The long term goal of this project is to define the possible roles of Ca2+ and cyclic nucleotides in the regulation of lens cell function. Specifically, this project will identify what lens cell membrane components make specific interactions with calmodulin, or are substrates for the lens cell cAMP-dependent protein kinase (cAMP-PK). The location of these components within the lens cell membrane should permit the development of models in which these components either regulate lens gap junction permeability, or regulate lens membrane ion pump/channel activities, or both. Affinity labeling procedures will be used to characterize the lens cell membrane components that make Ca-dependent interactions with calmodulin. The calmodulin-binding components, isolated using calmodulin affinity chromatographic procedures, will be used to generate antibodies in rabbits. These antibodies will be used to locate immunocytochemically, the calmodulin binding components in the lens cell membrane. The role of cAMP in regulating lens membrane activities will be achieved in four sections. The first will be to characterize the adenylate cyclase present in the lens cell membranes. The location of this enzyme in the lens cell will be determine dutilizing cytochemical procedures. Such identification could implicate the cyclase in junctional versus non-junctional lens membrane activities. The second section will consider the relationship between the lens cell phosphoproteins and lens membrane activities. This will be achieved using either crosslinking (to determine with which lens membrane components these phosphoproteins interact) or immunocytochemistry (to determine the locations of these phosphoproteins in the lens membrane). The third section will characterize the cyclic nucleotide phosphodiesterase activities present in the lens. The fourth section will characterize the lens cell protein phosphatase(s) that dephosphorylate the membrane substrates for cAMP-PK. Characterization of these different enzyme activities should indicate the role of cAMP in the regulation of lens cell-to-cell permeability, or lens membrane ion pump/channel activities, or both. Many manifestations of cataracts are exaggerations of changes found in the aging lens. The amount of insoluble protein increases in most forms of cataracts and the role of the lens membrane in this process remains to be explained. By defining the role of some of the lens membrane proteins, it is anticipated that this project will lead to an improved understanding of lens fiber regulation and thus a better basis for interpreting the nature of cataract formation.