Syunsuke Matsushita and Kuniyuki Hatori
Department of Biosystem Engineering,
Graduate School of Science and Engineering,
Yamagata University, Yonezawa, 992-8510 Japan
The sliding movement of actin filaments, consisting of heterogeneous components, on skeletal muscle myosin molecules was examined to specifically evaluate the effect of internal modulation of the actin filaments for force transmission on the sliding movement. Inactive actin molecules were prepared by conjugation with indocarbocyanine fluorescent dyes (IC3-OSu or Cy3-NHS) in molar ratios greater than a 3-fold excess. IC3-OSu is an analogue of Cy3-NHS, and it can bind to primary amino groups. IC3-conjugated actin (IC3-actin) monomers were polymerized into the filaments which led to complete impairment of both motile activity and myosin-ATPase activation. Filaments of Cy3-conjugated actin (Cy3-actin) exhibited a decrease in velocity to a third of the value (33%) observed for intact actin filaments. In the absence of ATP, dissociation rates of IC3- and Cy3-actin filaments from myosin molecules were greater than those of intact actin filaments, indicating that IC3- and Cy3-actin act as smaller resistance components against sliding movement compared to intact filaments. Subsequently, two types of copolymer filaments were prepared. The first type of copolymer were filaments copolymerized homogeneously with intact actin monomers and IC3-actin monomers, while the second kind were block copolymer filaments composed of two short filaments of intact actin and IC3-actin. The sliding velocities of these copolymer filaments hyperbolically decreased as the fraction of IC3-actin monomer increased. In practice, 75% IC3-actin within homogeneous copolymer was required to reduce the velocity by half. In the case of block copolymer 65% IC3-actin led to the same decrease in velocity. For Cy3-actin copolymer filaments similar differences between homogeneous and block copolymer filaments were also observed. Drag ratio between IC3-actin (or Cy3-actin) and intact actin was estimated by consideration of the force balance between the power force and the drag force imposed on a filament during steady movement. Consequently, the drag ratios of IC3-actin to intact actin were 0.31 (homogeneous copolymer) and 0.47 (block copolymer), respectively. Thus, IC3-actin incorporated homogeneous copolymers exhibits a smaller resistance to sliding movement than IC3-actin modified block copolymers.