CHOLESTEROL METABOLISM AND MITOCHONDRIAL CYTOCHROME P450

促肾上腺皮质激素刺激大鼠肾上腺束状细胞 用复杂阵列激活胆固醇侧链裂解 脂肪和蛋白质的因素。提出了一些实验，目的是 解决对胆固醇刺激的贡献 两种蛋白质--放线菌酮敏感肽的代谢 促肾上腺皮质激素(ACTH)(类固醇调节肽)和 对ACTH和ACTH不敏感的甾醇载体蛋白(SCP2) 环己亚胺。A蛋白E和OF的可能参与 还将检测脂肪酸结合蛋白。这个 5-羟基过氧基对刺激的贡献 二十碳四烯酸(5-HPETE)和多不饱和脂肪酸 酸性磷脂，两者都被ACTH和ACTH升高 直接刺激类固醇激素的生成，将被研究。重点 将放在SRP的作用机制上 肾上腺线粒体内膜间胆固醇的转移 以及这些其他肾上腺成分在促进这一过程中所起的作用 进程。由细胞活动产生的线粒体的变化 ACTH(由SRP介导)和SRP意志的直接作用 以理解这一过程为特征。这个 存在可比的刺激机制 牛肾上腺细胞原代培养中的胆固醇代谢 将会被评估。将开发几种新技术来 研究这些介质在可见细胞和完整细胞中的作用 线粒体。这包括特定蛋白质的转移和 抗体进入细胞，胶体金免疫组织化学染色细胞 和线粒体定位SRP、SCP2和P-450scc，以及 活性线粒体的分光光度定位方法 胆固醇(使用非利福平和外源P-450scc)。膜 细胞色素P-450scc的处置将通过一组 针对细胞色素产生的单抗。这个 多不饱和脂肪酸促进胆固醇代谢的研究 纯化的P-450scc重组囊泡中的磷脂 用这项技术和其他技术与刺激性 这些磷脂在完整的线粒体中的作用。这些 实验解决了细胞内的几个新方面 胆固醇的运动和代谢是类固醇的中心 在所有类固醇生成组织中的合成。

The stimulation of rat adrenal fasciculate cells by ACTH involves activation of cholesterol side-chain cleavage by a complex array of lipid and protein factors. Experiments are proposed that aim to resolve contributions to the stimulation of cholesterol metabolism by two proteins, a cycloheximide-sensitive peptide that is elevated by ACTH (sterol regulatory peptide (SRP)) and a sterol carrier protein (SCP2) that is insensitive to ACTH and cycloheximide. The possible involvement of A poprotein E and of fatty acid binding protein will also be examined. The contributions to stimulation from 5-hydroperoxy eicosatetroaenoic acid (5-HPETE) and from polyunsaturated acidic phospholipids, both of which are elevated by ACTH and directly stimulate steroidogenesis, will be investigated. Emphasis will be placed on the mechanism of action of SRP on intermembrane cholesterol transfer within adrenal mitochondria and the role of these other adrenal constituents in facilitating this process. Changes in mitochondria produced by the cellular action of ACTH (mediated by SRP) and by the direct action of SRP will be characterized with the aim of understanding this process. The presence of comparable mechanisms in the stimulation of cholesterol metabolism in primary culture of bovine adrenal cells will be evaluated. Several new techniques will be developed to study the actions of these mediators in virable cells and intact mitochondria. These include transfer of specific proteins and antibodies into cells, colloidl-gold immunohistochemistry of cells and mitochondria to localize SRP, SCP2, and P-450scc, and spectrophotometric methods for locating reactive mitochondrial cholesterol (using filipin and exogenous P-450scc). The membrane disposition of cytochrome P-450scc will be probed with a set of monoclonal antibodies raised against the cytochrome. The enhancement of cholesterol metabolism by polyunsaturated phospholipids in vesicles reconstituted with purified P-450scc will be compared by this and other techniques with the stimulatory effect of these phospholipids in intact mitochrondia. These experiments address several new aspects of intracellular cholesterol movement and metabolism that are central to steriod synthesis in all steroidogenic tissues.