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 (Kd = 0.5 microM) than amoeba actin filaments (Kd = 5 microM) even though the affinity for muscle and amoeba Mg-ADP-actin monomers (Kd = 0.1 microM) is the same (Blanchoin, L., and Pollard, T. D. (1998) J. Biol. Chem. 273, 25106-25111). Actophorin binds slowly (k+ = 0.03 microM-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 (Kd = 0.1 microM) than ADP-actin filaments provides the thermodynamic basis for driving disassembly of filaments that have hydrolyzed ATP and dissociated gamma-phosphate.
Blanchoin
L
L
Structural Biology Laboratory, The Salk Institute for Biological Studies, La Jolla, California 92037, USA.
Pollard
T D
TD
D000048:Acanthamoeba / Q000378:metabolism*; D051339:Actin Depolymerizing Factors; D000199:Actins / Q000378:metabolism*; D000818:Animals; D001608:Beryllium / Q000494:pharmacology; D005454:Fluorescence Polarization; D005459:Fluorides / Q000494:pharmacology; D007700:Kinetics; D008840:Microfilament Proteins / Q000378:metabolism*; D018482:Muscle, Skeletal / Q000378:metabolism; D010710:Phosphates / Q000378:metabolism; D011485:Protein Binding / Q000187:drug effects; D015800:Protozoan Proteins / Q000378:metabolism*; D011721:Pyrenes; D011817:Rabbits; D012235:Rhodamines
D051339:Actin Depolymerizing Factors; D000199:Actins; D008840:Microfilament Proteins; D010710:Phosphates; D015800:Protozoan Proteins; D011721:Pyrenes; D012235:Rhodamines; C047604:actophorin protein, Acanthamoeba; C035982:beryllium fluoride; D001608:Beryllium; D005459:Fluorides