Two Low-Affinity Ca2+-Binding Sites of Gelsolin that Regulate Association with Actin

Abstract

The time course of binding of actin to gelsolin or 1:1 gelsolin-actin complex was measured at defined Ca2+ concentrations in the range 0.5–500 μM. The rate of association was fol10wed by the fluorescence increase of a fluorescent label covalently linked to actin. Free Ca2+ was determined by titration with EGTA in the presence of Fura-2 as indicator. The experimental data were quantitatively evaluated by calculations of the kinetics of association of actin with gelsolin thereby taking into account the equilibrium of binding of Ca2+ ions to gelsolin. It was found that association of gelsolin with one actin monomer is regulated by a Ca2+-binding site with a dissociation constant Ka1= 25 μM. Binding of the second actin monomer was found to be controlled by a Ca2+-binding site of which the dissociation constant Kd2 was 200 μM. Mg2+ ions in the concentration range 0–1 mM did not compete with Ca2+ for binding to gelsolin. More complex interactions of gelsolin with actin such as nucleated actin polymerization were found to occur even at Ca2+ concentrations below Kdl (e.g. 10 μM) at almost maximal rates.