的量的函数。动力学数据和重组研究证实了肾脏线粒体细胞色素P450在25-羟基胆钙素1α羟化反应中作为末端氧化酶的作用。
Abstract A novel method employing the direct reduction of cytochrome oxidase in the presence of antimycin A has provided the means to reveal the presence of cytochrome P450 in chick kidney mitochondria as characterized by the carbon monoxide difference spectrum. This mitochondrial pigment has been found to have properties distinctly different from that of the kidney microsomal cytochrome P450. The mitochondrial content of cytochrome P450 has been found to be about 0.20 nmole per mg of protein. Reconstitution studies with the solubilized cytochrome have indicated that this carbon monoxide-binding hemoprotein is involved in the activity of the renal 25-hydroxycholecalciferol-1α-hydroxylase. The reconstitution required an added flavoprotein and iron-sulfur protein, as well as NADPH and the solubilized hemoprotein. Kinetic experiments on the competitive inhibition of 25-hydroxycholecalciferol-1α-hydroxylase activity of intact mitochondria by metyrapone and aminoglutethimide have shown that the inhibition constants, Ki, for the drugs are 1.6 x 10-4 m and 1.1 x 10-3 m, respectively. The spectral dissociation constants, Ks, for metyrapone and aminoglutethimide as determined from difference spectra are 1.4 x 10-4 m and 4.5 x 10-4 m, respectively. The microsomal spectral dissociation constant for metyrapone is 3.3 x 10-5 m. The similarity between the mitochondrial Ki and Ks values for metyrapone clearly indicates that the extent of the observed mitochondrial 1α-hydroxylase inhibition is a function of the amount of metyrapone bound to cytochrome P450. The kinetic data and the reconstitution studies have substantiated the role of the kidney mitochondrial cytochrome P450 as a terminal oxidase in the 1α hydroxylation of 25-hydroxycholecalciferol.