Sucrose Increases the Activation Energy Barrier for Actin-Myosin Strong Binding

摘要

To determine the mechanism by which sucrose slows in vitro actin sliding velocities, V, we used stopped flow kinetics and a single molecule binding assay, SiMBA. We observed that in the absence of ATP, sucrose (880 mM) slowed the rate of actin-myosin (A-M) strong binding by 71 ± 8% with a smaller inhibitory effect observed on spontaneous rigor dissociation (21 ± 3%). Similarly, in the presence of ATP, sucrose slowed strong binding associated with Pi release by 85 ± 9% with a smaller inhibitory effect on ATP-induced A-M dissociation, kT (39 ± 2%). Sucrose had no noticeable effect on any other step in the ATPase reaction. In SiMBA, sucrose had a relatively small effect on the diffusion coefficient for actin fragments (25 ± 2%), and with stopped flow we showed that sucrose increased the activation energy barrier for A-M strong binding by 37 ± 3%, indicating that sucrose inhibits the rate of A-M strong binding by slowing bond formation more than diffusional searching. The inhibitory effects of sucrose on the rate of A-M rigor binding (71%) are comparable in magnitude to sucrose’s effects on both V (79 ± 33% decrease) and maximal actin-activated ATPase, kcat, (81 ± 16% decrease), indicating that the rate of A-M strong bond formation significantly influences both kcat and V.