Kurzawa S E, Geeves M A
Max-Planck-Institut für Molekulare Physiologie, Dortmund, Germany.
J Muscle Res Cell Motil. 1996 Dec;17(6):669-76. doi: 10.1007/BF00154061.
The dissociation constant for actin binding to myosin and its subfragments (S1 & HMM) is <<1 microM at physiological ionic strength. Many of the methods used to measure such affinities are unreliable for a Kd below 0.1 microM. We show here that the use of phalloidin to stablise F-actin and fluorescently labelled proteins allows the affinity of actin for myosin S1 to be measured in a simple transient kinetic assay. The method can be used for Kd's as low as 10 nM and we demonstrate that the Kd's can be estimated using only microgram quantities of material. Furthermore we suggest how this method may be adapted for ng quantities of protein. This will allow the affinity of actin for myosin fragments to be estimated for proteins which are difficult to obtain in large quantities i.e. from biopsy material or from proteins expressed in baculovirus.
在生理离子强度下，肌动蛋白与肌球蛋白及其亚片段（S1和重酶解肌球蛋白）结合的解离常数远小于1微摩尔。许多用于测量此类亲和力的方法对于低于0.1微摩尔的解离常数是不可靠的。我们在此表明，使用鬼笔环肽稳定F-肌动蛋白和荧光标记的蛋白质，可以通过简单的瞬态动力学测定法测量肌动蛋白对肌球蛋白S1的亲和力。该方法可用于低至10纳摩尔的解离常数，并且我们证明仅使用微克量的材料就可以估算解离常数。此外，我们还提出了如何将此方法适用于纳克量的蛋白质。这将使得对于难以大量获得的蛋白质，即来自活检材料或杆状病毒中表达的蛋白质，能够估算肌动蛋白对肌球蛋白片段的亲和力。
