The long-term objective of this research is to understand the role of smooth muscle tropomyosin in the regulation of contraction. Although this role is presently unclear, it is thought that tropomyosin's end-to-end interaction plays a key part in the cooperative nature of regulation. In order to better understand the molecular basis of this end-to-end interaction, a specific goal of this proposal is to investigate the source of the large heterogeneity in gizzard tropomyosin's end-to-end interaction observed by hydroxylapatite chromatography. This question will be addressed by using several techniques including viscosity, chromatography, electrophoresis, amino acid analysis, and sedimentation. A second specific aim is to test the hypothesis that tropomyosin's end-to-end interaction is responsible for its cooperative behavior. This will be accomplished by determining the effect of gizzard tropomyosin with different end-to-end interactions on its cooperative binding to actin and its cooperative regulation of actomyosin ATPase activity. Caldesmon, a new smooth muscle protein, imparts a thin filament Ca2+- sensitivity to regulation which requires tropomyosin. In order to understand the mechanism thereby these two proteins work in concert, another goal of this project is to determine the effect of tropomyosin, with different end-to-end interactions, on the binding of caldesmon to the thin filament and correlate this with caldesmon's regulation of actomyosin ATPase activity. Finally, since the direct interaction between tropomyosin and caldesmon may play a key role in regulation, this grant proposes to investigate the direct interaction between tropomyosin, with different end-to- end interactions, and caldesmon with viscosity, spin labels, fluorescence labels, sedimentation, and chemical cross-linking.
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