Effect of ADP and ionic strength on the kinetic and motile properties of recombinant mouse myosin V

Mouse myosin V is a two-headed unconventional myosin with an extended neck that binds six calmodulins. Double-headed (heavy meromyosin-like) and single-headed (subfragment 1-like) fragments of mouse myosin V were expressed in Sf9 cells, and intact myosin V was purified from mouse brain. The actin-activated MgATPase of the tissue-purified myosin V, and its expressed fragments had a high V-max and a low K-ATPase. Calcium regulated the MgATPase of intact myosin V but not of the fragments, Both the MgATPase activity and the in vitro motility were remarkably insensitive to ionic strength. Myosin V and its fragments translocated actin at very:low myosin surface densities. ADP markedly inhibited the actin-activated MgATPase activity and the in vitro motility, ADP dissociated from myosin V subfragment 1 at a rate of about 11.5 s(-1) under conditions where the V-max was 3.3 s(-1), indicating that, although not totally rate-limiting, ADP dissociation was close to the Fate-limiting step. The high affinity for actin and the slow rate of ADP release helps the myosin head to re main attached to actin for a large fraction of each ATPase cycle and allows actin filaments to be moved by only a few myosin V molecules in vitro.