2 LOW-AFFINITY CA2-BINDING SITES OF GELSOLIN THAT REGULATE ASSOCIATION WITH ACTIN()

The time course of binding of actin to gelsolin or 1:1 gelsolin-actin complex was measured at defined Ca2+ concentrations in the range 0.5-5 00 mu M. The rate of association was followed by the fluorescence incr ease of a fluorescent label covalently linked to actin. Free Ca2+ was determined by titration with EGTA in the presence of Fura-2 as indicat or. The experimental data were quantitatively evaluated by calculation s 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 regul ated by a Ca2+-binding site with a dissociation constant K-d1 = 25 mu M Binding of the second actin monomer was found to be controlled by a Ca2+-binding site of which the dissociation constant K-d2 was 200 mu M . 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 K-d1 (e.g. 10 mu M) at almost maximal rat es.