CHOLESTEROL METABOLISM AND MITOCHONDRIAL CYTOCHROME P450

促肾上腺皮质激素对大鼠肾上腺束状细胞的刺激作用涉及 通过复杂阵列激活胆固醇侧链切割 脂质和蛋白质因素。 实验提出，目的 解决刺激胆固醇的因素 通过两种蛋白质代谢，一种环己酰亚胺敏感肽 促肾上腺皮质激素（固醇调节肽（SRP））和 固醇载体蛋白（SCP 2），对ACTH不敏感， 放线菌酮。 A蛋白E和 还将检查脂肪酸结合蛋白。 的 5-羟基过氧物对刺激的贡献 二十碳四烯酸（5-HPETE）和多不饱和脂肪酸 酸性磷脂，这两者都是由ACTH升高， 直接刺激类固醇生成，将进行研究。 重点 将放在SRP的作用机制上， 肾上腺线粒体胆固醇膜间转运 以及其他肾上腺成分在促进这一过程中的作用 过程 细胞活动产生的线粒体变化 促肾上腺皮质激素（由SRP介导）和SRP的直接作用将 以理解这个过程为目的。 的 在刺激中存在类似的机制 原代培养牛肾上腺细胞的胆固醇代谢 将被评估。 将开发几种新技术， 研究这些介质在病毒细胞和完整细胞中的作用， 线粒体 这些包括特定蛋白质的转移， 抗体进入细胞，胶体金免疫组化细胞 和线粒体定位SRP、SCP 2和P-450 SCC，以及 反应性线粒体定位的分光光度法 胆固醇（使用菲律宾和外源性P-450 SCC）。 膜 细胞色素P-450 SCC的处置将用一组 抗细胞色素的单克隆抗体。 的 多不饱和脂肪酸促进胆固醇代谢 用纯化的P-450 SCC重构的囊泡中的磷脂将 通过这种技术和其他技术与刺激相比， 这些磷脂在完整线粒体中的作用。 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核心 在所有类固醇合成组织中的合成。

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.