Human Plasma Gelsolin Reversibly Binds Mg-ATP in Ca²⁺-Sensitive Manner

Abstract

Gelsolin is a Ca²⁺-regulated actin-modulating protein found in a variety of cellular cytoplasm and also in blood plasma. Affinity separation of human plasma gelsolin was successfully accomplished by eluting the protein with a low concentration of nucleoside polyphosphate from immobilized Cibacron Blue F3GA (1, 2). This finding was followed by the demonstration that the protein had one class of ATP binding site with K_d=2.8×10^-7_M, which saturated at an ATP/gelsolin ratio of 0.6 in the absence of Ca²⁺ (3). To obtain further information on the nucleotide binding properties of gelsolin, binding studies were done in the presence of EGTA with GTP, ADP, and GDP by equilibrium dialysis. Incubation of plasma gelsolin with GTP resulted in binding of 0.6 mol of GTP per mol of protein with a dissociation constant of 1.8×10^-6_M, indicating that ATP binds to gelsolin with higher affinity than GTP. Neither ADP nor GDP at up to 100μM appreciably bound to gelsolin at a physiological salt concentration. Then, the effects of divalent metal ions on the ATP binding to plasma gelsolin were examined. Gelsolin bound to ATP with K_d=2.4×10^-6_M in a solution containing 2mM MgCl₂, whereas micromolar free Ca²⁺ concentrations inhibited ATP binding. Furthermore, addition of Ca²⁺ rapidly reversed the preformed nucleotide binding to gelsolin, suggesting that Ca²⁺ binding to gelsolin leads to a conformational change which disrupts a nucleotide binding fold in the protein molecule.