Equilibrium and Kinetics of the Assembly of Capping Proteins with the Ends of Actin Filaments

Abstract

We report on the determination of equilibrium and rate constants and of the stoichiometry of binding of capping proteins to the barbed ends of actin filaments. Equilibrium constants can be measured by the depolymerizing effect of capping proteins on actin filaments. When a capping protein blocks monomer consumption at the polymerizing barbed ends of treadmilling actin filaments, monomers continue to be produced at the depolymerizing pointed ends until a new steady state is reached in which monomer production at the pointed ends is balanced by monomer consumption at uncapped barbed ends. This method was applied to determination of the equilibrium constants K for binding of the gelsolin-actin complex in the presence of micromolar Ca2+ -concentrations (Kg above 1011 M−1) and in excess EGTA (Kg = 1010 M−1) and for binding of the capping protein from bovine brain (Kg = 2×109 M −1). Retardation of nucleated actin polymerization brought about by highly purified vinculin was analyzed in terms of the number of vinculin molecules bound to the ends of actin filaments. The fraction of barbed ends of actin filaments occupied by vinculin was determined based on the decrease of the rate of polymerization. A plot of the fraction of barbed ends occupied by vinculin versus the free vinculin concentration was sigmoidal, indicating that vinculin binds cooperatively to the barbed ends of actin filaments. According to a Hill plot representation at least three vinculin molecules were found to bind to a barbed end in a cooperative manner. Rates of capping can be measured by inhibition of polymerization of actin filaments. Possible formation by capping proteins of new filaments which polymerize toward the pointed ends, can be prevented by keeping the monomer concentration below the critical monomer concentration of the pointed ends where the barbed ends of treadmilling filaments polymerize and the pointed ends depolymerize. By using this method the gelsolin-actin complex was found to bind fourfold faster to the barbed ends in the presence of micromolar ca2×(10×106 M−1s−1) than in excess EGTA (2.5×106M−1s−1).KeywordsEquilibrium ConstantActin FilamentMonomer ConcentrationActin PolymerizationBovine BrainThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.