MODULATION OF MEMBRANE FUNCTION BY COMPLEMENT

拟议研究的长期目标是 描述两国之间相互作用的性质和程度 末端补体蛋白(C5、C6、C7、C8、C9)， 共同形成的膜攻击复合体(MAC) 人工和天然中的补体、脂类和蛋白质 靶膜。我们想要了解它的机制 这种复合体在细胞表面的组装，并阐明 MAC可以通过哪些机制导致免疫 细胞溶解和细胞死亡或保护宿主细胞免受自体 毁灭。 我们希望通过使用组合来实现我们的长期目标 生物物理、生化和免疫化学技术 调查MAC的组装及其对目标的影响 薄膜。我们的研究将主要集中在C9上，因为 这种蛋白质提供了主要的裂解和细胞毒功能 麦克。我们将使用合成肽方法来生成一个 天然C9和C9与靶结合时的表位图 薄膜。圆二色谱与电子自旋共振(ESR) 光谱学将用于分析构象的变化 当蛋白质聚集在膜上并穿透到膜中时。 相应的脂质双层结构的变化将是 用血沉和差示扫描量热法进行监测。这个 由此产生的MAC-膜复合体将通过冷冻- 断口电子显微镜。活动中心的局部化 将通过产生蛋白水解性片段来尝试C9 裂解或细胞毒活性。此类活动将通过以下方式进行分析 测量脂质体中标志物的释放，或改变 跨平面脂类双层的电导，并通过测试肽 直接因为它们对血细胞和细菌的存活的影响。

The long range objectives of the proposed research are to characterize the nature and the extent of interaction between the terminal complement proteins (C5, C6, C7, C8, C9), which together form the membrane attack complex (MAC) of complement, and lipids and proteins in artificial and natural target membranes. We want to understand the mechanism of assembly of this complex on a cell's surface and elucidate the mechanisms through which the MAC can either cause immune cytolysis and cell death or protect host cells from auto- destruction. We expect to attain our long range goals by using a combination of biophysical, biochemical and immunochemical techniques to investigate the assembly of the MAC and its effect on the target membrane. Our studies will focus predominantly on C9 because this protein provides the main lytic and cytotoxic functions of the MAC. We will use the synthetic peptide approach to generate an epitope map of native C9 and of C9 when bound to target membrane. Circular dichroism and electron spin resonance (ESR) spectroscopy will be used to analyze conformational changes in the protein as they assemble on and penetrate into the membrane. The corresponding changes in lipid bilayer structure will be monitored by ESR and differential scanning calorimetry. The resulting MAC-membrane complex will visualized by freeze- fracture electron microscopy. Localization of the active center of C9 will be attempted by producing proteolytic fragments with lytic or cytotoxic activity. Such activities will be assayed by measuring release of markers from liposomes, or changes in conductance across planar lipid bilayers, and by testing peptides directly for their effects on survival of blood cells and bacteria.