SPECTRIN-LIKE PROTEIN IN BRAIN

我们已经完成了最初提案的所有目标，以及 发现脑蛋白4.1，证明Brain 4.1为 突触素I，并开始研究小鼠脑内血影蛋白的表达 神经母细胞瘤细胞和背根神经节神经元的原代培养。 在本应用程序中，我们描述了旨在为我们提供 更好地了解大脑的幽灵蛋白的结构、功能、位置和 与突触素I的相互作用本方案的目的是(1) 结合多肽确定脑影蛋白的结构域结构 用单抗技术作图；(2)定量检测 脑影蛋白235k Dal Beta亚基上的磷酸基团。本地化 这些磷酸盐存在于大脑的血影蛋白结构域中，并决定 磷酸基团的功能；(3)定量测定两者的量 小鼠脑内血影蛋白亚型定量免疫斑点分析 测定；(4)确定脑影蛋白亚型的准确位置， 免疫电子显微镜观察特定神经元和神经胶质细胞内 利用单抗；(5)确定其结构域 突触素I，定位血影蛋白和突触小泡结合区域。 定量测定脑影蛋白-突触素I的亲和力和化学计量 并确定这种相互作用是否受突触蛋白的调节 I磷酸化。研究突触素I在大脑中的作用 光影蛋白-肌动蛋白的相互作用；(6)研究光影蛋白与肌动蛋白的相互作用 培养的S20Y小鼠体内血影蛋白亚基的合成和组装 神经母细胞瘤细胞与背根神经节(DRG)初级神经元 (7)用微量注射法研究脑影蛋白的功能 仅识别脑影蛋白的单抗(240/235)或 脑影蛋白(240/235E)亚型，神经母细胞瘤或原发背根节 神经元。观察血影蛋白抗体微量注射对慢性阻塞性肺疾病的影响 神经元形态和轴突延伸。确定对…的影响 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.