Abstract
Regulation of the F-actin severing activity of gelsolin by Ca 2+ has been investigated under physiologic ionic conditions. Tryptophan fluorescence intensity measurements indicate that gelsolin contains at least two Ca 2+ binding sites with affinities of 2.5 × 10 7 M −1 and 1.5 × 10 5 M −1. At F-actin and gelsolin concentrations in the range of those found intracellularly, gelsolin is able to bind F-actin with half-maximum binding at 0.14 μM free Ca 2+ concentration. Steady-state measurements of gelsolin-induced actin depolymerization suggest that half-maximum depolymerization occurs at ∼0.4 μM free Ca 2+ concentration. Dynamic light scattering measurements of the translational diffusion coefficient for actin filaments and nucleated polymerization assays for number concentration of actin filaments both indicate that severing of F-actin occurs slowly at micromolar free Ca 2+ concentrations. The data suggest that binding of Ca 2+ to the gelsolin-F-actin complex is the rate-limiting step for F-actin severing by gelsolin; this Ca 2+ binding event is a committed step that results in a Ca 2+ ion bound at a high-affinity, EGTA-resistant site. The very high affinity of gelsolin for the barbed end of an actin filament drives the binding reaction equilibrium toward completion under conditions where the reaction rate is slow.
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