ARE ACTIN-FILAMENTS MOVING UNDER UNLOADED CONDITIONS IN THE IN-VITRO MOTILITY ASSAY

With sliding actin-filament motility assays, filament velocity should be independent of changes in the level of actomyosin activation under unloaded conditions. Using a simple modification of the motility assay to measure relative changes in isometric force (activation), we deter mined that isometric force increased 200-fold with thiophosphorylation of the myosin regulatory light chain, and that with thiophosphorylate d myosin, isometric force was further increased by the addition of sat urating smooth-muscle tropomyosin (100%) or tropomyosin plus calponin (500%), and decreased with the addition of saturating caldesmon (-100% ). Under ''reducing conditions,'' filament velocity (2.0 mu m/s) was c onstant for mixtures of dephosphorylated and thiophosphorylated myosin containing >5% thiophosphorylated myosin, and was unaffected by the a ddition of saturating concentrations of tropomyosin or caldesmon. In c ontrast, ''standard assay conditions'' were found to be nonreducing. W ith fully thiophosphorylated smooth-muscle myosin, saturating smooth-m uscle tropomyosin increased velocity to 150% of control, and caldesmon halted all filament motion; with fully dephosphorylated myosin (<0.00 2 mol/mol) filaments only moved when tropomyosin or tropomyosin plus c alponin was added. Taken together, these observations suggest that und er ''standard conditions'' a mechanical load is present that is elimin ated by ''reducing conditions.'' Filament velocity was insensitive to changes in crossbridge density, under all conditions, suggesting that noncycling cross-bridges, generated by photochemical oxidation of myos in, is a likely source of mechanical loading.