Kinetics of gelsolin interaction with phalloidin-stabilized F-actin, rate constants for binding and severing

The kinetics of gelsolin interaction with actin filaments have been investigated using two fluorescent probes, tetramethylrhodamine isothiocyanate-labeled phalloidin bound to F-actin and N-(1-pyrenyl)iodoacetamide-labeled actin. we have also analyzed the F-actin severing by gelsolin using an assay for actin filaments which measures the polymerization rate of monomeric actin added to the gelsolin-severed filaments. Phalloidin-stabilized actin filaments were used in order to minimize the depolymerization reaction and thus simplify the kinetic analysis. Because gelsolin activity is Ca2+-activated, experiments were conducted in the presence of 0.5 mM CaCl2 to ensure maximal activity, We show that the interaction of gelsolin with F-actin may be separated into two distinct kinetic phases which correspond to binding and severing events. Using a two-step model of gelsolin activity, we have determined that gelsolin binds to F-actin with an association rate constant of 2 x 10(7) M(-1) s(-1), dissociates with a rate constant in the range 0.4-1.2 s(-1), and subsequently severs phalloidin-stabilized F-actin with a first-order rate constant of 0.25 s(-1). Characterization of the binding and severing reactions will facilitate further investigation of gelsolin activity and its regulation.