Blanchoin L, Pollard T D
Structural Biology Laboratory, The Salk Institute for Biological Studies, La Jolla, California 92037, USA.
J Biol Chem. 1998 Sep 25;273(39):25106-11. doi: 10.1074/jbc.273.39.25106.
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.
棘阿米巴肌动蛋白结合蛋白是ADF/丝切蛋白家族的成员，它既能结合肌动蛋白单体，也能结合肌动蛋白丝。我们利用荧光各向异性研究了肌动蛋白单体与在丝氨酸88被半胱氨酸取代的位置用罗丹明标记的重组肌动蛋白结合蛋白之间的相互作用。标记后的肌动蛋白结合蛋白保留了其对肌动蛋白的亲和力以及降低肌动蛋白丝低剪切粘度的能力。在生理离子强度下，肌动蛋白结合蛋白结合Mg-ADP-肌动蛋白单体（Kd = 0.1微摩尔）的能力比Mg-ATP-肌动蛋白单体强40倍。当与肌动蛋白单体结合时，肌动蛋白结合蛋白对Mg-ATP-肌动蛋白使肌动蛋白丝两端伸长的过程没有影响，而对Mg-ADP-肌动蛋白使两端伸长的速率略有增加。因此，肌动蛋白结合蛋白不会隔离肌动蛋白单体。沉降平衡超速离心表明，肌动蛋白结合蛋白和胸腺素β4竞争结合肌动蛋白单体。肌动蛋白结合蛋白和胸腺素β4对结合在肌动蛋白单体上的核苷酸交换速率有相反的影响。尽管肌动蛋白结合蛋白对ADP-肌动蛋白有高亲和力，但生理浓度的胸腺素β4能克服肌动蛋白结合蛋白对ADP交换的抑制作用。胸腺素β4能迅速将ADP-肌动蛋白循环回胸腺素β4-ATP-肌动蛋白池，为肌动蛋白丝的伸长做好准备。
