Lee S, Li M, Pollard T D
Department of Cell Biology and Anatomy, Johns Hopkins Medical School, Baltimore, Maryland 21205.
Anal Biochem. 1988 Jan;168(1):148-55. doi: 10.1016/0003-2697(88)90022-x.
We have used a fluorescence assay to measure the binding of Acanthamoeba profilin to monomeric Acanthamoeba and rabbit skeletal muscle actin labeled on cysteine-374 with pyrene iodoacetamide. The wavelengths of the pyrene excitation and emission maxima are constant at 346 and 386 nm, but the fluorescence is enhanced up to 50% by profilin. The higher fluorescence is largely due to higher absorbance in the presence of profilin. The fluorescence enhancement has a hyperbolic dependence on the concentration of profilin, suggesting a single class of binding sites. Linear Scatchard plots yield an estimate of the dissociation constant, Kd, of the complex of profilin with pyrenyl-actin. In low-ionic-strength buffers with 2 to 6 mM imidazole (pH 7.0) and 0.1 mM CaCl2 the Kd is 9 microM for both muscle and Acanthamoeba actin. In 50 mM KCl the Kd for the complex with Acanthamoeba actin is 16 microM, while the Kd for the complex with muscle actin is greater than 50 microM.
我们使用了一种荧光测定法来测量棘阿米巴肌动蛋白单体结合蛋白与用芘碘乙酰胺标记在半胱氨酸-374位点的棘阿米巴肌动蛋白单体和兔骨骼肌肌动蛋白的结合情况。芘的激发和发射最大波长在346和386纳米处保持恒定，但肌动蛋白单体结合蛋白可使荧光增强高达50%。较高的荧光很大程度上是由于在存在肌动蛋白单体结合蛋白的情况下吸光度更高。荧光增强对肌动蛋白单体结合蛋白浓度呈双曲线依赖性，表明存在单一类别的结合位点。线性斯卡查德图可得出肌动蛋白单体结合蛋白与芘基肌动蛋白复合物的解离常数Kd的估计值。在含有2至6 mM咪唑（pH 7.0）和0.1 mM氯化钙的低离子强度缓冲液中，肌肉肌动蛋白和棘阿米巴肌动蛋白的Kd均为9 microM。在50 mM氯化钾中，与棘阿米巴肌动蛋白形成的复合物的Kd为16 microM，而与肌肉肌动蛋白形成的复合物的Kd大于50 microM。
