Isoenzymes of Soybean Calcium-Dependent Protein Kinase

来自大豆的钙依赖性蛋白激酶是 一类新的蛋白激酶，其具有调节结构域， 类似于钙调素。 这种酶的生化特性 表明它在信号转导通路中起作用 利用钙作为第二信使。 最近的证据表明 大豆中存在多种CDPK同工酶。 支持 这一结论是观察到两个cDNA的序列 编码大豆CDPK的基因大约70%相同。 同工酶 具有不同的对钙的敏感性，底物特异性， 而组织分布可以提供差异的基础 细胞对钙的反应。 建议的目标 研究的目的是鉴定和表征CDPK的同工酶。 的 分子生物学、生物化学和免疫学技术将 用于研究结构、功能、调节和 CDPK同工酶的分布。 编码该蛋白的cDNA 同工酶将在大肠埃希氏菌中鉴定和表达。 表达的同工酶将被纯化， 表征了 识别特定同工酶的抗体将 被制造。 将使用抗体和cDNA探针来确定 同工酶在植物中的分布。 此外 催化和调节结构域的功能和相互作用 通过在E.杆菌和 随后对各个功能结构域进行表征。

Calcium-dependent protein kinase from soybean is the prototype for a new class of protein kinases having regulatory domains that are similar to calmodulin. The biochemical properties of this enzyme indicate that it plays a role in signal transduction pathways employing calcium as a second messenger. Recent evidence suggests that several isoenzymes of CDPK are present in soybean. Supporting this conclusion is the observation that the sequences of two cDNAs encoding soybean CDPK are approximately 70% identical. Isoenzymes having differing sensitivities to calcium, substrate specificities, and tissue distributions could provide the basis for differences in cellular responses to calcium. The objective of the proposed research is to identify and characterize isoenzymes of CDPK. The techniques of molecular biology, biochemistry, and immunology will be used to study the structure, function, regulation, and distribution of the CDPK isoenzymes. The cDNAs encoding the isoenzymes will be identified and expressed in Escherechia coli. The expressed isoenzymes will be purified and biochemically characterized. Antibodies that recognize specific isoenzymes will be made. Antibodies and cDNA probes will be used to determine the distribution of the isoenzymes in the plant. In addition the function and interaction of the catalytic and regulatory domains of CDPK will be studied through the expression in E. coli and subsequent characterization of the individual functional domains.