Smooth muscle myosin isolated from mammalian vascular tissue or from avian gizzard has a number of unique properties. Addition of approximately stoichiometric amounts of MgATP causes dephosphorylated filaments to depolymerize. The disassembled monomers adopt a conformation in which the 1500 Angstrom long myosin tail is folded into thirds, in contrast to the extended, asymmetric shape characteristic of skeletal myosin. Upon phosphorylation of the regulatory light chain in the myosin head the myosin reassembles into filaments. A major focus of this proposal is to understand the mechanism of these conformational transitions. The role of the light chains and of nucleotide in the folded to extended transition, and the question of whether events originating in the globular head can be directly detected in the rod will be investigated. The myosin conformation in solution will be determined by sedimentation velocity, while electron microscopy of metal-shadowed molecules will be used to resolve structural details. Our observations of rapid subunit exchange between minifilaments will be extended to larger synthetic and native filaments; exchange could be a mechanism by which myosin disassembles from the filament upon MgATP addition. A second major aspect of the proposal is concerned with the enzymatic activity of smooth muscle myosin. Do the two heads of myosin act independently or cooperatively, which kinetic steps are regulated by phosphorylation, and are there any differences between mammalian and avian smooth muscle myosins? The long-term goal of this project is to determine if the folded conformation exists in a smooth muscle cell and, if so, how it affects the function of these cells. Antibodies specific for the folded conformation will be prepared and used as probes to investigate the dynamics of the living muscle cell. It is hoped that through the combined approaches of hydrodynamic analysis, enzymatic activity, electron microscopy and immunology, some insight will be gained about how smooth muscle myosin functions in both normal and diseased vascular tissues.
