SPECTRIN-LIKE PROTEIN IN BRAIN

大脑中的血影蛋白样蛋白质

• 批准号: 3399424
• 负责人: STEVEN Richard GOODMAN
• 金额: $ 14.29万
• 依托单位: UNIVERSITY OF SOUTH ALABAMA
• 依托单位国家: 美国
• 财政年份: 1989
• 资助国家: 美国
• 起止时间: 1989-01-01 至 1991-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3399424
• 关键词: actins; binding proteins; brain; brain cell; calmodulin; chemical group; dorsal root; gel electrophoresis; glia; high performance liquid chromatography; immunochemistry; immunoelectron microscopy; immunofluorescence technique; laboratory mouse; laboratory rabbit; laboratory rat; membrane proteins; microtubules; monoclonal antibody; neuroblastoma; neurofilament; phosphates; phosphorylation; protein biosynthesis; protein sequence; protein structure; radiotracer; spectrin; stoichiometry; synapsins; tissue /cell culture

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.

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，证明脑 4.1 的身份 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、位置和 与突触蛋白 I 的相互作用。该提案的目的是 (1) 通过结合肽确定脑血影蛋白的结构域结构 利用单克隆抗体技术进行绘图； (2) 定量数量 脑血影蛋白 235k Dal Beta 亚基上的磷酸基团。 本地化 这些磷酸盐在大脑血影蛋白结构域内，并确定 磷酸基团的功能； (3) 定量两者的量 通过定量免疫点检测小鼠大脑内的脑血影蛋白亚型 化验； (4)确定脑血影蛋白亚型的精确位置， 通过免疫电子显微镜观察特定神经元和神经胶质细胞内的情况 利用单克隆抗体； (5) 确定域结构 突触蛋白 I，定位血影蛋白和突触小泡结合域。 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 我磷酸化。 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) 研究速率和化学计量 培养的 S20Y 小鼠中血影蛋白亚基的合成和组装 神经母细胞瘤细胞和背根神经节 (DRG) 初级神经元 文化； (7)通过显微注射研究脑血影蛋白的功能 仅识别脑血影蛋白 (240/235) 的单克隆抗体或 脑血影蛋白 (240/235E) 亚型，分为神经母细胞瘤或原发性 DRG 神经元。 观察血影蛋白抗体显微注射对血影蛋白抗体的影响 神经元形状和神经突延伸。 确定对的影响 微管、神经丝和肌动蛋白微丝组织。