Pollard T D, Cooper J A
Biochemistry. 1984 Dec 18;23(26):6631-41. doi: 10.1021/bi00321a054.
The current view of the mechanism of action of Acanthamoeba profilin is that it binds to actin monomers, forming a complex that cannot polymerize [Tobacman, L. S., & Korn, E. D. (1982) J. Biol. Chem. 257, 4166-4170; Tseng, P., & Pollard, T. D. (1982) J. Cell Biol. 94, 213-218; Tobacman, L. S., Brenner, S. L., & Korn, E. D. (1983) J. Biol. Chem. 258, 8806-8812]. This simple model fails to predict two new experimental observations made with Acanthamoeba actin in 50 mM KC1, 1 mM MgCl2, and 1 mM EGTA. First, Acanthamoeba profilin inhibits elongation of actin filaments far more at the pointed end than at the barbed end. According, to the simple model, the Kd for the profilin-actin complex is less than 5 microM on the basis of observations at the pointed end and greater than 50 microM for the barbed end. Second, profilin inhibits nucleation more strongly than elongation. According to the simple model, the Kd for the profilin-actin complex is 60-140 microM on the basis of two assays of elongation but 2-10 microM on the basis of polymerization kinetics that reflect nucleation. These new findings can be explained by a new and more complex model for the mechanism of action that is related to a proposal of Tilney and co-workers [Tilney, L. G., Bonder, E. M., Coluccio, L. M., & Mooseker, M. S. (1983) J. Cell Biol. 97, 113-124]. In this model, profilin can bind both to actin monomers with a Kd of about 5 microM and to the barbed end of actin filaments with a Kd of about 50-100 microM. An actin monomer bound to profilin cannot participate in nucleation or add to the pointed end of an actin filament. It can add to the barbed end of a filament. When profilin is bound to the barbed end of a filament, actin monomers cannot bind to that end, but the terminal actin protomer can dissociate at the usual rate. This model includes two different Kd's--one for profilin bound to actin monomers and one for profilin bound to an actin molecule at the barbed end of a filament. The affinity for the end of the filament is lower by a factor of 10 than the affinity for the monomer, presumably due to the difference in the conformation of the two forms of actin or to steric constraints at the end of the filament.
目前关于棘阿米巴肌动蛋白结合蛋白作用机制的观点是，它与肌动蛋白单体结合，形成一种无法聚合的复合物[Tobacman, L. S., & Korn, E. D. (1982) J. Biol. Chem. 257, 4166 - 4170; Tseng, P., & Pollard, T. D. (1982) J. Cell Biol. 94, 213 - 218; Tobacman, L. S., Brenner, S. L., & Korn, E. D. (1983) J. Biol. Chem. 258, 8806 - 8812]。这个简单模型无法预测在50 mM KCl、1 mM MgCl₂和1 mM EGTA条件下对棘阿米巴肌动蛋白进行的两项新实验观察结果。首先，棘阿米巴肌动蛋白结合蛋白在肌动蛋白丝的尖端比在刺端更能抑制肌动蛋白丝的伸长。根据简单模型，基于在尖端的观察，肌动蛋白结合蛋白 - 肌动蛋白复合物的解离常数（Kd）小于5 μM，而对于刺端则大于50 μM。其次，肌动蛋白结合蛋白抑制成核作用比抑制伸长作用更强。根据简单模型，基于两项伸长分析，肌动蛋白结合蛋白 - 肌动蛋白复合物的Kd为60 - 140 μM，但基于反映成核作用的聚合动力学，其Kd为2 - 10 μM。这些新发现可以用一个新的、更复杂的作用机制模型来解释，该模型与Tilney及其同事的提议有关[Tilney, L. G., Bonder, E. M., Coluccio, L. M., & Mooseker, M. S. (1983) J. Cell Biol. 97, 113 - 124]。在这个模型中，肌动蛋白结合蛋白既能以约5 μM的Kd与肌动蛋白单体结合，也能以约50 - 100 μM的Kd与肌动蛋白丝的刺端结合。与肌动蛋白结合蛋白结合的肌动蛋白单体不能参与成核作用，也不能添加到肌动蛋白丝的尖端。它可以添加到丝的刺端。当肌动蛋白结合蛋白与丝的刺端结合时，肌动蛋白单体不能与该端结合，但末端肌动蛋白原聚体可以以通常的速率解离。这个模型包括两个不同的Kd值——一个是肌动蛋白结合蛋白与肌动蛋白单体结合的Kd值，另一个是肌动蛋白结合蛋白与丝刺端的肌动蛋白分子结合的Kd值。对丝端的亲和力比对单体的亲和力低10倍，这可能是由于两种形式肌动蛋白的构象差异或丝端的空间位阻造成的。
