Northrop J, Weber A, Mooseker M S, Franzini-Armstrong C, Bishop M F, Dubyak G R, Tucker M, Walsh T P
J Biol Chem. 1986 Jul 15;261(20):9274-81.
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.
绒毛蛋白封闭肌动蛋白丝倒刺端所需的游离钙离子浓度比其切割活性所需的浓度低约4个数量级。在约10 - 30 nM Ca²⁺时封闭作用完成50%，这是静息细胞中的预期水平，而切割速率在约200 μM时达到半最大，从而有可能将丝封闭与丝切割完全分开。根据钙离子与绒毛蛋白结合以及钙离子 - 绒毛蛋白与肌动蛋白丝倒刺端结合之间的耦合平衡来分析封闭作用，得到两个结合常数的乘积为10¹⁶ - 10¹⁷ M⁻²。相比之下，报道的绒毛蛋白上快速交换钙离子位点的结合常数为2×10⁵ M⁻¹，钙离子饱和的绒毛蛋白与倒刺端结合的结合常数最小值为10¹¹ M⁻¹，乘积为2×10¹⁶ M⁻¹。这两组值的密切相似性表明封闭作用受绒毛蛋白上快速交换钙离子位点的调节。就耦合平衡而言，丝封闭所需的钙离子浓度随游离绒毛蛋白浓度的增加而降低。关于切割机制的信息匮乏，仅能估计调节切割的位点的钙离子结合常数的最大值约为2 - 5×10³ M⁻¹。切割之后新释放的倒刺端会迅速被封闭。
