CHEMISTRY AND FUNCTION OF DICTYOSTELIUM CALMODULIN

盘基菌钙调蛋白的化学性质和功能

• 批准号: 3277361
• 负责人: MARGARET B CLARKE
• 金额: $ 19.53万
• 依托单位: ALBERT EINSTEIN COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 1981
• 资助国家: 美国
• 起止时间: 1981-07-01 至 1988-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3277361
• 关键词: Dictyostelium; adenylate cyclase; affinity chromatography; antibody formation; cell differentiation; cell motility; chemical structure function; cyclic AMP; enzyme induction /repression; eukaryote; fluorescence microscopy; gel electrophoresis; immunochemistry; monoclonal antibody; mutant; nucleic acid sequence; phagocytosis; pinocytosis; radioimmunoassay; radiotracer; spectrometry; tissue /cell culture

Calmodulin is a calcium-binding protein found in all eukaryotic cells; it appears to be responsible for mediating the regulatory effects of calcium on numerous biological processes. The protein has been highly conserved, so its structure and functions can be conveniently examined in lower eukaryotes as well as vertebrate cells. This project would characterize calmodulin from the eukaryotic microorganism Dictyostelium discoideum and explore its roles, particularly in the regulation of motility. Earlier work has revealed that this protein, although functionally similar to vertebrate calmodulins, has several amino acid differences, including the absence of trimethylated lysine residue. Determination of the primary structure will be completed, and the consequences of the amino acid differences will be explored. Interaction with Ca++, troponin I, and vertebrate enzymes will be examined in this regard. Monoclonal antibodies will be raised against Dictyostelium calmodulin; the site (or sites) toward which these antibodies are directed will be determined, and mutants producing calmodulin altered at this site will be sought. Proteins that interact with Dictyostelium calmodulin will be identified and characterized, with emphasis on proteins that may be involved in regulating the structural states or interaction of myosin and actin. The ability of calmodulin to modulate myosin force production through a light chain kinase or other means will be tested directly. Antibodies against Dictyostelium calmodulin, myosin, and selected calmodulin-binding proteins will be used to visualize the localization of these proteins in mitotic and chemotaxing cells; for some of these proteins, monoclonal antibodies will be raised to facilitate purification and to map functional domains. The gene(s) encoding Dictyostelium calmodulin will be isolated and sequenced, as an initial step toward a long range goal of analyzing the function of the protein by genetically modifying it.

钙调素是一种存在于所有真核细胞中的钙结合蛋白， 似乎负责介导钙的调节作用 许多生物过程。 该蛋白质高度保守， 因此，可以方便地在较低温度下检查其结构和功能， 真核生物以及脊椎动物细胞。 该项目将体现 来自真核微生物盘基网柄藻的钙调蛋白， 探索它的作用，特别是在调节运动性方面。 早些时候 研究表明，这种蛋白质，虽然功能相似， 脊椎动物钙调素，有几个氨基酸的差异，包括 不存在三甲基化赖氨酸残基。 确定主要 结构将被完成，氨基酸的后果 将探讨差异。 与Ca++、肌钙蛋白I和 脊椎动物酶将在这方面进行检查。 单克隆抗体 将提高对网骨藻钙调蛋白;该网站（或网站）， 将确定这些抗体所针对的，以及突变体 将寻找在该位点改变的产生钙调蛋白的基因。 的蛋白质 将鉴定与网骨藻钙调蛋白相互作用， 特征，重点是可能参与调节的蛋白质 肌球蛋白和肌动蛋白的结构状态或相互作用。 的能力 钙调蛋白通过轻链激酶调节肌球蛋白力的产生 或其他手段将被直接测试。 抗网骨藻抗体 将使用钙调蛋白、肌球蛋白和选定的钙调蛋白结合蛋白 为了使这些蛋白质在有丝分裂和化学趋化中的定位可视化， 细胞;对于其中一些蛋白质，将产生单克隆抗体， 有助于纯化和绘制功能结构域。 所述基因 编码网骨藻钙调蛋白的基因将被分离和测序，作为 这是朝着分析其功能的长期目标迈出的第一步。 蛋白质通过基因改造。