Effects of KCl, MgCl2, and CaCl2 concentrations on the monomer-polymer equilibrium of actin in the presence and absence of cytochalasin D.

Critical metal concentrations of KCl, MgCl2, and CaCl2 below which actin cannot exist in filamentous form at equilibrium were estimated to be approximately 8 mM, 0.2 mM, and 0.4 mM, respectively, when actin, 0.5 mg/ml, was incubated for a long time at pH 8.0 and 25 degrees C. These values were obtained by viscosity, UV absorbance and pyrene-labeled actin fluorescence measurements. In the case of CaCl2, viscosity measurements resulted in a larger critical concentration, 0.7 mM. It turned out that actin filaments formed in a low concentration of CaCl2 were easily fragmented by shearing force. The higher the actin concentration, the lower the critical metal concentration was. The critical concentrations of actin above which actin can exist as a polymer at equilibrium became larger as the KCl, MgCl2, and CaCl2 concentrations were lower. It is advisable to add 0.05-0.1 mM CaCl2 to a depolymerization solution to obtain a concentrated monomeric solution. In the presence of cytochalasin D, which preferentially blocks the elongation at the barbed end of actin filaments, the critical concentrations of KCl and CaCl2 were the same as in its absence. On the other hand, the critical concentration of MgCl2 was increased and the extent of polymerization was decreased by cytochalasin D. In the presence of 50 mM KCl and 1 microM cytochalasin D, 0.01-0.1 mM MgCl2 markedly decreased the extent of polymerization of actin at equilibrium, where the critical actin concentration was tenfold increased. It is suggested that Mg2+ enhances dissociation of actin monomers at the pointed end of an actin filament.