Isolation and chemical characterization of Alzheimer's disease paired helical filament cytoskeletons: differentiation from amyloid plaque core protein.
Isolation and chemical characterization of Alzheimer's disease paired helical filament cytoskeletons: differentiation from amyloid plaque core protein.
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阿尔茨海默氏病配对螺旋丝细胞骨架的分离和化学表征：与淀粉样蛋白斑核心蛋白的分化。
DOI：
10.1083/jcb.107.6.2703
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发表时间：
1988
期刊：
影响因子：
--
通讯作者：
Ball,MJ
中科院分区：
文献类型：
--
作者：
Roher,AE;Palmer,KC;Chau,V;Ball,MJ
阿尔茨海默病的成对螺旋丝（PHFs）的纯化策略，其中神经元和淀粉样蛋白斑块核心（APCP）最初分离。这是通过增加摩尔浓度和不连续等渗Percoll密度梯度的等度蔗糖离心的几个步骤来实现的。在胶原酶消除污染的血管后，通过SDS处理产生神经元溶解。通过蔗糖密度梯度将释放的PHF细胞骨架与污染的APCP和脂褐质分离。最后一步是通过甲酸向盐酸胍的过渡对高度富集的PHF和APCP组分进行化学纯化。PHF和APCPs通过尺寸排阻HPLC分级分离，并通过自动氨基酸分析进一步表征和定量。我们还介绍了PHF多肽和APCP的一些形态学和免疫化学特征。我们的研究表明，除了在定位和形态上的差异，PHF和APCP显着不同的（a）化学结构（肽和氨基酸组成）;（B）表位特异性（抗泛素，antitau，抗神经丝）;（c）物理化学性质（盐酸胍中的结构构象）;和（d）硫代黄素T荧光发射。这些参数强烈表明重要的组成差异，并可能在阿尔茨海默病的PHF和APCP的发病机制。
The paired helical filaments (PHFs) of Alzheimer's disease were purified by a strategy in which the neurons and amyloid plaque cores of protein (APCP) were initially isolated. This was achieved by several steps of isocratic sucrose centrifugations of increasing molarity and a discontinuous isotonic Percoll density gradient. After collagenase elimination of contaminating blood vessels, lysis of neurons was produced by SDS treatment. The released PHF cytoskeletons were separated from contaminating APCP and lipofuscin by sucrose density gradient. A final step consisted in the chemical purification of highly enriched PHFs and APCP components via a formic acid to guanidine hydrochloride transition. PHFs and APCPs were fractionated by size exclusion HPLC and further characterized and quantitated by automatic amino acid analysis. We also present some of the morphological and immunochemical characteristics of PHF polypeptides and APCP. Our studies indicate that apart from differences in localization and morphology, PHF and APCP significantly differ in (a) chemical structure (peptide and amino acid composition); (b) epitope specificity (antiubiquitin, antitau, antineurofilament); (c) physicochemical properties (structural conformation in guanidine hydrochloride); and (d) thioflavine T fluorescence emission. These parameters strongly suggest important differences in the composition and, probably, in the etiopathology of PHF and APCP of Alzheimer's disease.