Purification and solubilization of paired helical filaments from Alzheimer brains.
Purification and solubilization of paired helical filaments from Alzheimer brains.
复制标题
阿尔茨海默病大脑中成对螺旋丝的纯化和溶解。
DOI：
10.1111/j.1471-4159.1990.tb13295.x
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发表时间：
1990
影响因子：
4.7
通讯作者：
Dean,GE
中科院分区：
文献类型：
--
作者：
Vogelsang,GD;Zemlan,FP;Dean,GE
本研究的目的是开发一种纯化和增溶方法，与现有的氨基酸测序技术兼容，用于来自阿尔茨海默病患者的成对螺旋丝（phf）。我们开发了一种温和的程序，将常规分离的PHF进行Tris/硼酸盐/十二烷基硫酸钠/2 -巯基乙醇电泳，结果将相对不溶性的PHF结构从共硫化污染蛋白和溶解的PHF相关蛋白中分离出来。在电泳4.5 h结束时，纯化的不溶性部分的氨基酸组成在随后的电泳中保持不变。电镜显示电泳前后均有完整的PHF结构，但在不溶性部分中未发现任何其他结构，这与从纤维结构中去除PHF相关蛋白的结果一致。与酶联免疫吸附试验中未进行电泳的部分相比，在其他实验室中分离的不溶性纤维结构对纯化phf产生的抗体显示出增强的免疫反应性。相对不溶性phf的增溶是通过延长电泳时间超过纯化所需的4.5 h来完成的。额外电泳34.5 h可溶解88%纯化后的相对不溶性phf。这导致在十二烷基硫酸钠-聚丙烯酰胺电泳凝胶分析中鉴定出四个主要的蛋白质条带，其mr值在~ 50,000和70,000之间，其中一个主要条带的mr值为~ 66,000。在这段时间内，PHF超微结构发生了缓慢的断裂，这是通过电子显微镜判断的。这种纯化技术将允许从溶解的phf中分离出一致可复制的蛋白质片段，可用于后续的序列分析。
The purpose of the present study was to develop a purification and solubilization method, compatible with current amino acid sequencing techniques, for paired helical filaments (PHFs) derived from patients with Alzheimer's disease. We have developed a mild procedure that subjects conventionally isolated PHFs to Tris/borate/sodium dodecyl sulfate/2‐mercaptoethanol electrophoresis and results in the separation of the relatively insoluble PHF structures from both copurifying contaminating proteins and solubilized PHF‐associated proteins. At the end of 4.5 h of electrophoresis, the purified insoluble fraction had an amino acid composition that was invariant during subsequent electrophoresis. Electron microscopy revealed an intact PHF structure before and after electrophoresis but no evidence of any other structures in the insoluble fraction, a result consistent with the removal of PHF‐associated proteins from the filament structure. Isolated insoluble filament structures displayed an enhanced immunoreactivity with antibodies raised against purified PHFs in other laboratories, when compared with the fraction not subjected to electrophoresis in enzyme‐linked immunosorbent assays. Solubilization of the relatively insoluble PHFs was accomplished by extending the time of electrophoresis beyond the 4.5 h required for purification. Additional electrophoresis for 34.5 h solubilized 88% of the purified, relatively insoluble PHFs. This resulted in the identification of four major protein bands between Mrvalues of ∼50,000 and 70,000 on sodium dodecyl sulfate‐polyacrylamide electrophoresis gel analysis, with a predominant band with an Mrof ∼66,000. A slow fragmentation of the PHF ultrastructure occurred during this time, as judged by electron microscopy. This purification technique will permit the isolation of consistently reproducible protein fragments from solubilized PHFs, which may be used for subsequent sequence analysis.