FACTORS AFFECTING MOVEMENT OF F-ACTIN FILAMENTS PROPELLED BY SKELETAL-MUSCLE HEAVY-MEROMYOSIN

The measurement of fluorescent-labeled actin filament movement driven by mechanoenzymes (e.g., myosin) is an important methodology for the study of molecular motors. It is assumed that the filament velocity (V(f)) is analogous to the unloaded shortening velocity (V(u)) seen in muscle fibers. Methods are described to reproducibly quantitate the movement of these filaments and to select uniformly moving filaments and specify their V(f). Use of these techniques allowed comparison of V(f) to literature values for V(u) with regard to [ATP], [ADP], [P(i)], pH, ionic strength (10-150 mM), and temperature (15-30-degrees-C). V(f) and V(u) are quantitatively similar with respect to the effects of substrate and product concentrations and temperatures > 20-degrees-C. However, V(f) is more sensitive to decreases in pH and temperatures < 20-degrees-C than V(u). At ionic strengths of 50-150 mM, V(f) and V(u) exhibit similar ionic strength dependencies (decreasing with ionic strength). At ionic strengths < 50 mM, V(f) is markedly reduced. Results of experiments using adenosine 5'-O-(3-thiotriphosphate) suggest that increasing the number of weakly bound cross bridges does not seriously affect V(f). Thus, although V(f) is a good analogue for V(u) under certain conditions (elevated ionic strength and temperatures > 20-degrees-C), under others it is not. The results of motility assays must be cautiously interpreted.