Calcium-mediated regulation of recombinant hybrids of full-length Physarum myosin heavy chain with Physarum/scallop myosin light chains

Physarum myosin is a Ca^2＋-binding protein and its activity is inhibited by Ca^2＋. In the present study, to clarify the light chains （LCs） from the different species （Physarum and scallop） and to determine the specific Ca^2＋-regulated effects, we constructed hybrid myosins with a Physarum myosin heavy chain （Ph.HC） and Physarum and/or scallop myosin LCs, and examined Ca^2＋-mediated regulation of ATPases and motor activities. In these experiments, it was found that Ca^2＋ inhibited motilities and ATPase activities of Physarum hybrid myosin with scallop regulatory light chain （ScRLC） and Physarum essential light chain （PhELC） but could not inhibit those of the Physarum hybrid myosin mutant Ph.HC/ScRLC/PhELC-3A which lacks Ca^2＋-binding ability, indicating that PhELC plays a critical role in Ca^2＋-mediated regulation of Physarum myosin. Furthermore, the effects of Ca^2＋ on ATPase activities of Physarum myosin constructs are in the following order： Ph.HC/PhRLC/PhELC 〉 Ph.HC/ScRLC/PhELC 〉 Ph.HC/PhRLC/ScELC 〉 Ph.HC/ScRLC/ScELC, sug- gesting that the presence of PhRLC and PhELC leads to the greatest Ca^2＋ sensitivity of Physarum myosin. Although we did not observe the motilities of Physarum hybrid myosin Ph.HC/PhRLC/ ScELC and Ph.HC/ScRLC/ScELC, our results suggest that Ca^2＋-binding to the PhELC may alter the flexibility of the regulatory domain and induce a ＇closed＇ state, which may consequently prevent full activity and force generation.