ANALYSIS OF THE INTERACTIONS OF ACTIN DEPOLYMERIZING FACTOR WITH G-ACTIN AND F-ACTIN

Chick actin depolymerizing factor (ADF) is an actin binding protein previously shown to rapidly depolymerize actin filaments in vitro, yielding a 1:1 complex of ADF and actin monomer. Here we show that ADF protects actin monomer from denaturation by EDTA by inhibiting the exchange of actin-bound nucleotide. Under low ionic strength conditions, the approximate dissociation constant (K(D)) for the ADF-actin complex determined from exchange of nucleotide (1,N6-etheno-ATP) is about 150 nM and is calcium-independent. Addition of ADF to monomeric actin inhibits actin assembly as well as the ATP hydrolysis that normally accompanies assembly. Complex formation is demonstrated between ADF and actin containing either ATP, ADP, or AMPPNP as the bound nucleotide. A K(D) of 0.1-0.2 muM was calculated for both the ADF-ATP-actin and ADF-AMPPNP-actin complexes, whereas the K(D) for the ADF-ADP-actin complex is about 1.3 muM. ADF can either depolymerize or cosediment with F-actin in a stoichiometric fashion, but these reciprocal activities are pH-dependent. At pHs between 6.5 and 7.1, ADF cosediments with F-actin and demonstrates only weak depolymerizing activity. ADF binding is cooperative and saturates at a 1:1 ADF:actin molar ratio. At pHs between 7.1 and 7.7, ADF shows increasing depolymerizing activity and less F-actin binding. At pH 8.0, ADF depolymerizes F-actin in a stoichiometric manner. Both the F-actin binding and the depolymerizing activities of ADF are inhibited by phalloidin. At pH 8.0, substoichiometric quantities of ADF, but not ADF-actin complex, transiently enhance the nucleating ability of F-actin solutions in a calcium-insensitive manner, suggesting that ADF has a weak severing activity, but does not cap the filament ends in a manner that inhibits the restoration of the original filament number. We conclude from these results that ADF is a noncapping, calcium-independent, pH sensitive F-actin binding/severing protein which is capable of forming moderate affinity complexes with actin monomers in which the nucleotide exchange is inhibited. The significance of these findings to a presumed cellular role for ADF is discussed.