PAIRED HELICAL FILAMENTS AND PLAQUE AMYLOID PROTEINS

成对的螺旋丝和斑块淀粉样蛋白

• 批准号: 2050783
• 负责人: GEORGE G GLENNER
• 金额: $ 17.77万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-05-01 至 1996-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2050783
• 关键词: Alzheimer's disease; SDS polyacrylamide gel electrophoresis; amyloid proteins; chemical cleavage; histopathology; human subject; human tissue; laboratory mouse; laboratory rabbit; molecular cloning; neurofibrillary tangles; paired helical filament; postmortem; posttranslational modifications; protein purification; protein sequence; protein structure

The paired helical filaments (PHFS) composing the neurofibrillary tangles have been the subject of chemical investigation for over fifteen years yet their nature still remains clouded. A method has been developed, which completely solubilizes and denatures the PHFs to make them amenable to chemical analysis. A solubilized concentrate of PHFs has been analyzed on SDS-PAGE gels and the peak PHF "core" or backbone protein fraction gives an apparent monomeric molecular weight of 35 kDa. The major protein eluted from the SDS gels and bound to a PVDF membrane is found by amino acid sequence analysis to have a blocked N-terminus. This protein was then subjected to tryptic digestion yielding 5 peptide peaks. A partial amino acid sequence of the major peptide has been obtained which fails to correspond to any known protein when compared to a sequence data bank.This investigation will provide a complete amino acid sequence of the PHF backbone protein by utilizing further chemical and enzymic cleavage to produce overlapping peptides for analysis. The PHF gene will be cloned based on these sequences. These studies and the analyses for polysaccharide and phosphate residues will determine if abnormalities in post-translational events are responsible for PHF formation. The amyloid core of senile plaques has never previously been reproducibly defined. A method employing sequentially anhydrous trifluoroacetic acid and 5.8 M guanidine isothiocyanate solubilizes a concentrate of plaques and SDS-PAGE reveals a major protein band at 6 kDa. Amino acid sequence analysis reveals a blocked N-terminus. This plaque amyloid fibril protein, which is believed to be composed of the beta protein, will be subjected to chemical and enzymic digestion in order to define its length and complete chemical composition. This analysis should reveal the cleavage sites of the beta protein precursor leading to the deposition of the plaque amyloid fibrils and aid in defining its pathogenesis.

组成神经纤维缠结的成对螺旋丝(PHF) 已经成为化学研究的对象超过15年了 然而，它们的性质仍然阴云密布。已经开发出一种方法， 使PHF完全溶解和变性 使它们易于进行化学分析。增溶的A PHF的浓缩物已经在SDS-PAGE凝胶和 峰值PHF“核心”或主干蛋白质组分给出了一个明显的 单体相对分子质量为35 kDa。洗脱出来的主要蛋白质 通过氨基酸序列分析发现，十二烷基硫酸钠凝胶与PVDF膜结合 分析显示有一个被阻断的N-末端。然后将这种蛋白质 以胰酶消化产生5个多肽峰。部分氨基酸 已获得不对应的主肽序列 当与序列数据库比较时，任何已知的蛋白质。这 调查将提供PHF的完整氨基酸序列 通过进一步利用化学和酶裂解主干蛋白质来 产生重叠的多肽以供分析。PHF基因将被克隆 根据这些序列。这些研究和分析 多糖和磷酸残留物将确定是否存在异常 翻译后事件是PHF形成的原因。淀粉样蛋白 老年斑块的核心以前从未被重复定义过。一个 方法依次采用无水三氟乙酸法和5.8M 异硫氰酸胍溶解浓缩的斑块和 SDS-PAGE显示在6 kDa处有一条主要的蛋白质条带。氨基酸序列 分析揭示了一个被阻断的N-末端。这个斑块中的淀粉样原纤维 据信由β蛋白组成的蛋白质将是 经过化学和酶消化以确定其长度 和完整的化学成分。这一分析应该揭示出 导致β蛋白前体沉积的裂解位点 斑块淀粉样蛋白纤维和有助于确定其发病机制。