Formation of 100 A filaments from purified glial fibrillary acidic protein in vitro.
Formation of 100 A filaments from purified glial fibrillary acidic protein in vitro.
复制标题
体外纯化的神经胶质原纤维酸性蛋白形成 100 个 A 丝。
DOI：
10.1016/0022-2836(79)90340-1
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发表时间：
1979
影响因子：
5.6
通讯作者：
A. Bignami
中科院分区：
文献类型：
--
作者：
D. Rueger;J. Huston;Doris Dahl;A. Bignami
胶质细胞酸性蛋白是中枢神经系统星形胶质细胞特有的，在体外聚合成类似于天然的~ 100 μ m纤维的纤维。通过免疫亲和色谱法从牛脑的水提取物中纯化后，GFA†蛋白在非常低的离子强度溶液中高度可溶。蛋白质溶液在预丝溶剂条件（2 mm-Tris · HCl，pH 7.8，20 °C，含有0.5 mm-二硫苏糖醇）下的沉降平衡分析表明，溶液中物质的少分散混合物具有约186，000至227，000的典型表观重均分子量范围。在pH 6.0和8.0之间，溶解度是pH和离子强度以及温度的函数，并且通过降低pH或温度以及通过提高离子强度来促进沉淀。GFA蛋白在窄的pH和离子强度范围内以细丝形式缔合;聚合0.1 mg/ml蛋白质溶液的最佳条件是100 mm-咪唑-HCl缓冲液（pH 6.8），温度为37 ℃，并且不需要辅因子。花丝主要表现为直径约100 μ m的光滑曲线结构缠结，长度不定，但也观察到一些横向关联的花丝成粗束。虽然细丝的形成不受还原剂的存在或不存在的影响，但在氧化条件下，蛋白质亚基之间形成二硫键。在pH8.5的2 mm-Tris · HCl缓冲液中透析可使GFA蛋白的组装和分解过程完全完成，而在组装和分解过程中加入0.5mM-二硫苏糖醇可显著增强GFA蛋白的组装和分解过程，这些实验进一步阐明了GFA蛋白作为星形胶质细胞特异性中间丝亚基的作用。此外，他们认为，100毫微米细丝，作为细胞骨架的其他组成部分，可以在胶质细胞质中组装和拆卸。
Glial fibrillary acidic protein, which is specific to astroglia in the central nervous system, polymerizesin vitrointo filaments similar to native ~ 100 Å filaments. Following purification from aqueous extracts of bovine brain by immunoaffinity chromatography, GFA†protein is highly soluble in very low ionic strength solutions. Sedimentation equilibrium analysis of protein solutions in prefilament solvent conditions (2 mm-Tris · HCl, pH 7.8, 20 °C, containing 0.5 mm-dithiothreitol) indicates a paucidisperse mixture of species in solution with a typical range of apparent weight-average molecular weights from about 186,000 to 227,000. Between pH 6.0 and 8.0 the solubility is a function of pH and ionic strength as well as temperature, and precipitation is favored by lowering the pH or temperature and by raising the ionic strength. GFA protein associates in the form of filaments over a narrow range of pH and ionic strength; optimal conditions for polymerization of a 0.1 mg/ml protein solution are 100 mm-imidazole-HCl buffer (pH 6.8), at a temperature of 37 °C, and there is no requirement for co-factors. Filaments appear primarily as tangles of smooth curvilinear structures approximately 100 Å in diameter and of indefinite length, although some lateral association of filaments into thick bundles is also observed. While the formation of filaments is not affected by the presence or absence of reducing agent, under oxidizing conditions disulfide linkages form between protein subunits. Disassembly is achieved by dialysis against 2 mm-Tris · HCl buffer (pH 8.5), but this process is significantly enhanced by the addition of 0.5 mM-dithiothreitol during assembly and disassembly.These experiments clarify the role of GFA protein as the subunit of astroglialspecific intermediate filaments. In addition, they suggest that the 100 Å filament, as other components of the cytoskeleton, may assemble and disassemble in the glial cytoplasm.