PAIRED HELICAL FILAMENT AND PLAQUE AMYLOID PROTEINS

神经纤维缠结由成对的螺旋丝构成 已经成为化学研究的主题超过十五年了 他们的本质仍然是模糊的。 一种方法，指定为无水， 沸腾甲酸（ABFA）技术，已被开发出来， 使PHF溶解和变性，使其易于化学降解， 分析. 已将PHF的增溶浓缩物在 琼脂糖-4B和分析的峰值PHF“核心”或骨架蛋白级分 通过十二烷基硫酸锂-PAGE（LDS-PAGE）。 这使得许多聚合物 谱带和表观单体分子量为74 kd的PHF 骨架蛋白，该值与β蛋白的值非常一致 基因产物，79 kd。 在8 M尿素中的电泳减少了 条带表明它们是由以下物质产生的非共价聚合物： 疏水相互作用：氨基酸分析表明，甲酸 该方法仅产生氨基酸半胱氨酸和胱氨酸的修饰。 初步氨基酸序列分析显示PHF的N-末端 待封闭的骨架蛋白。 因此，这项调查将 提供PHF骨架蛋白的完整氨基酸序列， 利用化学和酶裂解产生重叠肽， 氨基酸序列分析和质谱分析，以确定氨基 末端封闭部分。 只有提供可接受的 将使用来自起始材料的定量产率。 本 关于构成“老年”的淀粉样纤维的组成的数据 斑块是冲突的，尽管β蛋白或β蛋白 免疫组织化学研究显示前体是主要成分。 因此，与上述调查类似的调查也将在 在“老年”斑的均质制备物上进行， 通过ABFA方法溶解，以确定 淀粉样蛋白的分子量、氨基酸组成和序列 纤维蛋白质组成。

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, designated the anhydrous, boiling formic acid (ABFA) technique, has been developed, which completely solubilizes and denatures the PHFs to make them amenable to chemical analysis. A solubilized concentrate of PHFs has been fractionated on Sepharose-4B and the peak PHF "core" or backbone protein fractions analyzed by lithium dodecyl sulfate-PAGE (LDS-PAGE). This gives numerous polymeric bands and an apparent monomeric molecular weight of 74 kd for the PHF backbone protein, a value in close agreement to that of the beta protein gene product, 79 kd. Electrophoresis in 8 M urea reduces the number of bands suggesting that they are non-covalent polymers resulting from hydrophobic interaction: Amino acid analyses show that the formic acid method produces modification of only the amino acids, cysteine and cystine. Initial amino acid sequence analysis revealed the N-terminus of the PHF backbone protein to be blocked. This investigation, therefore, will provide a complete amino acid sequence of the PHF backbone protein by utilizing chemical and enzymic cleavage to produce overlapping peptides for amino acid sequence analyses and mass spectroscopy to identify the amino terminal blocking moiety. Only methodology that provides acceptable quantitative yields from starting material will be employed. The present data on the composition of the amyloid fibrils composing the "senile" plaque is conflicting although the beta protein or the beta protein precursor appears by immunohistochemical studies to be a major component. An investigation similar to that of the above will therefore, also be performed on homogeneous preparations of "senile" plaques which are also solubilized by the ABFA method in order to determine definitively the molecular weight and amino acid composition and sequence of the amyloid fibril protein composing them.