Orthovanadate Slows Kinetics Of The Acto-Myosin Interaction In Skinned Muscle Fibers By Competition Between Myosin-ADP-Pi and Myosin-ADP-Vi Cross-Bridges For Actin Sites

Abstract

Orthovanadate (Vi) is a phosphate analog that has been used to study the relationship between biochemical reactions and structural and mechanical transitions in the cross-bridge cycle, with great success. In solution, Vi binds to myosin with high affinity, forming a stable myosin-ADP-Vi complex. In permeabilized muscle fibers, Vi binds to an actin-myosin-ADP state during cross-bridge cycling, and suppresses isometric tension, isometric stiffness and unloaded shortening velocity. We investigated the effects of Vi on the mechanics and kinetics of the actin-myosin interaction in skinned fibres from rabbit psoas (sarcomere length 2.4 μm, temperature 12 °C). After correction for myofilament compliance, the observed changes in stiffness indicate that the reduction in T0 isdue to a proportional reduction in the number of myosin cross-bridges attached to actin (Caremani et al., Biophys J 480a/2289-Pos, 2007). The effect of [Pi] (range 0-15 mM added [Pi]) on T0 and the rate constant of force development following a period of unloaded shortening (kD) in the presence of Vi (0.1 mM) suggest that Vi acts as a competitive inhibitor of Pi for the myosin-ADP state (Caremani et al., Biophys J 128a/621-Pos, 2008). Simulations show that the effects of Vi on force, stiffness and rate of force redevelopment, as well as on the actomyosin ATPase (Wilson et al, Biophys J 68:216-226,1995) and velocity of unloaded shortening(Chase et al, J Physiol 460:231-246, 1993), can be explained with a straightforward modification of the kinetic scheme of Dantzig et al, J Physiol 451:247-278, 1992 to include myosin-ADP-Vi heads competing with myosin-ADP- Pi heads for actin sites. Supported by NIH (R01 AR049033.03) and MiUR (Italy).