Wischik C M, Novak M, Thøgersen H C, Edwards P C, Runswick M J, Jakes R, Walker J E, Milstein C, Roth M, Klug A
Medical Research Council Laboratory of Molecular Biology, Cambridge, United Kingdom.
Proc Natl Acad Sci U S A. 1988 Jun;85(12):4506-10. doi: 10.1073/pnas.85.12.4506.
A substantially enriched preparation of Alzheimer paired helical filaments (PHFs) has been used as a starting point for biochemical studies. Pronase treatment, which strips off adhering proteins, leaves a resistant core that is structurally intact. This has been used to raise a monoclonal antibody that decorates the filament core. The antibody has been used to follow the extraction of two peptide fragments (9.5 and 12 kDa) by immunoblotting. The link between the PHF as a morphological entity and these peptides has been established independently by photoaffinity labeling with a chemical ligand to the PHF core. Sequence analysis of these peptides was used to design oligonucleotide probes for cloning a cognate cDNA, which leads to its identification as human microtubule-associated tau protein. The sequencing of the 9.5- and 12-kDa peptides shows they are derived from a conserved region of tau containing three repeating segments. Since these fragments have been copurified with the Pronase-resistant core and are only released by subsequent steps, the corresponding part of the tau molecule must be tightly bound in the PHF core.
阿尔茨海默病配对螺旋丝（PHF）的一种显著富集的制剂已被用作生化研究的起点。蛋白酶K处理可去除附着的蛋白质，留下结构完整的抗性核心。这已被用于制备一种可识别丝状体核心的单克隆抗体。该抗体已用于通过免疫印迹追踪两个肽片段（9.5 kDa和12 kDa）的提取。通过用与PHF核心的化学配体进行光亲和标记，独立建立了作为形态实体的PHF与这些肽之间的联系。这些肽的序列分析用于设计寡核苷酸探针以克隆同源cDNA，这导致其被鉴定为人微管相关tau蛋白。9.5 kDa和12 kDa肽的测序表明它们来自tau的一个包含三个重复片段的保守区域。由于这些片段已与蛋白酶K抗性核心共纯化，并且仅在后续步骤中释放，因此tau分子的相应部分必须紧密结合在PHF核心中。
