Actin polymerization. The effect of brevin on filament size and rate of polymerization

Abstract

Fluorescent probes covalently bound to actin or to the actin binding protein, brevin, have been utilized to provide information about actin filaments formed in the presence of brevin as well as about the effect of brevin on the rate of polymerization. At actin to brevin ratios of 10:1 to 100:1, the observed diffusion coefficients of filaments, as measured by fluorescence photobleaching recovery using rhodamine-labeled actin or fluorescein-labeled brevin, are similar to those calculated from theoretical considerations for rigid rods. At lower brevin concentrations, the observed diffusion coefficients for actin filaments are lower than predicted, indicating that the filament structure is closer to that observed in the absence of brevin where filaments are immobilized due to interactions between them. The fluorescein-labeled brevin was found to be about as effective in influencing actin polymerization as unlabeled brevin. Using pyrene-labeled actin, we show that brevin binds 2 mol of monomeric actin. We conclude that at sufficiently high brevin concentration there is one brevin molecule per actin filament. From measurements of the initial rate of polymerization at 5.9 microM actin in the presence of brevin, we calculate both the apparent elongation rate constant and dissociation rate constant from one end (presumably the slow-growing end) of the actin filament. The former is highly dependent on Mg2+ concentration while the latter is not.