Enzymatic characterization and functional domain mapping of brain myosin-V

The actin binding and ATPase properties, as well as the functional domain structure of chick brain myosin-V, a two-headed, unconventional myosin, is reported here, Compared to conventional myosin from skeletal muscle, brain myosin-V exhibits low K-EDTA- and Ca-ATPase activities (1.8 and 0.8 ATP/s per head), The physiologically relevant Mg-ATPase is also low (similar to 0.3 ATP/s), unless activated by the presence of both F-actin and Ca2+ (V-max of 27 ATP/s), Ca2+ stimulates the actin-activated Mg-ATPase over a narrow concentration range between 1 and 3 mu M. In the presence of saturating Ca2+ and 75 mM KCl, surprisingly low concentrations of F-actin activate the Mg-ATPase in a hyperbolic manner (K-ATPase of 1.3 mu M). Brain myosin-V also binds with relatively high affinity (compared to other known myosins) to F-actin in the presence of ATP, as assayed by cosedimentation, Digestion of brain myosin-V with calpain yielded a 65-kDa head domain fragment that cosediments with actin in an ATP-sensitive manner and a 80-kDa tail fragment that does not interact with F-actin, The 80-kDa fragment results from cleavage one residue beyond the proline-, glutamate-, serine-, threonine-rich region, Our data indicate that the Mg-ATPase cycle of brain myosin-V is tightly regulated by Ca2+, probably via direct binding to the calmodulin light chains in the neck domain, which like brush border myosin-I, results in partial (similar to 30%) dissociation of the calmodulin associated with brain myosin-V, The effect of Ca2+ binding, which appears to relieve suppression by the neck domain, can be mimicked by calpain cleavage near the head/neck junction.