NON-MUSCLE MYOSIN ACTIVITY AND ASSEMBLY

非肌肉肌球蛋白活性和组装

• 批准号: 3755736
• 负责人: JIMMY H COLLINS
• 金额: --
• 依托单位: NORFOLK STATE UNIVERSITY
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3755736
• 关键词: adenosinetriphosphatase; antibody specificity; brush border membrane; calcium binding protein; calmodulin dependent protein kinase; chemical binding; chickens; enzyme inhibitors; enzyme mechanism; gastrointestinal epithelium; laboratory rabbit; membrane activity; myosin light chain kinase; myosins; phosphorylation; protein biosynthesis; protein reconstitution; protein sequence; radionuclides; tissue /cell culture

Myosin, actin and associated cytoskeletal proteins interact to form and maintain the large brush border membrane surface necessary for nutrient absorption in intestinal epithelial cells. In addition, actomyosin-based movements of microvili and contraction of the terminal web region of the brush border may be important in the absorptive process itself. To define and characterize the molecular basis of these events, the role of chicken brush border myosin phosphorylation and of calcium binding to myosin will be studied in regulation of: a) myosin actin-activated ATPase activity, b) myosin filament assembly, c) the conformation of the myosin molecule, and brush border myosin-directed movement of latex beads in an in vitro motility assay. A brush border calmodulin-dependent heavy chain kinase, calmodulin-independent light chain kinase that we have recently identified and isolated will used in these studies to phosphorylate purified brush border myosin at specific, well-defined sites. The regulation of the activity of the calmodulin-dependent myosin kinases by calcium and calmodulin and of the heavy chain kinase by autophosphorylation will also be characterized. The heavy chain kinase, which is also heavy chain kinase known to be regulated by calmodulin, will be assayed for in extracts of other tissues and compared immunologically to the apparently similar and widely distributed calmodulin-dependent protein kinase II. Our previous characterization of the myosin phosphorylation sites by phosphoamino acid analysis and peptide mapping will be extended to the determination of the sequence around each site. In addition, the heavy chain phosphorylation site will be localized within the heavy chain. The regulation of contraction of isolated brush borders by myosin phosphorylation and by calcium will also be characterized by identification of the phosphorylation myosin, and by use of specific antibody and peptide inhibitors of each kinase. In vivo phosphorylation sites will also be identified in chicken intestinal explants. These studies should enhance our understanding of myosin function and organization and their regulation in intestinal epithelial and other non-muscle cells.

肌球蛋白，肌动蛋白和相关的细胞骨架蛋白相互作用，形成和 保持养分所需的大刷边框膜表面 肠上皮细胞的吸收。 另外，基于肌动蛋白 微维利的运动和终端Web区域的收缩 在吸收过程本身中，刷边框可能很重要。 定义 并表征这些事件的分子基础，鸡的作用 刷子边界肌球蛋白磷酸化和钙结合与肌球蛋白的结合将 对以下调节进行研究：a）肌球蛋白肌动蛋白激活的ATPase活性，b） 肌球蛋白细丝组件，c）肌球蛋白分子的构象， 刷子肌球蛋白指导的乳胶珠的运动在体外 运动性测定。 刷边界钙调蛋白依赖性重链激酶， 我们最近确定的独立于钙调蛋白的轻链激酶 在这些研究中使用孤立的将用于磷酸化纯化的刷子 在特定的，定义明确的地点接壤。 调节 钙和钙依赖性肌球蛋白激酶的活性 自磷酸化的钙调蛋白和重链激酶也将 被描述。 重链激酶也是重链激酶 已知由钙调蛋白调节，将在提取的提取物中进行测定 其他组织并在免疫学上与显然相似的人进行比较 广泛分布的钙调蛋白依赖性蛋白激酶II。 我们的先前 磷酸氨基酸对肌球蛋白磷酸化位点的表征 分析和肽映射将扩展到确定 每个站点周围的序列。 另外，重链磷酸化 站点将位于重链中。 调节 通过肌球蛋白磷酸化收缩孤立的刷子边界，并通过 钙也将以鉴定磷酸化的特征 肌球蛋白，并使用每种特异性抗体和肽抑制剂 激酶。 体内磷酸化位点也将在鸡肉中鉴定 肠外植物。 这些研究应该增强我们对 肌球蛋白功能和组织及其在肠道中的调节 上皮和其他非肌肉细胞。