Expression and characterization of Calcium-sensitive myosin II

Abstract

Purpose: Myosin II is one of the typical motor proteins and is classified as non-regulated, phosphorylatable and Ca-binding myosins. Physarum myosin II belongs to Ca-binding one. Myosin II regulated by Ca-binding has not yet expressed as a recombinant protein. Here, we report the expression of heavy mero-myosin of physarum myosin II together with preliminary characterizations. Method: We used baculovirus expression system. Sf9 cells were infected with the virus constructs. Result: When baculovirus of heavy chain(HC) fragments was infected together with those of phosphorylated light chain (PLC) and Ca-binding light chain (CaLC), Sf9 cells produced soluble HMM, which were recovered in the supernatant together with PLC and CaLC. The HMM showed Mg-ATPase activity of 0.21 (s⁻¹head⁻¹), and actin-activated ATPase activity with Vmax=1.27 (s⁻¹head⁻¹), and Km=1.8μM. The movement of actin filaments on the HMM-coated glass surface was sensitive to Ca²⁺. We will show the effect of Ca²⁺ on the movement of HMM associated with various kinds of light chains. [J Physiol Sci. 2006;56 Suppl:S109]