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

This is the authors' abstract. We don't add key points for this paper.

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 microM. The rate of association was followed 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 Ca(2+)-binding site with a dissociation constant Kd1 = 25 microM. Binding of the second actin monomer was found to be controlled by a Ca(2+)-binding site of which the dissociation constant Kd2 was 200 microM. 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 Kd1 (e.g. 10 microM) at almost maximal rates.