FUNCTIONAL ASPECTS OF C-GMP-DEPENDENT PROTEIN KINASE

环磷酸腺苷和环磷酸鸟苷似乎都引起平滑肌松弛 通过增加它们各自的蛋白激酶活性。 而 普遍认为，环GMP可能通过 环GMP依赖性蛋白激酶活性增加， 这种酶及其特异性底物才刚刚开始被确定。 这项研究建议是针对更充分地表征循环 GMP依赖性蛋白激酶，特别强调了解 这两种类型的酶的特定环GMP结合位点的作用。 这些位点在亲和力上明显不同（超过30倍）， 因此导致一种独特的变构调节。 第一个具体目标将试图更多地了解 酶 已知该酶是二硫键连接的二聚体，但 两种类型的环GMP结合位点的相对位置 酶的底物结合位点是未知的。 该特定 目的是利用共价标记，部分蛋白水解和肽 测序以更精确地描述酶的组织。 的 使用的探针包括用8-叠氮基环[32 P]GMP标记， 8-叠氮基[α-32 P]ATP，用[γ-32 P]ATP磷酸化酶 用1-[1- 14 C]甘氨酸乙酯标记暴露的羧基。 将通过HPLC色谱法分离标记的肽。 的相对 位点的位置将通过肽的共标记 用两种不同的探针，并通过比较标记的 具有酶的氨基酸序列的肽。 第二个具体目标将研究这两个网站在 控制天然蛋白质底物的磷酸化。 可溶性和 颗粒状牛气管提取物将被磷酸化， SDS-聚丙烯酰胺凝胶电泳分离的磷酸化蛋白质 或2维聚丙烯酰胺凝胶电泳。 的 观察到的磷酸化将与蛋白质的活化相关 激酶通过环GMP结合到一个或两个特定的环GMP 结合位点。 环磷酸鸟苷已被认为是参与控制平滑肌 在许多组织中。 很有吸引力的推测是， 环GMP系统可能与哮喘等疾病有关， 高血压 本提案中所述的研究将提供 更好地验证这些假设。

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.