Lind S E, Janmey P A, Chaponnier C, Herbert T J, Stossel T P
J Cell Biol. 1987 Aug;105(2):833-42. doi: 10.1083/jcb.105.2.833.
This paper documents the reversible appearance of high-affinity complexes of profilin and gelsolin with actin in extracts of platelets undergoing activation and actin assembly. Sepharose beads coupled to either monoclonal anti-gelsolin antibodies or to polyproline were used to extract gelsolin and profilin, respectively, from EGTA-containing platelet extracts and determine the proportion of these molecules bound to actin with sufficient affinity to withstand dilution (high-affinity complexes). Resting platelets (incubated for 30 min at 37 degrees C after gel filtration) contained nearly no high-affinity actin/gelsolin or actin/profilin complexes. Thrombin, within seconds, caused quantitative conversion of platelet profilin and gelsolin to high-affinity complexes with actin, but these complexes were not present 5 min after stimulation. The calcium-dependent actin filament-severing activity of platelet extracts, a function of free gelsolin, fell in concert with the formation of EGTA-stable actin/gelsolin complexes, and rose when the adsorption experiments indicated that free gelsolin was restored. The dissociation of high-affinity complexes was temporally correlated with the accumulation of actin in the Triton-insoluble cytoskeleton.
本文记录了在经历激活和肌动蛋白组装的血小板提取物中，肌动蛋白结合蛋白和凝溶胶蛋白与肌动蛋白形成的高亲和力复合物的可逆出现。分别使用偶联有单克隆抗凝溶胶蛋白抗体或聚脯氨酸的琼脂糖珠，从含乙二醇双乙胺四乙酸（EGTA）的血小板提取物中提取凝溶胶蛋白和肌动蛋白结合蛋白，并确定这些分子中以足够亲和力与肌动蛋白结合以抵抗稀释的比例（高亲和力复合物）。静息血小板（凝胶过滤后在37℃孵育30分钟）几乎不含高亲和力的肌动蛋白/凝溶胶蛋白或肌动蛋白/肌动蛋白结合蛋白复合物。凝血酶在数秒内使血小板肌动蛋白结合蛋白和凝溶胶蛋白定量转化为与肌动蛋白的高亲和力复合物，但刺激后5分钟这些复合物不存在。血小板提取物的钙依赖性肌动蛋白丝切断活性（游离凝溶胶蛋白的一种功能）与EGTA稳定的肌动蛋白/凝溶胶蛋白复合物的形成同步下降，当吸附实验表明游离凝溶胶蛋白恢复时活性上升。高亲和力复合物的解离在时间上与肌动蛋白在不溶于曲拉通的细胞骨架中的积累相关。
