IN-VITRO MOTILITY ANALYSIS OF ACTIN-TROPOMYOSIN REGULATION BY TROPONIN AND CALCIUM - THE THIN FILAMENT IS SWITCHED AS A SINGLE COOPERATIVE UNIT

In striated muscles, contractility is controlled by Ca2+ binding to the regulatory protein complex troponin, which is a component of the thin filaments. Troponin is an allosteric inhibitor acting on tropomyosin to switch the thin filament between ''on'' and ''off'' states. We have used an in vitro motility assay to examine troponin regulation of individual actin-tropomysin filaments moving over immobilized skeletal muscle heavy meromyosin. The most striking observation is that the actin-tropomyosin filament appears to be regulated as a single unit. At pCa 9.0, addition of up to 4 nM troponin causes the proportion of filaments motile to decrease from >85% to 20% with no dissociation of the filaments from the heavy meromyosin surface or change in velocity. Increasing Ca2+ concentration causes the filaments to be switched back on the half-maximal increase in the proportion of filaments motile at pCa 5.8-6.0 and a modest increase in filament velocity. This is an ''all or none'' process in which an entire filament, up to 15 mu M long, switches rapidly as a single cooperative unit. Thus, the effect of Ca2+ upon the thin filament is to recruit motile filaments.