Quantitative analysis of the effect of Acanthamoeba profilin on actin filament nucleation and elongation

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Abstract
The current view of the mechanism of action of Acanthamoeba profilin is that it binds to actin monomers, forming a complex that cannot polymerize. This simple model fails to predict 2 new experimental observations made with Acanthamoeba actin in 50 mM KCl, 1 mM MgCl2 and 1 mM EGTA [ethylene glycol bis(.beta.-aminoethyl ether)-N,N,N'',N''-tetraacetic acid]. 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 .mu.M on the basis of observations at the pointed end and greater than 50 .mu.M 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 .mu.M on the basis of 2 assays of elongation but 2-10 .mu.M 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. In this model, profilin can bind both to actin monomers with a Kd of about 5 .mu.M and to the barbed end of actin filaments with a Kd of about 50-100 .mu.M. 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 2 different Kd: 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 2 forms of actin or to steric constraints at the end of the filament.