MOLECULAR BASIS OF THE STEROIDOGENIC ACTION OF ACTH

建议使用Y-1小鼠肾上腺肿瘤细胞和牛肾上腺细胞 研究促肾上腺皮质激素的作用机制，以达到两个具体的 目的： 1. 胆固醇细胞内转运的调节 途径：质膜大于囊泡大于溶酶体 大于线粒体将研究（a）脉冲的[3 H]胆固醇 亚油酸-LDL;（B）胆固醇转运蛋白的光亲和标记 使用[3 H]叠氮基胆固醇，该胆固醇在 暴露于UV光;和（c）通过跟随荧光的移动， 胆固醇通过活细胞与视频增强显微镜。 胆固醇载体蛋白将在细胞中被鉴定和测量 在有和没有ACTH的情况下孵育。 载体蛋白将在 暴露于紫外光后，由细胞提取物制备SDS凝胶， [3H]内容将被测量。 2. 胆固醇从线粒体外膜到线粒体内膜的运动 将通过（a）孵育[3 H]标记的囊泡进行检查 胆固醇（外膜作为供体，内膜作为受体）。 将通过离心分离囊泡，并将[3 H]转移至 （B）通过用[3 H]叠氮基胆固醇进行光亲和标记， 识别线粒体内的胆固醇转运蛋白。 的 将通过制备的SDS凝胶上的氚标记检测蛋白质 从线粒体提取物孵育细胞后， [3 H]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.