PAIRED HELICAL FILAMENTS AND PLAQUE AMYLOID PROTEINS

成对的螺旋丝和斑块淀粉样蛋白

• 批准号: 3121257
• 负责人: GEORGE G GLENNER
• 金额: $ 14.77万
• 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/3121257
• 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.

神经纤维缠结由成对螺旋丝构成 15年来一直是化学研究的主题 但他们的本质仍然模糊不清 已经开发了一种方法， 其完全溶解和变性PHF 使其易于进行化学分析。 溶解的 在SDS-PAGE凝胶上分析了PHF的浓缩物， 峰值PHF“核心”或骨架蛋白级分给出了明显的 单体分子量为35 kDa。 主要的蛋白质洗脱从 通过氨基酸序列发现SDS凝胶并结合到PVDF膜上 分析以具有封闭的N-末端。 然后将这种蛋白质 胰蛋白酶消化产生5个肽峰。 的部分氨基酸 已经获得了主要肽的序列， 与序列数据库相比， 研究将提供PHF的完整氨基酸序列 通过利用进一步的化学和酶切， 产生重叠肽用于分析。 PHF基因将被克隆 基于这些序列。 这些研究和分析， 多糖和磷酸盐残留将决定是否异常， 翻译后事件是PHF形成的原因。 淀粉样 老年斑的核心以前从未被可重复地确定。 一 方法依次使用无水三氟乙酸和5.8M 异硫氰酸胍溶解噬斑的浓缩物， SDS-PAGE显示在6 kDa处的主要蛋白条带。 氨基酸序列 分析显示封闭的N-末端。这种斑块淀粉样纤维 蛋白质，这被认为是由β蛋白质，将是 进行化学和酶消化，以确定其长度 和完整的化学成分。 这一分析将揭示 β蛋白前体的切割位点导致 斑块淀粉样纤维，并帮助确定其发病机制。