NONMUSCLE MYOSIN ACTIVITY AND ASSEMBLY

非肌肉肌球蛋白活性和组装

• 批准号: 2176650
• 负责人: JIMMY H COLLINS
• 金额: $ 18.43万
• 依托单位: TEMPLE UNIV OF THE COMMONWEALTH
• 依托单位国家: 美国
• 财政年份: 1988
• 资助国家: 美国
• 起止时间: 1988-08-01 至 1996-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2176650
• 关键词: adenosinetriphosphatase; brush border membrane; calcium; calmodulin; calmodulin dependent protein kinase; conformation; enzyme activity; gastrointestinal absorption /transport; gastrointestinal epithelium; membrane reconstitution /synthesis; microfilaments; myosin light chain kinase; myosins; phosphorylation; protein sequence; tissue /cell culture

Myosin, actin and associated cytoskeletal proteins interact to form and maintain the large brush border membrane surface necessary for nutrients absorption in intestinal epithelial cells. In addition, actomyosin-based movements of microvilli and contractions 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, calmoudulin-dependent light chain kinase and calmoudulin- independent light chain kinase that we have recently identified and isolated will be used in these studies to phosphorylate purified brush 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 the only 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 calmoulin-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 sites, by reconstitution of nyosin-depleted brush borders with phosphorylated 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 形成并维持大刷状缘膜表面 肠上皮细胞吸收营养所必需的。 在 此外，基于肌动球蛋白的微绒毛运动和 刷子边缘末端网状区域的收缩可能 在吸收过程本身中很重要。 定义和 描述这些事件的分子基础， 鸡刷状缘肌球蛋白磷酸化和钙的影响 将研究与肌球蛋白的结合：a) 肌球蛋白 肌动蛋白激活的 ATP 酶活性，b) 肌球蛋白丝组装，c) 肌球蛋白分子的构象和刷状缘 体外运动中乳胶珠的肌球蛋白定向运动 化验。 刷状缘钙调蛋白依赖性重链激酶， 钙调蛋白依赖性轻链激酶和钙调蛋白- 我们最近发现的独立轻链激酶 并分离将用于这些研究中以磷酸化纯化的 在特定的、明确的部位刷肌球蛋白。 的监管 钙对钙调蛋白依赖性肌球蛋白激酶的活性 和钙调蛋白和重链激酶 自身磷酸化也将得到表征。重链 激酶是唯一已知受到调节的重链激酶 通过钙调蛋白，将在其他组织的提取物中进行检测 在免疫学上与明显相似且广泛的 分布式钙调蛋白依赖性蛋白激酶 II。 我们之前的 肌球蛋白磷酸化位点的表征 磷酸氨基酸分析和肽图分析将得到扩展 以确定每个位点周围的序列。 在 此外，重链磷酸化位点将被定位 重链内。 孤立收缩的调节 刷状缘通过肌球蛋白磷酸化和钙也会 通过磷酸化位点的鉴定来表征， 重建Nyosin耗尽的刷状缘 磷酸化肌球蛋白，并通过使用特异性抗体和 每种激酶的肽抑制剂。 体内磷酸化位点 也将在鸡肠道外植体中进行鉴定。 这些 研究应该增强我们对肌球蛋白功能的理解 肠上皮等的组织及其调节 非肌肉细胞。