Characterization of a Calmodulin-like Protein from Arabidopsis

作为第二信使的钙离子的主要细胞内靶标是钙结合蛋白。在植物中，钙调蛋白是唯一被识别并具有生物化学特征的钙结合蛋白。另一方面，动物含有许多额外的钙结合蛋白（钙调蛋白样蛋白），它们介导细胞或组织特异性钙调节过程。基于cDNA克隆和直接生化分析，本研究的工作假设是植物也含有钙调素样蛋白，它可能介导钙调节的过程，不同于钙调素介导的过程。为了检验和进一步扩展这一假设，提出了以下具体目标：(1)从拟南芥中分离得到一个cDNA克隆，该克隆编码的蛋白与其他高等植物钙调素具有约60%的氨基酸序列同源性，并将其与拟南芥幼苗中纯化的钙调素蛋白的生化特性进行比较；(2)建立拟南芥cDNA编码蛋白的antobody6探针，利用该探针确定拟南芥发育过程中钙调素样多肽表达的组织特异性，并检测可能与钙调素样多肽相互作用的靶蛋白的亚细胞分布；(3)以NAD激酶激活为模型靶酶系统，研究拟南芥钙调素与钙调素样蛋白的功能关系。本研究的长期目标是确定植物中钙结合蛋白家族的成员，并确定这些蛋白在植物生长发育中的生理作用。由于已知类似的蛋白质在动物的许多细胞内功能的调节中起重要作用，因此对植物中这些假定的调节分子的表征将大大提高我们对钙对植物细胞功能的调节的理解。

A primary intracellular target for calcium ions acting as second messengers are calcium binding proteins. In plants, calmodulin is the only recognized and biochemically characterized calcium binding protein. Animals, on the other hand, contain a number of additional calcium-binding proteins (calmodulin-like proteins), which mediate cell- or tissue- specific calcium-regulated processes. The working hypothesis for this research, based on cDNA cloning and direct biochemical assays, is that plants also contain calmodulin-like proteins which may mediate calcium- regulated processes distinct from those mediated by calmodulin. To test and further extend this hypothesis the following specific goals are proposed: (1) to complete the characterization of a cDNA clone, isolated from Arabidopsis thaliana, which encodes a polypeptide that shares approximately 60% amino acid sequence identity with other higher plant calmodulins, by comparing the biochemical properties of the protein encoed by this clone with authentic calmodulin purified from Arabidopsis seedlings; (2) to develop an antobody6 probe for the protein encoded by the Arabidopsis cDNA, and to use this probe to ascertain the tissue- specificity of expression of the calmodulin-like polypeptide during Arabidopsis development, and to examine the subcellular distribution of target proteins that potentially interact with the calmodulin-like polypeptide; and (3) to examine the functional relationship between calmodulin and the Arabidopsis calmodulin-like protein using NAD kinase activation as a model target enzyme system. The long range goals of this research are to identify in plants the members of the family of calcium binding proteins, and to determine what physiological roles these proteins play in plant growth and development. Since similar proteins are known to be important in the regulation of many intracellular functions in animals, the characterization of these putative regulatory molecules in plants should greatly enhance our understanding of the regulation of plant cell function by calcium.