KINETIC-ANALYSIS OF THE INTERACTION OF ACTIN-DEPOLYMERIZING FACTOR (ADF) COFILIN WITH G-ACTIN AND F-ACTIN - COMPARISON OF PLANT AND HUMAN ADFS AND EFFECT OF PHOSPHORYLATION/

The thermodynamics and kinetics of actin interaction with Arabidopsis thaliana actin-depolymerizing factor (ADF)(1), human ADF, and S6D muta nt ADF(1) protein mimicking phosphorylated (inactive) ADF are examined comparatively. ADFs interact with ADP.G-actin in rapid equilibrium (k (+) = 155 mu M-1.s(-l) and k(-) = 16 s(-1) at 4 degrees C under physio logical ionic conditions). The kinetics of interaction of plant and hu man ADFs with F-actin are slower and exhibit kinetic cooperativity, co nsistent with a scheme in which the initial binding of ADF to two adja cent subunits of the filament nucleates a structural change that propa gates along the filament, allowing faster binding of ADF in a ''zipper '' mode. ADF binds in a non-cooperative faster process to gelsolin-cap ped filaments or to subtilisin-cleaved F-actin, which are structurally different from standard filaments (Orlova, A, Prochniewicz, E,, and E gelman, E, H. (1995) J. Mel. Biol. 245, 598-607), In contrast, the bin ding of phalloidin to F-actin cooperatively inhibits its interaction w ith ADF. The ADF-facilitated nucleation of ADP actin self-assembly ind icates that ADF stabilizes lateral interactions in the filament, Plant and human ADFs cause only partial depolymerization of F-actin at pH 8 , consistent with identical functions in enhancing F-actin dynamics, P hosphorylation does not affect ADF activity per se, but decreases its affinity for actin by 20-fold.