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.
