Groesch M E, Otto J J
Department of Biological Sciences, Purdue University, West Lafayette, Indiana.
Cell Motil Cytoskeleton. 1990;15(1):41-50. doi: 10.1002/cm.970150107.
Vinculin and talin are adhesion plaque proteins which have been shown to interact with each other in vitro. In order to begin to investigate where the talin-binding domain is in vinculin, vinculin was digested with Staphylococcus aureus V8 protease to generate two major fragments of 85 and 30 kDa, and these fragments were purified. Nitrocellulose overlays with 125I-talin and the 125I-85 kDa vinculin fragment and sucrose density gradient centrifugation demonstrated that the talin-binding domain was localized to the 85 kDa vinculin fragment. Quantification of 125I-talin binding in the overlays showed that four times more talin bound to the 85 kDa fragment as compared to intact vinculin. Competitive immunoprecipitation experiments demonstrated that unlabeled 85 kDa fragment was about three-fold more effective at competing for 125I-85 kDa binding to talin than was unlabeled vinculin. These results suggest that the 30 kDa fragment inhibits the vinculin-talin interaction even though the talin-binding domain is localized in the 85 kDa fragment.
纽蛋白和踝蛋白是粘着斑蛋白，已证实在体外它们可相互作用。为了开始研究纽蛋白中踝蛋白结合结构域的位置，用金黄色葡萄球菌V8蛋白酶消化纽蛋白，产生了两个主要片段，分别为85 kDa和30 kDa，然后对这些片段进行了纯化。用125I-踝蛋白与125I-85 kDa纽蛋白片段进行硝酸纤维素膜覆盖实验以及蔗糖密度梯度离心实验表明，踝蛋白结合结构域定位于85 kDa的纽蛋白片段。对覆盖实验中125I-踝蛋白结合的定量分析显示，与完整纽蛋白相比，结合到85 kDa片段上的踝蛋白是其四倍。竞争性免疫沉淀实验表明，未标记的85 kDa片段在竞争125I-85 kDa与踝蛋白结合方面比未标记的纽蛋白有效约三倍。这些结果表明，尽管踝蛋白结合结构域定位于85 kDa片段中，但30 kDa片段会抑制纽蛋白与踝蛋白的相互作用。
