| Abstract: | The binding constants of Acanthamoeba profilin to fluorescein-labeled actin from Acanthamoeba and from rabbit skeletal muscle have been determined by measuring the reduction in the actin tracer diffusion coefficients, determined by fluorescence photobleaching recovery, as a function of added profilin concentration. Data were analyzed using a two-parameter nonlinear regression analysis to determine the profilin-actin dissociation constant Kd and the profilactin diffusion coefficient, DPA. For fluorescein-labeled Acanthamoeba actin, the least-squares estimates for Kd and DPA, along with approximate single standard deviation confidence intervals, are Kd = 48 (36, 63) microM and DPA = 6.72 (6.62, 6.81) X 10(-7) cm2s-1. For fluorescein-labeled skeletal muscle actin, the corresponding values are Kd = 147 (94, 225) microM and DPA = 6.7 (6.3, 7.0) X 10(-7) cm2s-1. These dissociation constants are the first to be determined from direct physical measurement; they are in agreement with values inferred from earlier studies on the effect of profilin on the assembly of actin that had been fluorescently labeled or otherwise modified at Cys 374. These results place important restrictions on the interpretation of experiments in which fluorescently labeled actin is used as a probe of living cytoplasm or cytoplasmic extracts that include profilin. |
