Interaction of actin monomers with Acanthamoeba actophorin (ADF/cofilin) and profilin.
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 (Kd = 0.1 microM) 40 times stronger than Mg-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.
Blanchoin
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Structural Biology Laboratory, The Salk Institute for Biological Studies, La Jolla, California 92037, USA.
Pollard
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D000048:Acanthamoeba / Q000378:metabolism*; D000199:Actins / Q000378:metabolism*; D000818:Animals; D001667:Binding, Competitive; D003285:Contractile Proteins; D005454:Fluorescence Polarization; D008840:Microfilament Proteins / Q000737:chemistry / Q000378:metabolism*; D051304:Profilins; D011485:Protein Binding; D015800:Protozoan Proteins; D011994:Recombinant Proteins / Q000378:metabolism; D012235:Rhodamines
D000199:Actins; D003285:Contractile Proteins; D008840:Microfilament Proteins; D051304:Profilins; D015800:Protozoan Proteins; D011994:Recombinant Proteins; D012235:Rhodamines; C047604:actophorin protein, Acanthamoeba