Our long range goal is to understand the regulatory mechanism(s) in smooth muscle. Smooth muscle are vital for many processes (eg. in arteries and veins) and in order to treat abnormal function an appreciation of the basic biochemistry is essential. An increase in intracellular Ca2+ induces contraction and this involves phosphorylation of the 20,000-dalton light chain of myosin via the calmodulin-dependent myosin light chain kinase (MLCK). Dephosphorylation occurs in relaxation. Our data suggests that phosphorylation controls contractile activity by changing the conformation of myosin (from an inactive to an active state) and that the changes occurring under physiological conditions occur also in the 6S-10S transition of monomeric myosin. Parts of this in-vitro transition therefore can serve as a model for the in-vivo process. Our initial objective is to challenge the shape-activity hypothesis and to confirm that enzymatic activity is directed by conformation. A subsequent emphasis will be to identify those changes that occur in the myosin molecule that modify ATPase activity and actin-binding, to define the changes that occur during the 10S-6S transition and to correlate these to the alterations induced by phosphorylation. Since little is known about the structures of the smooth muscle myosin molecule a preliminary characterization of tertiary and quaternary structure is essential. Those changes that might fulfil a regulatory function will be identified. Some of the factors that influence myosin conformation will also be studied, including actin and tropomyosin, with the objective of relating altered conformation to a modificaiton of biological properties. It will also be determined if myosin conformation can direct or influence kinetic parameters associated with phosphorylation and with ATPase activities. Additional sites on the light chain are phosphorylated by both MLCK and protein kinase C. However, the biological consequences of these phosphorylations are not established and this will be studied and correlated to conformation. Our studies on MLCK will be continued and we will probe the functional organization of the molecule using limited proteolysis and test the hypothesis that the apoenzyme is inactive because of interaction with an inhibitory zone. Synthetic peptides, based on the known sequences of calmodulin-binding sites, will be used as models and will be tested for inhibitory activity with the Ca2+-independent form of MLCK. Caldesmon is proposed as a regulatory protein in smooth muscle and studies will be carried out to evaluate this hypothesis.
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