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.
