Modification of the bi-directional sliding movement of actin filaments along native thick filaments isolated from a clam

Summary The properties of bi-directional sliding of F-actin prepared from rabbit skeletal muscle moving along clam thick filaments have been characterized in the presence of agents known to modify unloaded shortening velocity in muscle to determine if the sliding characteristics of actin are similar in the two directions of movement. Actin filaments moved at a fast velocity towards the central bare zone (11.1±0.2 m s^-1) and at a slower velocity away from the bare zone (3.9±0.3 m s^-1). Movement of filaments at the slow sliding velocity is thought to be sustained by a change in orientation of the myosin head. The Michaelis Menten constant (K_m values) of 0.3 mm in the presence of MgATP concentrations of 0.01–2.0 mm at an ionic strength of 43.5 mm were reduced to 0.1 mm at low ionic strength (18.5 mm) although the K_m values at the fast and slow sliding velocities at each ionic strength were similar. In the presence of constant concentrations of MgATP, increasing the MgADP concentrations from 0.5 to 2 mm, decreased the bi-directional sliding velocity of actin. The data were well fitted with an equation described by Michaelis Menten kinetics yielding mean absolute K_m and K_i values of 0.41±0.01 and 0.44±0.05 mm for the fast velocity and 0.29±0.07 and 0.45±0.02 mm for the slow velocity of sliding, respectively. The K_m and K_i values were not significantly different from each other at either the fast or slow sliding, velocities. The actin filament sliding velocity appeared to be controlled through the thick filament as actin was devoid of regulatory proteins and the presence of Ca²⁺ modified the MgATP dependent movement of actin. The pCa value for half maximal sliding velocity was 7.0 for both fast and slow velocities. The K_m and K_i values and the Ca²⁺ sensitivity of the actin movement at the fast and slow sliding velocity are similar suggesting that no major biochemical changes have occurred in the myosin head as a result of a change in orientation.