thin filament4inked regulatory protein caldesmon and to determine how the interplay between thin-filament-linked and thick-filament-linked systems regulates the level of cross bridge activation (force production) and the rate of cross bridge cycling (unloaded shortening velocity). The proposed studies will test the hypothesis that the actin-binding protein, caldesmon, is a major component of an actin-linked regulatory system which meddles the activation of contraction which occurs in response to myosin phosphorylation. The proposed studies will specifically test the hypothesis that caldesmon modulates both cross bridge cycling rates and the number of attached cross bridges at any given level of myosin phosphorylation. These studies will also test the hypothesis that both calcium-calmodulin and caldesmon phosphorylation regulate the effects of caldesmon. The experimental approach will involve measurement of caldesmon phosphorylation and mechanical parameters for pharmacologically activated smooth muscle. In addition, the levels of caldesmon and myosin phosphorylation will be manipulated in chemically permeabilized (skinned) muscles while measuring the effects on contractile properties (i.e. shortening velocity and force development). Phosphopeptide mapping, phosphoamino analysis, and gas phase microsequencing will be used to analyze the phosphorylation sites on caldesmon. In collaboration with project #3, an in vitro actin-motility assay will also be used to determine what effects caldesmon, phosphocaldesmon, and calcium-calmodulin have on the translation velocity of actin filaments over a myosin surface. Lastly, the distribution of caldesmon among native actin filaments isolated from tonic, vascular, and pha3ic, nonvascular smooth muscle will be measured to determine d there are caldesmon-free populations of actin in smooth muscles. These studies will not only verity that caldesmon plays a regulatory role in smooth muscle but will also provide significant new information about the mechanisms underlying this regulatory process and about the involvement of caldesmon in the formation of latch bridges in vascular smooth muscles.
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