Different calcium dependence of the capping and cutting activities of villin

Abstract

The concentration of ionized calcium required for the capping of barbed filament ends by villin is about 4 orders of magnitude lower than that required for the cutting activity of villin. Capping was 50% complete at about 10-30 nM Ca2+, a level expected in resting cells, whereas the cutting rate was half-maximal at about 200 microM, making it possible to completely separate filament capping from filament cutting. Analysis of capping in terms of coupled equilibria between calcium binding to villin and calcium-villin binding to the barbed ends of actin filaments gives a value of 10(16)-10(17) M-2 for the product of the two binding constants. By comparison the binding constant reported for the rapidly exchanging calcium sites on villin is 2 X 10(5) M-1 and that for binding of calcium-saturated villin to barbed ends has a minimum value of 10(11) M-1 giving a product of 2 X 10(16) M-1. The close similarity of the two sets of values suggests that capping is regulated by the rapidly exchanging calcium sites on villin. In terms of coupled equilibria the calcium requirement for filament capping decreases with increasing concentrations of free villin. The scant information on the mechanism of cutting allows only an estimate of the maximal value for the calcium-binding constant of the site regulating cutting which is about 2-5 X 10(3) M-1. Cutting is followed by rapid capping of the newly released barbed ends.