ASYMMETRIC DISTRIBUTION OF CHOLESTEROL IN MEMBRANES

这项拟议中的研究将考察调节 胆固醇在细胞内的分布。为此，域和域 膜内胆固醇的结构与胆固醇的转移 将检查膜之间的相互作用。具体来说，这项工作将集中在 固醇载体蛋白SCP和SCP-2在调节中的作用 膜胆固醇结构域结构及其在赋予特异性 膜间胆固醇转移。初步证据表明， 这些蛋白质在促进固醇转移的能力上有明显的不同。 在膜之间，显然是通过完全不同的机制实现的。 单细胞胞外多糖对膜甾醇结构域及甾醇交换的影响 生物膜之间的分离将在体外使用SCP进行检测 从大鼠肝脏和从大肠杆菌中获得的重组SCP中，表达 克隆了SCP基因。膜上的甾醇结构域和甾醇交换 用荧光胆固醇类似物测量，一种新的分析方法不需要 施主和受主膜的分离，单色He/Cd激光器 激励和计算机化的多频相位和调制 荧光测定法。具体目标是审查： 1)SCP在调节体内胆固醇结构域结构中的作用 膜。 2)自发性和SCP介导的甾醇转移的特异性。 3)机制(S)(载体、碰撞转移、聚变或直接作用于 膜)，从而SCP可增强和/或赋予对甾醇的专一性 有动静。 要了解SCP在细胞内固醇运动中的作用需要 膜中胆固醇结构的知识。此域 结构可能会对胆固醇的运输和 微粒体酶的调节，如酰基辅酶A胆固醇酰基 转移酶。这项研究的长期目标是检查 SCPS在细胞内胆固醇运动中的作用及其可能的机制 参与胆固醇积聚的过程。了解这些知识 这些因素应该有助于增加我们对一些问题的理解 涉及细胞内类固醇异常转运和/或积聚的疾病 (骨刺细胞性贫血、肿瘤、动脉粥样硬化和衰老)。

The proposed research will examine fundamental factors that regulate cholesterol distribution within the cell. To this end both the domain structure of cholesterol within membranes and the transfer of cholesterol between membranes will be examined. specifically the work will focus on the role of the sterol carrier proteins, SCP and SCP-2, in regulation membrane cholesterol domain structure and in conferring specificity to intermembrane cholesterol transfer. Preliminary evidence indicates that these proteins differ markedly in their ability to enhance sterol transfer between membranes and apparently do so by entirely different mechanisms. The effect of SCPs on membrane sterol domains and on exchange of sterols between biological membranes will be examined in vitro using SCPs isolated from rat liver and recombinant SCPs obtained from E.coli, expressing the cloned SCP genes. Membrane sterol domains and sterol exchange will be measured with a fluorescent cholesterol analogue, a new assay not requiring separation of donor and acceptor membranes, monochromatic He/Cd laser excitation, and computerized multifrequency phase and modulation fluorometry. The specific aims are to examine: 1) Role of SCPs in regulating the domain structure of cholesterol within membranes. 2) Specificity of spontaneous and SCP-mediated sterol transfer. 3) Mechanism(s) (carrier, collisional transfer, fusion, or direct effect on membranes) whereby SCPs may enhance and/or confer specificity to sterol movement. Understanding the role of SCPs in intracellular sterol movement requires a knowledge of the domain structure of cholesterol in membranes. This domain structure may have important consequences for cholesterol transport and the regulation of microsomal enzymes such as acyl CoA cholesteryl acyl transferase. The long term goal of this research is to examine the role of SCPs in the intracellular movement of cholesterol and how they may be involved in the process of cholesterol accumulation. Knowledge of these factors should be helpful in increasing our understanding of a number of diseases involving abnormal sterol trafficking and/or accumulation in cells (spur-cell anemia, neoplasia, atherosclerosis, and aging).