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

We have studied the effect of an internal load on the movement of actin fil aments over a bed of heavy meromyosin (HMM) in the in vitro motility assay. Immobilized alpha-actinin can bind to actin filaments reversibly and ultim ately stop the filaments from moving. Above a critical concentration of alp ha-actinin, thin filament velocity rapidly diminished to zero. The fraction of thin motile filaments decreased linearly to zero with increasing alpha- actinin concentration. The concentration of alpha-actinin needed to stop al l filaments from moving (0.8 mu g/ml with actin) was very consistent both w ithin and between experiments. In the present study we have defined the 'in dex of retardation' as the concentration of alpha-actinin needed to stop al l filament movement, and we propose that this index is a measure of the iso metric force exerted by HMM on actin filaments. When we measured the effect of immobilized alpha-actinin on motility in the presence of 10 mM P-i we f ound that the index of retardation was 0.62 +/- 0.07 (n = 3) times that in the absence of P-i. This observation is in agreement with the reduction of isometric tension in chemically-skinned muscle due to P-i. In a series of c omparative experiments we observed that tropomyosin and troponin increase t he index of retardation and that the degree of increase depends upon the tr opomyosin isoform studied. The index of retardation of actin is increased 1 .8-fold by skeletal-muscle tropomyosin, and 3-fold by both cardiac-muscle a nd smooth-muscle tropomyosin. In the presence of troponin the index of reta rdation is 2.9-3.4-fold greater than that of actin with all tropomyosin iso forms.