The kinetics of actin nucleation and polymerization.

The polymerization kinetics of rabbit skeletal muscle actin was studied by following the increase in fluorescence of tracer amounts of actin conjugated to N-pyrenyl iodoacetamide. The observed polymerization kinetics could be precisely fit by numerical integration of equations describing a nucleation-elongation process. Under all conditions tested, the rate of nucleation was proportional to the fourth power of the actin concentration; therefore, the actin nucleus is a tetramer. In buffers containing either MgCl, or CaC12, but not both, the observed kinetics accurately fits the unique polymerization time course derived from the actin concentration, the critical concentration, and the product of the nucleation and filament elongation rate constants. When MgClz was added to G-actin in a buffer also containing CaCl,, polymerization did not follow simple nucleation-elongation kinetics because divalent cation exchange preceded nucleation. Relative filament elongation rate constants were 12,24,31,79,100, and 67 for actin in 1 l l l ~ CaCl,, 0.1 m CaClz + 0.1 M KC1, 1 m MgC1~,2 m MgCl,, 1 n m MgCl, + 0.1 M KC1, and 50 CM MgClz + 0.1 M KCl, respectively. The relative rate constants for filament-monomer dissociation differed less than 2-fold, but the relative nucleation rate constants varied dramatically: 1.7, 46, 550, 10,000, 10,000, and 2,900, respectively, under these conditions of polymerization. These data strongly support the validity of the nucleation-elongation theory of actin polymerization and establish the nucleus size as four. The rate of nucleus formation is the major variable determining the rate of actin polymerization. The high degree of sensitivity of the nucleation rate to the concentration of actin and to ionic conditions may indicate the way in which intracellular polymerization kinetics are regulated.