Lal A A, Korn E D
J Biol Chem. 1985 Aug 25;260(18):10132-8.
In buffer containing 50 mM KCl, 1 mM MgCl2, 1 mM EGTA, 5 mM imidazole, pH 7.5, 0.1 mM CaCl2, 0.2 mM dithiothreitol, 0.01% NaN3, and 0.2 mM ATP, the KD for the formation of the 1:1 complex between Acanthamoeba actin and Acanthamoeba profilin was about 5 microM. When the actin was modified by addition of a pyrenyl group to cysteine 374, the KD increased to about 40 microM but the critical concentration (0.16 microM) was unchanged. The very much lower affinity of profilin for modified actin explains the anomalous critical concentrations curves obtained for 5-10% pyrenyl-labeled actin in the presence of profilin and the apparently weak inhibition by profilin of the rate of filament elongation when polymerization is quantified by the increase in fluorescence of pyrenyl-labeled actin. Light-scattering assays of the polymerization of unmodified actin in the absence and presence of profilin gave a similar value for the KD (about 5-10 microM) when determined by the increase in the apparent critical concentration of F-actin at steady state at all concentrations of actin up to 20 microM and by the inhibition of the initial rates of polymerization of actin nucleated by either F-actin or covalently cross-linked actin dimer. In the same buffer, but with ADP instead of ATP, the critical concentration of actin was higher (4.9 microM) and the KD of the profilin-actin complex was lower for both unmodified (1-2 microM) and 100% pyrenyl-labeled actin (4.9 microM).
在含有50 mM氯化钾、1 mM氯化镁、1 mM乙二醇双四乙酸、5 mM咪唑（pH 7.5）、0.1 mM氯化钙、0.2 mM二硫苏糖醇、0.01%叠氮化钠和0.2 mM三磷酸腺苷的缓冲液中，棘阿米巴肌动蛋白与棘阿米巴肌动蛋白结合蛋白形成1:1复合物的解离常数（KD）约为5微摩尔。当肌动蛋白通过在半胱氨酸374处添加一个芘基进行修饰时，KD增加到约40微摩尔，但临界浓度（0.16微摩尔）不变。肌动蛋白结合蛋白对修饰后肌动蛋白的亲和力大大降低，这解释了在肌动蛋白结合蛋白存在的情况下，5 - 10%芘基标记的肌动蛋白所获得的异常临界浓度曲线，以及当通过芘基标记的肌动蛋白荧光增加来定量聚合时，肌动蛋白结合蛋白对丝状体伸长速率的明显弱抑制作用。在不存在和存在肌动蛋白结合蛋白的情况下，对未修饰肌动蛋白聚合的光散射分析表明，当通过在高达20微摩尔的所有肌动蛋白浓度下稳态时F - 肌动蛋白表观临界浓度的增加以及通过抑制由F - 肌动蛋白或共价交联的肌动蛋白二聚体引发的肌动蛋白聚合初始速率来测定时，KD值相似（约5 - 10微摩尔）。在相同缓冲液中，但用二磷酸腺苷代替三磷酸腺苷时，肌动蛋白的临界浓度更高（4.9微摩尔），未修饰的（1 - 2微摩尔）和100%芘基标记的肌动蛋白（4.9微摩尔）的肌动蛋白结合蛋白 - 肌动蛋白复合物的KD更低。
