Department of Biochemistry and Molecular Biology, The George Washington University School of Medicine and Health Sciences, Washington DC, USA.
Ann N Y Acad Sci. 2012 Oct;1270:86-92. doi: 10.1111/j.1749-6632.2012.06671.x.
A high-performance liquid chromatography (HPLC) methodology is presented. Using bovine and rabbit F- and G-actin, this methodology results in both fractions as being well-resolved peaks, which were confirmed by dot blot immunoassay and fluorescence microscopy. F- and G-actin were incubated with thymosin β4 (Tβ4) and DNase and then analyzed by HPLC, which indicated that Tβ4 and DNase inhibit G-actin polymerization and that Tβ4 depolymerizes F-actin in a dose- and time-dependent manner. The F- and G-actin content in sputum from healthy controls and cystic fibrosis (CF) patients were measured by HPLC before and after incubation with Tβ4, DNase, and gelsolin. These data demonstrate higher quantities of F-actin in the sputum of CF patients compared to healthy individuals, and also demonstrate a significantly increased F/G-actin ratio in CF sputum. Further, Tβ4, DNase, and gelsolin each increase the depolymerization of F-actin in CF sputum in a dose-dependent fashion that is additive when these agents are combined.
介绍了一种高效液相色谱（HPLC）方法。使用牛和兔 F-肌动蛋白和 G-肌动蛋白，该方法可将两种蛋白均分离为峰形良好的组分，这一点通过斑点印迹免疫检测和荧光显微镜得到了证实。F-肌动蛋白和 G-肌动蛋白与胸腺素β4（Tβ4）和脱氧核糖核酸酶孵育后，通过 HPLC 进行分析，结果表明 Tβ4 和脱氧核糖核酸酶抑制 G-肌动蛋白聚合，Tβ4 以剂量和时间依赖的方式使 F-肌动蛋白解聚。通过 HPLC 测量健康对照者和囊性纤维化（CF）患者痰液中的 F-肌动蛋白和 G-肌动蛋白含量，然后在孵育 Tβ4、脱氧核糖核酸酶和凝胶蛋白前后进行测量。这些数据表明 CF 患者痰液中的 F-肌动蛋白含量高于健康个体，并且 CF 患者痰液中的 F/G-肌动蛋白比值显著增加。此外，Tβ4、脱氧核糖核酸酶和凝胶蛋白各自以剂量依赖的方式增加 CF 患者痰液中 F-肌动蛋白的解聚，当这些试剂联合使用时，其作用具有加性。
