FUNCTIONAL ASPECTS OF C-GMP-DEPENDENT PROTEIN KINASE

环状AMP和环状GMP似乎都能使平滑肌肉松弛 通过增加它们各自的蛋白激酶活性。而当 有一种普遍的看法是，环状GMP可能通过 环状GMP依赖的蛋白激酶活性的增加， 这种酶及其特定的底物才刚刚开始被定义。 这项研究建议旨在更全面地表征循环 GMP依赖的蛋白激酶，特别强调了解 两类特异性环状GMP结合位点在酶上的作用。 这些站点在亲和力方面存在显著差异(超过30倍)，并且可能 因此导致了一种独特的变构调节。 第一个具体目标将试图了解更多关于 这种酶。已知这种酶是二硫键连接的二聚体，但 两种类型的环状GMP结合位点在 关于底物结合部位的酶是未知的。这个特定的 AIM将利用共价标记、部分蛋白分解和多肽 测序以更准确地描述酶的组织。这个 使用的探针包括8-叠氮环[32P]GMP标记和 8-叠氮基[Alpha-32P]ATP，酶与[Gamma-32P]ATP的磷酸化 以及用1-[1-14C]甘氨酸乙酯标记暴露的羧基。 用高效液相色谱分离标记的多肽。相对的 位点的位置将通过多肽的标记来确定 使用两种不同的探针，并通过比较标记的序列 带有酶的氨基酸序列的多肽。 第二个具体目标是研究这两个地点在 天然蛋白质底物的磷酸化控制。可溶的和 颗粒牛气管提取物将被磷酸化，并 十二烷基硫酸钠-聚丙烯酰胺凝胶电泳法分离磷酸化蛋白质 或根据需要进行双向聚丙烯酰胺凝胶电泳法。这个 观察到的磷酸化将与蛋白质的激活相关。 通过环GMP与特定的环GMP中的一个或两个结合来激活 结合部位。 环状GMP被认为参与了对平滑肌的控制 许多组织中的色调。很有吸引力的是，我们推测 环状GMP系统可能与哮喘和哮喘等疾病有关 高血压。本提案中描述的研究将提供一个 检验这些假说的更坚实的基础。

Both cyclic AMP and cyclic GMP appear to cause relaxation of smooth muscles through an increase of their respective protein kinase activities. While there is a general concensus that cyclic GMP probably acts through an increase in cyclic GMP-dependent protein kinase activity, the enzymology of this enzyme and its specific substrates are just beginning to be defined. This research proposal is directed toward more fully characterizing cyclic GMP dependent protein kinase with a special emphasis on understanding the role of the two types of specific cyclic GMP binding sites on the enzyme. These sites differ markedly in affinity (by more than 30-fold) and may therefore result in a unique type of allosteric regulation. The first specific aim will attempt to learn more about the structure of the enzyme. The enzyme is known to be disulfide linked dimer but the relative location of the two types of cyclic GMP binding sites on the enzyme with respect to substrate binding sites is unknown. This specific aim will utilize covalent labeling, partial proteolysis and peptide sequencing to more precisely describe the organization of the enzyme. The probes to be used include the labeling with 8-azido cyclic [32P]GMP and 8-azido [Alpha-32P]ATP, phosphorylation of the enzyme with [Gamma-32P]ATP and labeling of exposed carboxyl groups with 1-[1-14C]glycine ethyl ester. Labeled peptides will be separated by HPLC chromatography. The relative position of the sites will be determined both by colabeling of peptides with two different probes and by comparison of the sequences of labeled peptides with the amino acid sequence of the enzyme. The second specific aim will study the role of these two sites in the control of phosphorylation of native protein substrates. Soluble and particulate bovine trachea extracts will be phosphorylated and the phosphorylated proteins separated by SDS-polyacrylamide gel electrophoresis or 2-dimensional polyacrylamide gel electrophoresis, as required. The phosphorylations observed will be correlated with activation of the protein kinase through cyclic GMP binding to one or both of the specific cyclic GMP binding sites. Cyclic GMP has been suggested to be involved in controlling smooth muscle tone in many tissues. It is attractive to speculate that alterations in the cyclic GMP system could be involved in such diseases as asthma and hypertension. The studies described in this proposal will provide a stronger basis upon which to test such hypotheses.