Mechanism of interaction of Acanthamoeba actophorin (ADF/cofilin) with actin filaments

We characterized the interaction of Acanthamoeba actophorin, a member of ADF/cofilin family, with filaments of amoeba and rabbit skeletal muscle actin. The affinity is about 10 times higher for muscle actin filaments (K-d = 0.5 mu M) than amoeba actin filaments (K-d = 5 mu M) even though the affinity for muscle and amoeba Mg-ADP-actin monomers (K-d = 0.1 mu M) is the same (Blanchoin, L., and Pollard, T. D. (1998) J, Biol, Chem. 273, 25106-25111), Actophorin binds slowly (k(+) = 0.03 mu M-1 s(-1)) to and dissociates from amoeba actin filaments in a simple bimolecular reaction, but binding to muscle actin filaments is cooperative. Actophorin severs filaments in a concentration-dependent fashion. Phosphate or BeF3 bound to ADP-actin filaments inhibit actophorin binding. Actophorin increases the rate of phosphate release from actin filaments more than 10-fold. The time course of the interaction of actophorin with filaments measured by quenching of the fluorescence of pyrenyl-actin or fluorescence anisotropy of rhodamine-actophorin is complicated, because severing, depolymerization, and repolymerization follows binding. The 50-fold higher affinity of actophorin for Mg-ADP-actin monomers (K-d = 0.1 mu M) than ADP-actin filaments provides the thermodynamic basis for driving disassembly of filaments that have hydrolyzed ATP and dissociated gamma-phosphate.