Actin-Activated Atpase and Actin Sliding Velocities are Similarly Influenced by Actin-Myosin Binding Kinetics

Abstract

Detachment-limited models of muscle shortening assume that with increasing myosin densities maximal velocities are achieved when at least one myosin head is bound to a given actin filament. However, numerous studies suggest that actin sliding velocities, V, are influenced by actin-myosin attachment kinetics, suggesting that V should saturate - along with actin-activated myosin ATPase activity, v - when myosin heads saturate binding sites on actin. Here we used an in vitro motility assay to measure both V and v in the same flow cell. We observed that both V and v exhibited similar saturation kinetics with increasing myosin densities and that increasing ionic strength similarly decreased the Km for both activation curves. Using the same in vitro assay, we measured the calcium-dependence of V for regulated thin filaments (actin filaments reconstituted with tropomyosin and troponin) and observed that the myosin-dependence of calcium sensitivity (pCa50) exhibited saturation kinetics similar to that of V and v. These results suggest that saturation of V, v, and pCa50 all occur when myosin saturates binding sites on actin. This is consistent with estimates showing that saturating myosin densities in a motility assay correspond to a linear spacing of myosin along an actin filament that is similar to the repeat of myosin binding sites along one side of the filament.