STRUCTURE AND FUNCTION OF ACTIN-MEMBRANE LINKAGE ATPASE

肌动蛋白-膜连接ATP酶的结构和功能

• 批准号: 3288227
• 负责人: JIMMY H COLLINS
• 金额: $ 7.75万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 1985
• 资助国家: 美国
• 起止时间: 1985-07-01 至 1989-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3288227
• 关键词: adenosinetriphosphatase; affinity chromatography; apical membrane; autoradiography; brush border membrane; cell cell interaction; cell motility; cytoskeleton; densitometry; electron microscopy; gastrointestinal epithelium; gel filtration chromatography; immunoprecipitation; intestinal villi; ion exchange chromatography; ion transport; liposomes; membrane activity; membrane structure; monoclonal antibody; nucleotides; phosphorylation; radiotracer

The overall goal of this proposed research is to understand the molecular basis of membrane-cytoskeletal interactions in vertebrate cells. The extensive plasma membrane formed by microvilli on the apical surface of the intestinal epithelial cell interacts along the length of the microvilli with the actin cytoskeleton through periodically spaced, ATP-sensitive cross bridges composed of a 110,000-dalton protein-calmodulin complex. This complex also has calcium-activated Mg2+ ATPase activity. To define and characterize the molecular basis of the association of the complex with both the membrane and cytoskeleton and to define the function of the ATPase, the ATPase activity and actin and membrane binding of the complex will be studied under well defined conditions by reconstitution with proteins purified from the microvillus, isolated microvilli and microvillar cytoskeletons and liposomes. Biochemical, biophysical, electron microscopic and immunochemical approaches will be used to study both native and reconstituted systems. The possible function of the ATPase in microvillar motility, in regulation of the cytoskeletal and membrane binding of the complex and in calcium ion transport will be determined. The possible association of the complex with the lipid bilayer of the membrane and with other membrane proteins will be studied. In addition, regulation of these events by calcium ion binding to the complex and by phosphorylation of the 110,000-dalton protein will also be characterized. These studies should enhance our understanding of cytoskeletal-membrane interactions and their regulation in intestinal epithelial and other cells.

这项研究的总体目标是了解分子 脊椎动物细胞膜-细胞骨架相互作用的基础。 这 顶端表面由微绒毛形成的广泛质膜 肠上皮细胞沿着微绒毛的长度相互作用 通过周期性间隔的、ATP 敏感的肌动蛋白细胞骨架 由 110,000 道尔顿的蛋白质-钙调蛋白复合物组成的交叉桥。 该复合物还具有钙激活的 Mg2+ ATP 酶活性。 定义 并表征该复合物与以下物质缔合的分子基础： 膜和细胞骨架并定义其功能 ATPase、ATPase 活性以及复合物的肌动蛋白和膜结合 将在明确的条件下通过重构进行研究 从微绒毛、分离的微绒毛和微绒毛中纯化的蛋白质 细胞骨架和脂质体。 生物化学、生物物理、电子 将使用显微镜和免疫化学方法来研究天然 并重构了系统。 ATP酶的可能功能 微绒毛运动，调节细胞骨架和膜 将确定复合物的结合和钙离子转运。 该复合物与脂质双层的可能关联 将研究膜和其他膜蛋白。 此外， 通过与复合物结合的钙离子和通过 110,000 道尔顿蛋白质的磷酸化也将得到表征。 这些研究应该增强我们对细胞骨架膜的理解 肠上皮细胞和其他细胞中的相互作用及其调节。