ACTIN - REGULATORY PROTEINS IN DICTYOSTELIUM

肌动蛋白 - 盘基网柄菌中的调节蛋白

• 批准号: 3286966
• 负责人: SUSAN S. BROWN
• 金额: $ 9.07万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 1986
• 资助国家: 美国
• 起止时间: 1986-02-01 至 1990-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3286966
• 关键词: Dictyostelium; affinity chromatography; antibody; antibody formation; autoradiography; blocking antibody; cell type; chemical binding; cross immunity; crosslink; gel electrophoresis; immunofluorescence technique; membrane activity; morphology; phosphorylation; proteolysis; radiotracer

A large number of actin regulatory proteins have been studied, but in general, they are from different organisms, making it difficult to know what fraction of these proteins might be necessary for the functioning of actin in a single type of cell. The long term objective of this proposal is to completely characterize all of the actin-regulatory proteins from a single cell type, Dictyostelium discoideum, including both soluble and membrane-associated proteins. The first soluble protein to be characterized is a 30,000 dalton protein which crosslinks actin in a calcium-independent fashion. This protein will be compared with the previously described actin-crosslinking proteins by immunological cross-reactivity and peptide mapping, to look for relatedness (which might explain why there are several proteins of overlapping function in this organism). Interactions among the various crosslinking proteins will also be looked for, and evidence that they are under separate regulation will be sought. The next step is to proceed with a systematic search for more soluble actin-regulatory proteins, using effects on the low shear viscosity of actin as an assay. Subfractionation of each fraction will continue until a single peak of activity is obtained. The first approach to be used with membranes is to make antibodies vs. whole membranes, and look for the ability of this antibody to block the effect of membranes on the low shear viscosity of actin. If blocking activity is obtained, the antibody will be fractionated by adsorbing it to portions of a lane of Dictyostelium membrane proteins run on a gel. If blocking activity is not obtained, several alternate approaches to the question of membrane actin-binding proteins are proposed.

已经研究了大量肌动蛋白调节蛋白，但 一般，它们来自不同的生物，使得很难知道 这些蛋白质中的哪一部分对于功能可能需要 肌动蛋白在单一类型的细胞中。 该提议的长期目标 是完全表征来自A的所有肌动蛋白调节蛋白 单细胞类型，dictyostelium discoideum，包括可溶性和 膜相关蛋白。 要表征的第一个可溶性蛋白是30,000道尔顿蛋白 它以独立于钙的方式交联肌动蛋白。 该蛋白会 与先前描述的肌动蛋白 - 跨链接蛋白进行比较 免疫交叉反应性和肽图，以寻找相关性 （这可能解释了为什么有几种重叠功能的蛋白质 在这个生物体中）。 各种交联蛋白之间的相互作用 还将寻找，并证明它们在分开 将寻求法规。 下一步是进行系统的搜索，以进行更多的可溶性 肌动蛋白调节蛋白，利用对低剪切粘度的影响 肌动蛋白作为测定法。 每个分数的亚分数将持续到 获得活动的单个峰。 与膜一起使用的第一种方法是制作抗体V。 整个膜，并寻找这种抗体阻止的能力 膜对肌动蛋白低剪切粘度的影响。 如果阻止 获得活性，将其吸附到 底层膜蛋白的一部分在凝胶上运行。 如果 无法获得阻止活动，几种替代方法 提出了膜肌动蛋白结合蛋白的问题。