An understanding of the mechanism of force production in smooth muscle and its regulation is essential in dealing with the very serious disorders associated with smooth muscle such as vascular disease. When fully understood, the differences between smooth and skeletal muscles can be exploited therapeutically to combat these disorders. Smooth muscle generates more tension per myosin molecule than does skeletal muscle. Smooth muscle can also maintain tension for long periods of time after stimulation (latch). These differences are probably due to differences in the subcellular organization, the structure of myosin, and the regulatory systems. The regulatory systems are of particular medical importance since only they can be modulated by an external influence. A particularly interesting component of the regulatory system of smooth muscle is the actin and calmodulin binding protein caldesmon. Caldesmon inhibits the actin activated hydrolysis of ATP by smooth and skeletal myosin and their proteolytic subfragments. The inhibition of the ATPase activity of skeletal myosin subfragment-1 is associated with an inhibition of the binding of subfragment-1 to actin. In contrast, the ATPase activity of heavy meromyosin from smooth muscle occurs with an increase in the binding of heavy meroymosin to actin. The present proposal is to determine how caldesmon can have opposite effects on binding and still produce inhibition of ATPase activity. In so doing the mechanism of caldesmon activity will be clarified and further differences between smooth and skeletal myosins will be explored. Three hypotheses for the increased binding of smooth heavy meromyosin in the presence of ATP will be explored: (1) The presence of two binding sites on actin-caldesmon - one productive and the other nonproductive. (2) The reduction in the rate of ADP release. (3) A calcium dependent crosslinking of the actin filaments. These studies will involve measurement of binding of caldesmon and myosin subfragments to actin, steady-state kinetics, rapid kinetic measurements using a stopped-flow spectrophotometer, chemically crosslinking actin, myosin and caldesmon, proteolytic digestion of various protein-protein complexes to determine sites of interaction, and affinity chromatography. Additional studies are designed to determine the role of uncharacterized caldesmon binding proteins in the regulatory process. Also, the mechanism of reversal of caldesmon's inhibitory activity will be investigated.
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