MEMBRANE-MICROFILAMENT CONNECTIONS

本项目的目标是确定该机制 以及微丝系统和 质膜。 之前我们已经分析了 非红细胞血影蛋白作为这种皮层下网络的模型。 我们现在建议将我们的研究扩展到分析36，000个 蛋白酪氨酸激酶底物称为钙调蛋白。 这些蛋白质 其以单体或具有10，000 Mr光的复合物形式存在， 链与细胞骨架蛋白肌动蛋白和血影蛋白相互作用 并受Ca++和磷脂调节。 我们将确定 这个家族的其他成员使用一种技术， 与肌动蛋白和磷脂相互作用的能力，以及详细的 这些蛋白质的结构和功能的比较将是 进行了 Ca++结合的详细分析，包括亲和力 将进行调制。 我们将分析功能 轻链与S-100蛋白的相关性。 单克隆 并且多克隆抗体将被提高到限定的区域 用于分析其功能的钙蛋白酶（肽）。 的 组织和亚细胞分布使用蛋白质印迹和 免疫荧光显微术。 钙调蛋白的体内靶点 将使用交联试剂测定， 免疫沉淀。 calpactin重链和轻链基因 将在细菌中表达并纯化蛋白质产物 并进行了生物活性测试 我们将尝试识别 钙结合所需的calpactin区域。 的影响 钙调蛋白的酪氨酸磷酸化将通过 确定是否有任何差异的能力， 磷酸化钙蛋白与磷脂、Ca++或 血影蛋白 这些研究将进一步加深我们对 皮层下纤维网及其与细胞关系 转型

The goal of this project is the determination of the mechanism and result of connections between the microfilament system and the plasma membrane. Previously we had analyzed the system of non-erythroid spectrins as a model for this subcortical network. We now propose to extend our research to the analysis of 36,000 Mr tyrosine kinase substrates termed calpactins. These proteins which exist as monomers or as complexes with a 10,000 Mr light chain interact with the cytoskeletal proteins actin and spectrin and are modulated by Ca++ and phospholipid. We will identify other members of this family using a technique which relies on its ability to interact with actin and phospholipid, and a detailed comparison of the structure and function of these proteins will be made. A detailed analysis of Ca++-binding, including affinity modulators will be undertaken. We will analyze the functional relatedness of the light chain with the S-100 proteins. Monoclonal and polyclonal antibodies will be raised to defined regions (peptides) of calpactin for use in analysis of its function. The tissue and subcellular distribution using Western Blotting and immunofluorescence microscopy. The in vivo target of calpactins will be determined using cross-linking reagents followed by immunoprecipitation. The calpactin heavy and light chain genes will be expressed in bacteria and protein products will be purified and tested for biological activity. We will attempt to identify the region of calpactin needed for Ca++-binding. The effect of tyrosine phosphorylation of calpactin will be tested by determining whether there is any difference in the ability of phosphorylated calpactin to interact with phospholipid, Ca++ or spectrin. These studies will further our understanding of the subcortical filament network and its relationship to cell transformation.