Measurement of rate constants for actin filament elongation in solution

Abstract

This paper describes a simple method to measure the rate constants for actin filament elongation using pyrene-actin fluorescence as a measure of the polymer concentration and unlabeled actin filaments as nuclei. With careful selection of conditions, the initial rate of polymerization is directly proportional to the actin monomer concentration above the critical concentration. Plots of initial rate versus actin concentration give the critical concentration (x intercept), the association rate constant, k+ (slope), and the dissociation rate constant, k-(y intercept). By calibrating the system under conditions where the absolute values of these rate constants are known from previous electron microscopic experiments [T. D. Pollard and M. S. Mooseker (1981) J. Cell Biol. 88, 654-659; J. A. Cooper, S. B. Walker, and T. D. Pollard (1983) J. Muscle Res. Cell Motil. 4, 253-262], one can calculate the absolute values of the rate constants under other conditions as well as the length of the filaments used as a nuclei. This approach has proven useful for evaluating the effect of actin-binding proteins on the polymerization process.