Walsh T P, Weber A, Davis K, Bonder E, Mooseker M
Biochemistry. 1984 Dec 4;23(25):6099-102. doi: 10.1021/bi00320a030.
"Cutting" of actin filaments by villin was evaluated from the time course of filament depolymerization. Depolymerization was initiated by diluting polymerized actin, labeled with a fluorescent probe on either lysine-374 or cysteine-375, to a concentration well below the critical into a medium containing free villin and various concentrations of calcium (in addition to potassium and magnesium). It was observed that at high calcium concentrations (200 microM) the time course of depolymerization could not be described by the single exponential that defines it at low calcium and low villin levels. Instead, at high calcium, the exponent increased with time and the rate of depolymerization became greater than that of controls in the absence of villin. This contrasts with the inhibition of depolymerization by villin at low calcium. The latter inhibition is a consequence of the capping of the barbed filament end by villin as are the inhibition of filament elongation and the elevation of the critical concentration. Evidence is presented that the effects of villin at high calcium are the result of cutting of the actin filaments by villin. It thus appears that different calcium binding sites control capping and cutting and that the calcium binding sites regulating cutting have a much lower affinity for calcium than the sites regulating capping of the barbed filament ends.
通过肌动蛋白丝的解聚时间进程来评估绒毛蛋白对肌动蛋白丝的“切割”作用。解聚过程通过将在赖氨酸-374或半胱氨酸-375上用荧光探针标记的聚合肌动蛋白稀释至远低于临界浓度，使其进入含有游离绒毛蛋白和各种浓度钙（以及钾和镁）的介质中来启动。观察到在高钙浓度（200微摩尔）下，解聚的时间进程不能用在低钙和低绒毛蛋白水平下定义它的单指数来描述。相反，在高钙条件下，指数随时间增加，解聚速率变得大于无绒毛蛋白时的对照。这与低钙时绒毛蛋白对解聚的抑制作用形成对比。后者的抑制作用是绒毛蛋白封闭肌动蛋白丝的带刺末端的结果，同样也是对肌动蛋白丝伸长的抑制和临界浓度升高的结果。有证据表明，高钙时绒毛蛋白的作用是绒毛蛋白切割肌动蛋白丝的结果。因此，似乎不同的钙结合位点控制着封闭和切割作用，并且调节切割的钙结合位点对钙的亲和力远低于调节带刺肌动蛋白丝末端封闭的位点。
