Smooth muscle myosin isolated from mammalian vascular tissue or from avian gizzard has a number of unique properties. Upon addition of MgATP, dephosphorylated filaments are dissociated to monomeric myosin. Moreover, the 1500 angstroms long smooth muscle myosin tail is folded into thirds, in contrast to the extended, asymmetric shape characteristic of skeletal myosin. Phosphorylation of the regulatory light chain is necessary to reassemble the myosin into filaments. The first objective of the proposed study is to determine how events in the globular head region, namely binding of MgATP and phosphorylation, affect filament assembly and cause conformational transitions between the extended (6S) and folded (10S) forms of the smooth muscle myosin molecule. Insight into a possible mechanism will be obtained by observing the behavior of myosin which has been modified by light chain removal or by phosphorylation at different sites on the light chain. Effects of nucleotide binding and phosphorylation will also be investigated through the use of heavy meromyosin, the proteolytic subfragment which lacks the region of the rod that folds. Sedimentation velocity will be used to determine the effect of these modifications on the conformation of monomeric myosin and its assembly into polymer in solution, while electron microscopy of metal-shadowed molecules will show structural details such as the position of the heads relative to the rod. A second major aspect of this proposal involves the enzymatic function of smooth muscle myosin. Are changes in myosin structure and assembly reflected in changes in the actin-activated ATPase activity, independent of the state of light chain phosphorylation? What is the enzymatic activity and conformation of myosin with only one of its two heads phosphorylated? Information obtained from gel filtration, native gel electrophoresis, and actin-activated ATPases will be used to answer these questions. The long-term goal of this project is to determine if the folded conformation actually exists in a smooth muscle cell and, if so, how it relates to 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 approach of hydrodynamic analysis, electron microscopy and immunology, new insights will be gained about the function of smooth muscle in both normal and diseased vascular tissues.
| Trybus, K M; Lowey, S (1987) Subunit exchange between smooth muscle myosin filaments. Prog Clin Biol Res 245:81-90 |
