PAIRED HELICAL FILAMENT AND PLAQUE AMYLOID PROTEINS

构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已经超过15年了 它们的性质仍然是阴云密布。一种名为无水的方法， 沸腾甲酸(ABFA)技术已经开发出来，完全可以 使PHF溶解和变性，使其对化学物质顺从 分析。一种溶解的PHF浓缩物已被分馏 Sepharose4B和峰PHF“核心”或主干蛋白质组分分析 十二烷基硫酸锂-PAGE(LDS-PAGE)。这给了无数的聚合物 PHF的条带和表观单体分子量为74kd 主干蛋白，与β蛋白的值非常一致 基因产物，79kd。在8M尿素中进行电泳会减少细胞的数量 表明它们是非共价聚合物的条带 疏水相互作用：氨基酸分析表明，甲酸 方法只对氨基酸、半胱氨酸和半胱氨酸进行修饰。 初步氨基酸序列分析揭示了PHF的N-末端 被阻断的主干蛋白质。因此，这项调查将 通过以下方式提供PHF主干蛋白的完整氨基酸序列 利用化学和酶裂解生产重叠多肽 氨基酸序列分析和质谱学鉴定氨基酸 终端阻塞部分。唯一提供可接受的方法 将使用来自起始材料的定量产量。现在 构成“老年人”的淀粉样蛋白纤维的组成数据 斑块是相互冲突的，尽管β蛋白或β蛋白 免疫组织化学研究表明，前体似乎是主要成分。 因此，与上述类似的调查也将是 在均质制剂上进行的“老年”斑块也 用ABFA法增溶，以便最终确定 淀粉样蛋白的相对分子质量、氨基酸组成和序列 构成它们的原纤维蛋白。

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.