SPECTRIN-LIKE PROTEIN IN BRAIN

我们已经完成了原提案的所有目标， 发现大脑蛋白4.1，证明大脑4.1是 突触蛋白I，并开始研究小鼠脑血影蛋白的表达 神经母细胞瘤细胞和背根神经节神经元的原代培养物。 在此应用程序中，我们描述了旨在为我们提供 更好地了解脑血影蛋白的结构，功能，位置， 与突触蛋白I的相互作用。 这项建议的目的是（1） 结合肽测定脑血影蛋白的结构域 用单克隆抗体技术作图;（2）定量 脑血影蛋白的235 k Dal β亚基上的磷酸基团。 本地化 这些磷酸盐在脑血影蛋白结构域的结构，并决定 磷酸基团的功能;（3）定量两种磷酸基团的量， 用定量免疫斑点法测定小鼠脑内脑血影蛋白亚型 测定;（4）确定脑血影蛋白亚型的精确位置， 在特定的神经元和神经胶质细胞内， 利用单克隆抗体;（5）确定结构域结构 突触蛋白I，定位血影蛋白和突触囊泡结合结构域。 定量脑血影蛋白-突触蛋白I的亲和力和化学计量 相互作用，并确定这种相互作用是否受突触蛋白调节 I磷酸化。 研究突触蛋白I在大脑中的作用 血影蛋白-肌动蛋白相互作用;（6）研究血影蛋白-肌动蛋白相互作用的速率和化学计量。 S20 Y小鼠血影蛋白亚基合成与组装 神经母细胞瘤细胞和背根神经节（DRG）初级神经元 （7）用显微注射法研究脑血影蛋白的功能。 仅识别脑血影蛋白的单克隆抗体（240/235），或 脑血影蛋白（240/235 E）亚型，分为神经母细胞瘤或原发性DRG 神经元 微量注射血影蛋白抗体观察其对 神经元形状和神经突延伸。 确定对 微管、神经丝和肌动蛋白微丝组织。

We have completed all of the aims of the original proposal, as well as discovering brain protein 4.1, demonstrating the identity of brain 4.1 as synapsin I, and beginning the study of brain spectrin expression in mouse neuroblastoma cells and primary cultures of dorsal root ganglion neurons. In this application we describe experiments which are designed to give us a better understanding of brain spectrin structure, function, location, and interaction with synapsin I. The aims of this proposal are (1) to determine the domain structure of brain spectrin by combining peptide mapping with monoclonal antibody technology; (2) Quantitate the number of phosphate groups on the 235k Dal Beta subunit of brain spectrin. Localize these phosphates within the brain spectrin domain structure, and determine the function of the phosphate groups; (3) Quantitate the amount of the two brain spectrin subtypes within mouse brain with a quantitative immunodot assay; (4) Determine the precise location of brain spectrin subtypes, within specific neurons and glial cells by immunoelectronmicroscopy utilizing monoclonal antibodies; (5) Determine the domain structure of synapsin I, localizing the spectrin and synaptic vesicle binding domains. Quantitate the affinity and stoichiometry of the brain spectrin-synapsin I interaction and determine whether this interaction is regulated by synapsin I phosphorylation. Study the role of synapsin I in the brain spectrin-actin interaction; (6) Study the rate and stoichiometry of spectrin subunit synthesis and assembly within cultured S20Y mouse neuroblastoma cells and dorsal root ganglion (DRG) primary neuronal cultures; (7) Study the function of brain spectrin by microinjection of monoclonal antibodies which recognize only the brain spectrin (240/235) or brain spectrin (240/235E) subtypes, into neuroblastoma or primary DRG neurons. Observe the effect of spectrin antibody microinjection upon neuronal shape and neurite extension. Determine the effect upon microtubule, neurofilament, and actin microfilament organization.