We propose to test the hypothesis that cyclic adenosine-monophosphate is involved in regulation of alternate pathways of cellular aging in Dictyostelium discoideum. We will define biochemically the prestalk and prespore cells of this organism with the goal of developing a model system for the study of cellular aging. We will test the involvement of cyclic AMP in stalk cell differentiation by a three phase approach utilizing both in vivo and in vitro probes. First, we will test the inducibility or reversal of differentiation and aging by perturbation of the system with components of the cyclic AMP regulatory pathway. We will perturb normal cell determination with substrates (cyclic AMP, 5'AMP, adenosine, inorganic phosphate, and ammonium ion) isolated proteins (cyclic AMP phosphodiesterase, phosphodiesterase inhibitor, and 5'AMP nucleotidase) and univalent antibodies against phosphodiesterase and 5'AMP nucleotidase. The extent of perturbation will be measured by its effect on the morphology, histology and biochemistry of the two cell types. Second we will continue our characterization of the cyclic AMP regulatory system in the two cell types. We will investigate (1) the ultrastructural localization of phosphodieserase, 5'AMP nucleotidase and adenyl cyclase (2) the metabolic fate of exogenous cyclic AMP (3) the localization of cyclic AMP, 5'AMP and adenosine in the two cell types and (4) the distribution of adenyl cyclase in stalk and spore cells. Finally we will probe three enzymes involved in the regulation of cyclic AMP levels, phosphodiesterase, adenyl cyclase and 5'AMP nucleotidase, for their mode of regulation in vivo by (1) isolating the enzymes and determining their kinetic properties (2) performing immunotitration experiments to determine the genetic or epigenetic mechanism for their accumulation in a specific cell type, (3) develop an immunotitration assay to test for latent enzyme activity in the cell type showing no activity, (4) characterize and determine the cellular origin of isozymes (5) use 125I-conconavalin A to determine the cellular location of membrane-bound enzymes and (6) develop antisera for immuno histochemical localization of two enzymes.