Acanthamoeba actophorin is a member of ADF/cofilin family that binds both actin monomers and filaments. We used fluorescence anisotropy to study the interaction of actin monomers with recombinant actophorin labeled with rhodamine on a cysteine substituted for Serine-88. Labeled actophorin retains its affinity for actin and ability to reduce the low shear viscosity of actin filaments. At physiological ionic strength, actophorin binds Mg-ADP-actin monomers (K d ‫؍‬ 0.1 M) 40 times stronger than

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... -ATP-actin monomers. When bound to actin monomers, actophorin has no effect on elongation at either end of actin filaments by Mg-ATP-actin and slightly increases the rate of elongation at both ends by Mg-ADP-actin. Thus actophorin does not sequester actin monomers. Sedimentation equilibrium ultracentrifugation shows that actophorin and profilin compete for binding actin monomers. Actophorin and profilin have opposite effects on the rate of exchange of nucleotide bound to actin monomers. Despite the high affinity of actophorin for ADP-actin, physiological concentrations of profilin overcome the inhibition of ADP exchange by actophorin. Profilin rapidly recycles ADP-actin back to the profilin-ATP-actin pool ready for elongation of actin filaments. Acanthamoeba actophorin (1, 2) belongs to a family of low molecular weight actin-binding proteins including vertebrate cofilin, destrin, and ADF (3-9), echinoderm depactin (10, 11), and yeast cofilin (12, 13). Cofilin is dephosphorylated in response to various agonists (Refs. 14 -16; and reviewed by Moon and Drubin, Ref. 17) suggesting that it may be part of a regulated signal transduction mechanism controlling the assembly of the actin cytoskeleton. The original protein of this type, ADF, 1 got its name "actin depolymerizing factor" by virtue of its ability to depolymerize actin filaments. Similar evidence led to the name depactin. However, cofilin copolymerizes with actin at pH Ͻ7.3, depolymerizing filaments only at higher pH (18, 19) . Subsequent work has not led to a consistent view of the mechanism of action of ADF/cofilin proteins. A variety of evidence suggested that ADF/ cofilin proteins sever actin filaments and hasten depolymeriza-tion by creating more ends to dissociated subunits (2, 18 -20). Carlier et al. (21) questioned the severing activity of the ADF/ cofilin family and proposed a "dynamizing" mechanism for plant cofilin. Their interpretation was that plant cofilin promotes actin filament turnover by increasing the rates of ATPactin association at the barbed end and ADP-actin dissociation at the pointed end. Either by severing or enhancing dissociation of subunits from the pointed end, ADF/cofilin proteins should release Mg-ADP-actin monomers, which bind actophorin and cofilin with higher affinity than ATP-actin (21, 22). Both porcine brain and yeast cofilin inhibit the exchange of Mg-ATP bound to actin (23, 24). The effect of ADF/cofilin proteins on ADP exchange is not known, but high affinity binding might trap ADP-actin bound to an ADF/cofilin protein. Using a new fluorescence anisotropy assay, we confirm the higher affinity of actophorin for Mg-ADP-actin than Mg-ATPactin and characterize in detail the effect of both actophorin and profilin on the exchange of the bound nucleotide. Without profilin, actophorin forms a stable complex with ADP-actin. Profilin competes with actophorin for binding actin monomers. Even with saturating concentrations of actophorin, a low concentration of profilin promotes the rapid exchange of ADP for ATP. Given the higher affinity of Mg-ATP-actin for profilin, these reactions rapidly transfer actin from actophorin to profilin, allowing actophorin to recycle back to actin filaments. We also show that actophorin bound to ADP-or ATP-actin does not inhibit actin filament elongation at either the barbed or pointed end of filaments. MATERIALS AND METHODS Reagents-Materials came from the following sources: Sigma, dithiothreitol (DTT), EDTA, Tris, sodium azide, Me 2 SO, hexokinase, ATP, ADP, phalloidin, Sephadex G-25 medium; Molecular Probes (Eugene, OR), 1,N 6 -ethenoadenosine-5Ј-diphosphate (⑀-ADP), TCEP (Tris-(2-carboxyethyl)phosphine, 5Ј-tetramethylrhodamine maleimide isomer; Whatman (Maidstone, United Kingdom), DEAE-cellulose DE-52. Preparation of Mutant Actophorins-Residues Asn-33, Glu-70, and Ser-88 of actophorin were changed to cysteine by reverse polymerase chain reaction mutagenesis (25), and the mutations were verified by sequencing. These cysteines were the only reactive sulfhydryls, because neither of the two native cysteines are accessible to solvent (26). Wild type and mutant actophorins in plasmid vector pMW172 were expressed in Escherichia coli strain BL21 (DE3) pLysS and purified according to Quirk et al. (27), except that 2 mM dithiothreitol was included in all buffers for mutant actophorins to avoid cysteine oxidation. Purified actophorins were stored in 10 mM Tris-Cl, pH 7.5, 1 mM EDTA, 2 mM DTT, 1 mM NaN 3 . Preparation of Fluorescent Conjugates-A 4-ml sample of 50 M actophorin in storage buffer was dialyzed 3 h into labeling buffer (10 mM Tris-Cl, pH 7.5, 1 mM EDTA, 2 mM TCEP, 1 mM NaN 3 ) and then reacted with 7-fold molar excess of 5Ј-tetramethylrhodamine maleimide isomer from a 20 mM stock in Me 2 SO for 2 h at 4°C with gentle stirring. Centrifugation removed precipitated dye. Two steps removed free dye: first, gel filtration on a 60 ϫ 1.5-cm column Sephadex G25 equilibrated with 10 mM Tris-Cl, pH 7.5, 1 mM EDTA, 2 mM DTT, 250 mM NaCl, 1 mM NaN 3 , at 4°C; and second, chromatography on a 13 ϫ 1.5-cm column of DEAE-cellulose DE-52 equilibrated with 10 mM Tris-HCl, pH