Measurement of Nucleotide Release from Synthetic Myosin Thick Filament along which Actin Fiaments Slide

Abstract

Actin filaments move along myosin thick filaments a fast speed towards the central bare zone and at a slower speed away from the bare zone. Sellers and Kachar speculated that myosin heads rotate180 degrees to face the actin in the correct configuration and the detachment of the rotated heads from actin occurs at a slower rate. Recently, we have found that the thermal activation energy of the backward movement was remarkably higher than that of the forward movement, suggesting that the backward movement causes the myosin heads to be constrained, and increases the energy required for the ADP release step. However, there is no direct evidence for the slow ADP release rate in the backward movement. Here, in order to examine whether rate of ADP release step depends on the direction of the actin movement, we measured nucleotide turnover rates of bipolar myosin thick filament along which actin filament slides by monitoring the displacement of prebound fluorescent ATP analog, Cy3-EDA-ATP on flash photolysis of caged ATP.