Biochemical Journal

Research article

A simple method for measuring the relative force exerted by myosin on actin filaments in the in vitro motility assay: evidence that tropomyosin and troponin increase force in single thin filaments

Wu BING, Adam KNOTT, Steven B. MARSTON

Abstract

We have studied the effect of an internal load on the movement of actin filaments over a bed of heavy meromyosin (HMM) in the in vitro motility assay. Immobilized α-actinin can bind to actin filaments reversibly and ultimately stop the filaments from moving. Above a critical concentration of α-actinin, thin filament velocity rapidly diminished to zero. The fraction of thin motile filaments decreased linearly to zero with increasing α-actinin concentration. The concentration of α-actinin needed to stop all filaments from moving (0.8µg/ml with actin) was very consistent both within and between experiments. In the present study we have defined the ‘index of retardation’ as the concentration of α-actinin needed to stop all filament movement, and we propose that this index is a measure of the isometric force exerted by HMM on actin filaments. When we measured the effect of immobilized α-actinin on motility in the presence of 10mM Pi we found that the index of retardation was 0.62±0.07 (n = 3) times that in the absence of Pi. This observation is in agreement with the reduction of isometric tension in chemically-skinned muscle due to Pi. In a series of comparative experiments we observed that tropomyosin and troponin increase the index of retardation and that the degree of increase depends upon the tropomyosin isoform studied. The index of retardation of actin is increased 1.8-fold by skeletal-muscle tropomyosin, and 3-fold by both cardiac-muscle and smooth-muscle tropomyosin. In the presence of troponin the index of retardation is 2.9–3.4-fold greater than that of actin with all tropomyosin isoforms.

  • contractility
  • isometric force
  • Ca2+-regulation
  • regulatory proteins
  • co-operativity