STRUCTURAL STUDIES OF ALZHEIMER 200K NEURO FILAMENT

在人类大脑的老化过程中，特别是在老年痴呆症中， 阿尔茨海默型（AD），存在异常纤维的积累， 由成对的螺旋缠绕的中间丝组成的细胞器 （PHF）。 最近，几个实验室提出，这种纤维 材料可以是后修饰的神经丝（NF）， 蛋白 迄今为止，NF蛋白的功能还没有得到很好的表征， 也没有太多关于NF 200 K分子结构的信息 或NF 160 K蛋白。 大多数结构工作都集中在NF 70 K上 亚单位 基于新的免疫化学和电子显微镜发现 PHF可能与NF有关，我们提出了一个完整的系列， 分子研究，其目标是完整的抗原表征 NF 200 K蛋白质的一部分。 多克隆和 与NF 200 K蛋白或PHF交叉反应的单克隆抗体（如 以及我们生产的单克隆抗体）将用于描述 NF 200 K蛋白在一级结构水平上的决定簇。 NF 200 K蛋白的有限蛋白水解片段将用于 确定各种抗原决定簇的位置， 研究NF 200 K的未知结构域。 以前都 用传统的液相色谱法很难纯化NF亚基， 大多数研究人员已经采取切除NF亚基的方法， SDS-PAGE凝胶。 我们建议尝试改善这些程序， 将HPLC技术引入神经化学领域。 几 将在这三种情况下尝试不同的列方法 NF亚基的分离以及从NF亚基中分离蛋白水解亚基 200 K亚基。 这一方法的改进对今后 测序和功能研究。 蛋白水解NF 200 K片段将 也可用作生产单克隆抗体的免疫原， 将测试其与PHF的交叉反应性。 我们亦建议 为了比较天冬酰胺、L-天冬氨酸和 阿尔茨海默病NF蛋白和年龄匹配的正常对照之间的D-天冬氨酸。 脑蛋白中D-天冬氨酸的存在最近被认为是 都破坏了蛋白质功能并参与衰老。 NF 200K 将来自阿尔茨海默氏症大脑受影响区域的蛋白质与NF 从未受影响的大脑中分离出200 K，试图观察NF 200 K蛋白是否 在整个大脑中是相同的，或者如果NF 200 K的局部修饰可以 发生.

During aging of the human brain and particularly in presenile dementia of the Alzheimer type (AD), there is an accumulation of abnormal fibrous organelles composed of paired, helically wound intermediate filaments (PHF). Recently, several laboratories have proposed that this fibrous material may be a post-translationally modified neurofilament (NF) protein. To date, the function of NF proteins is not well characterized, nor is there much information about the molecular structure of the NF 200K or NF 160K proteins. Most structural work has centered on the NF 70K subunit. Based on the new immunochemical and electron microscopic findings of PHF possibly being related to NF, we propose an integrated series of molecular studies which has as its goal the full antigenic characterization of the NF 200K protein from Alzheimer brain. Both polyclonal and monoclonal antibodies which cross-react with the NF 200K protein or PHF (as well as the monoclonals we produce) will be used to describe the determinants on the NF 200K protein at the primary structural level. Limited proteolytic fragments of the NF 200K protein will be used to identify the location of the various antigenic determinants and to investigate the still unknown domain structure of NF 200K. In the past, it has been very difficult to purify the NF subunits by conventional liquid chromatography, and most researchers have resorted to excising NF subunits from SDS-PAGE gels. We propose to try to improve these procedures by introducing HPLC techniques to this area of neurochemistry. Several different column methodologies will be tried in the isolation of the three NF subunits and also in the isolation of proteolytic subunits from the NF 200K subunit. This improvement in methodology is necessary for future sequencing and functional studies. The proteolytic NF 200K fragments will also be used as immunogens for the production of monoclonal antibodies, which will be tested for their cross-reactivity with PHF. We also propose to compare the relative amounts of asparagine, L-aspartic acid and D-aspartate between Alzheimer NF proteins and age-matched normal controls. The presence of D-aspartate in brain proteins has been recently proposed as both disrupting protein function and involved in aging. The NF 200K protein from affected areas of Alzheimer brain will be compared with NF 200K isolated from unaffected brain in an attempt to see if NF 200K protein is identical throughout the brain or if local modifications of NF 200K can occur.