Kinetics of actin elongation and depolymerization at the pointed end

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Abstract
We measured the rate of elongation at the pointed filament end with increasing concentrations of G-actin [J(c) function] using villin-capped actin filaments of very small (actin/villin = 3, VA3) and relatively large size (actin/villin = 18, VA18) as nuclei for elongation. The measurements were made under physiological conditions in the presence of both Mg2+ and K+. In both cases the J(c) function was nonlinear. In contrast to the barbed filament end, however, the slope of the J(c) function sharply decreased rather than increased when the monomer concentration was lowered to concentrations near and below the critical concentration c.infin.. At zero monomer concentration, depolymerization at the pointed end was very slow with a rate constant of 0.02 s-1 for VA18. When VA3 was used, the nonlinearity of the J(c) function was greatly exaggerated, and the nuclei elongated at actin concentrations below the independently measured critical concentration for the pointed end. This is consistent with and confirms our previous finding [Weber, A., Northrop, J., Bishop, M. F., Ferrone, F. A., and Mooseker, M. S. (1987) Biochemistry (preceding paper in the issue)] that at an actin-villin ratio of 3 a significant fraction of the villin is free and that a series of steady states exist between villin-actin complexes of increasing size and G-actin. The rate constant of elongation seems to increase with increasing G-actin concentrations because of increasing conversion of free villin into villin-actin oligomers during the period of the measurement of the initial elongation rate. The villin-actin oligomers have a much higher rate constant of actin binding than does free villin. By contrast, villin-actin oligomers of an average size of 18 actin molecules per villin (VA18) do no coexist with significant free villin (and consequently do not elongate at G-actin concentrations below the independently measured c.infin.), and hence, another explanation is required for the curvature of their J(c) function. After a number of possible causes for the nonlinearity of the J(c) function not related to a change in the properties of the pointed end were ruled out, we conclude that under physiological salt conditions the changes in the apparent rate constants at the pointed end of actin filaments may be due to a change in the nucleotide content of the terminal actin molecule from ADP to ATP. We successfully fit the data with a slightly simplified version of the model by Pantaloni et al. [Pantoloni, D., Carlier, M. F., and Korn, E. D. (1985) J. Biol. Chem. 260, 6572-6578].