The long-term goal of this project is to quantitatively characterize Ca++ regulation of cyclic nucleotide metabolism in heart muscle with particular emphasis on the role played by calmodulin (CaM). CaM mediates Ca++ stimulation of several important enzymes include the Ca++-sensitive phosphodiesterase (PDE), phosphorylase kinase (PK), and myosin light chain kinase (MLK). The affinity of CaM for these proteins is significantly enhanced upon binding of Ca++ to CaM. This grant proposal is based on two related working hypotheses. First, we propose that CaM-protein complexes may exist under physiological conditions even in the absence of free Ca++, and that the rate limiting step for Ca++ stimulation may be the diffusion of Ca++ to preformed complexes. Secondly, we propose that binding of Ca++ to CaM results in expression of a hydrophobic domain which is a major component of CaM-protein interfaces. It is predicted that various hydrophobic ligands of physiological interest may therefore antagonize binding of CaM to various CaM binding proteins. It is one of the major objectives of this proposal to rigorously quantitate the free energy of coupling for binding of Ca++ and CaM binding proteins to CaM. In addition, the influence of various hydrophobic ligands on CaM-protein associations will also be examined. These general objectives will be accomplished using fluorescent CaM (AEDANS.CaM) and fluorescence anisotropy to directly quantitate CaM binding to the PDE, MLK, PK and the guanyl nucleotide regulatory complex (G/F) of adenylate cyclase. The influence of CaM(Ca2+)4 on the sensitivity of heart adenylate cyclase to various effectors (norepinephrine, glucagon, guanyl nucleotides and cholera toxin) will also be examined since we have recently discovered that CaM binds to G/F in response to Ca++. The major focus of this project is to quantitate and characterize binding of CaM to these protein systems since these interactions lie as the basis for CaM regulation of enzyme activities.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
Research Project (R01)
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Molecular Cytology Study Section (CTY)
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University of Washington
Schools of Medicine
United States
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