The main objective of this research is to investigate the biochemical interrelationship and mechanism of regulation of the multiple forms and factors of the cyclic nucleotide phosphodiesterase enzyme system. The direction of these studies is to test the hypothesis that a recently purified high affinity enzyme form is the basic catalytic subunit of mammalian cyclic nucleotide phosphodiesterase and that lower affinity forms are aggregates of this enzyme with itself and calcium dependent regulation (calmodulin), inhibitory binding protein, or other protein effectors. Biochemical, immunological, and pharmacological techniques will be employed for this purpose. Purified components of selected portions of the phosphodiesterase systems from kidney, brain, skeletal muscle, uterus, and acute myelogenous leukemic cells will be studied for their similarities and differences including kinetics, susceptibility to activation/inhibition, immunological crossreactivity and identity, peptide maps, drug binding, and substrate affinity labeling. Also, previously defined conditions for an integrated and systematic comparative study with linear sucrose gradient fractionation, behavior on ion-exchange celluloses, gel filtration, and isoelectric focusing will be used. Studies will be continued with cultured, synchronized Hela cells to define the role of cyclic nucleotide phosphodiesterases in the cell cycle and their susceptibility to cellular regulation. In addition, studies will be continued on the resposes of cyclic nucleotide phosphodiesterases of normal and leukemic leukocytes to serum, methylxanthines, and plant mitogens. These studies are designed to test our hypothesis that activation/inhibition occurs by protein synthetic-dependent and-independent mechanisms through the production of factors that modulate the activities of high and low affinity forms of cyclic nucleotide phosphodiesterases. Knowledge gained from these studies will be used to initiate additional studies on the regulation of high affinity, particulate cyclic nucleotide phosphodiesterase by insulin in normal and diabetic tissues and during the growth of cells.
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