MOLECULAR BASIS OF THE STEROIDOGENIC ACTION OF ACTH

建议用Y-1小鼠肾上腺肿瘤细胞和牛肉肾上腺细胞 研究促肾上腺皮质激素的作用机制，以实现两个特异性 目标： 1.细胞内胆固醇转运的调节 途径：浆膜大于囊泡大于溶酶体 将通过(A)脉冲的[~3H]胆固醇来研究比线粒体更大的 亚油酸-低密度脂蛋白；(B)胆固醇运输蛋白的光亲和标记 使用与蛋白质共价结合的[~3H]叠氮胆固醇 暴露在紫外光下；以及(C)通过跟随荧光的运动 通过视频增强显微镜观察活细胞中的胆固醇。 胆固醇载体蛋白将在细胞中被识别和测量 与ACTH一起孵育和不与ACTH孵育。载体蛋白将在 由细胞提取物经紫外线照射后制备的十二烷基硫酸钠凝胶 [3H]含量将被测量。 2.胆固醇从线粒体膜外向内的移动 将通过(A)孵育标记有[~3H]的囊泡来检测 胆固醇(外膜为供体，内膜为受体)。 囊泡将通过离心法分离并转移[~3H] (B)用[~3H]叠氮胆固醇进行光亲和标记，以 识别线粒体中的胆固醇运输蛋白。这个 蛋白质将通过在制备的十二烷基硫酸钠凝胶上看到的氚标记来检测 与细胞孵育后的线粒体提取液 [~3H]叠氮胆固醇。

It is proposed to use Y-1 mouse adrenal tumor cells and beef adrenal cells to study the mechanism of action of ACTH in order to achieve two specific aims: 1. The regulation of intracellular transport of cholesterol through the pathway: plama membrane greater than vesicles greater than lysosomes greater than mitochondria will be studied by (a) pulses of [3H]cholesteryl linoleate-LDL; (b) photoaffinity labeling of cholesterol transport proteins using [3H]azidocholesterol which binds covalently to proteins during exposure to UV light; and (c) by following movement of fluorescent cholesterol through the living cell with video intensification microscopy. Cholesterol carrier proteins will be identified and measured in cells incubated with and without ACTH. The carrier proteins will be isolated on SDS gels prepared from cell extracts following exposure to UV light and [3H] content will be measured. 2. The movement of cholesterol from outer to inner mitochondrial membranes will be examined by (a) incubation of vesicles labeled with [3H] cholesterol (outer membrane as donor and inner membrane as acceptor). Vesicles will be separated by centrifugation and transfer of [3H] will be measured; and (b) by photoaffinity labeling with [3H]azidocholesterol to identify cholesterol transport proteins within the mitochondria. The proteins will be detected by tritium labeling seen on SDS gels prepared from mitochondrial extracts after incubation of cells with [3H]azidocholesterol.