CHEMISTRY AND FUNCTION OF DICTYOSTELIUM CALMODULIN

盘基菌钙调蛋白的化学性质和功能

• 批准号: 3277359
• 负责人: MARGARET B CLARKE
• 金额: $ 18.8万
• 依托单位: OKLAHOMA MEDICAL RESEARCH FOUNDATION
• 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 1993-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3277359
• 关键词: Dictyostelium; calmodulin; cell differentiation; cell motility; chemical structure function; chemotaxis; cyclic AMP; density gradient ultracentrifugation; eukaryote; fluorescence microscopy; fungal genetics; genetic manipulation; immunochemistry; immunofluorescence technique; molecular cloning; monoclonal antibody; mutant; myosins; nucleic acid sequence; phagocytosis; pinocytosis; point mutation; protein sequence; radioimmunoassay; 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.

钙调蛋白是一种存在于所有真核细胞中的钙结合蛋白；它 似乎负责调节钙离子的调节作用 在众多的生物过程中。蛋白质是高度保守的， 因此，可以方便地对其结构和功能进行研究 真核生物以及脊椎动物细胞。这个项目的特点是 真核微生物盘基网柄菌和盘基网柄菌中的钙调蛋白 探索它的作用，特别是在运动调节方面。早些时候 研究表明，这种蛋白质虽然在功能上类似于 脊椎动物的钙调蛋白有几个氨基酸差异，包括 没有三甲基化赖氨酸残基。初级数的确定 结构将完成，而氨基酸的后果 我们将探讨不同之处。与钙离子、肌钙蛋白I和钙离子的相互作用 脊椎动物的酶将在这方面进行检测。单克隆抗体 将针对网柄菌钙调素提高；该部位(或多个部位)朝向 将确定这些抗体针对的是哪种抗体，以及突变体 将寻求在该位置产生改变的钙调蛋白。蛋白质是指 与网柄苔藓相互作用的钙调蛋白将被确定并 特征的，重点是可能参与调控的蛋白质 肌球蛋白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量的产生 或其他手段将直接进行测试。抗网囊线虫抗体 将使用钙调蛋白、肌球蛋白和选定的钙调蛋白结合蛋白 可视化这些蛋白质在有丝分裂和趋化作用中的定位 细胞；对于这些蛋白质中的一些，单抗将被提高到 便于纯化和绘制功能结构域。基因(S) 将分离和测序Dictyostelialcalmodin的编码基因，作为一个 朝着长期目标迈出的第一步，即分析 通过对蛋白质进行基因修饰。