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 determined that isometric force increased 200-fold with thiophosphorylation of the myosin regulatory light chain, and that with thiophosphorylated myosin, isometric force was further increased by the addition of saturating 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 microns/s) was constant for mixtures of dephosphorylated and thiophosphorylated myosin containing > 5% thiophosphorylated myosin, and was unaffected by the addition of saturating concentrations of tropomyosin or caldesmon. In contrast, "standard assay conditions" were found to be nonreducing. With fully thiophosphorylated smooth-muscle myosin, saturating smooth-muscle tropomyosin increased velocity to 150% of control, and caldesmon halted all filament motion; with fully dephosphorylated myosin (< 0.002 mol/mol) filaments only moved when tropomyosin or tropomyosin plus calponin was added. Taken together, these observations suggest that under "standard conditions" a mechanical load is present that is eliminated by "reducing conditions." Filament velocity was insensitive to changes in cross-bridge density, under all conditions, suggesting that noncycling cross-bridges, generated by photochemical oxidation of myosin, is a likely source of mechanical loading.
Haeberle
J R
JR
Department of Molecular Physiology and Biophysics, University of Vermont, Burlington 05405, USA.
Hemric
M E
ME
D000199:Actins / Q000502:physiology*; D000205:Actomyosin / Q000502:physiology; D000818:Animals; D001696:Biomechanical Phenomena; D055592:Biophysical Phenomena; D001703:Biophysics; D002148:Calmodulin-Binding Proteins / Q000494:pharmacology; D066298:In Vitro Techniques; D007537:Isometric Contraction / Q000502:physiology; D009068:Movement / Q000187:drug effects / Q000502:physiology; D009130:Muscle, Smooth / Q000502:physiology; D009218:Myosins / Q000737:chemistry / Q000502:physiology; D010766:Phosphorylation; D010777:Photochemistry; D014335:Tropomyosin / Q000502:physiology
D000199:Actins; D002148:Calmodulin-Binding Proteins; D014335:Tropomyosin; D000205:Actomyosin; D009218:Myosins