Ksiezak-Reding H, Liu W K, Yen S H
Department of Pathology, Albert Einstein College of Medicine, Bronx, NY 10461.
Brain Res. 1992 Dec 4;597(2):209-19. doi: 10.1016/0006-8993(92)91476-u.
We performed phosphate analysis of tau proteins isolated from normal human brain, tau proteins associated with paired helical filaments (PHF-tau), and Alzheimer tau not associated with PHF. These tau fractions were of high purity. Normal and Alzheimer tau were purified by heat treatment, acid extraction and calmodulin-affinity chromatography with or without HPLC. Fractions containing primarily PHF-tau polypeptides of 60, 64 and 68 kDa and their degraded fragments were purified either on a sucrose density gradient as filaments (PHF) or by heat treatment and acid extraction as amorphous proteins (PHF-tau). PHF and PHF-tau were found to contain 6-8 mol phosphate/mol protein while normal and Alzheimer tau proteins contained 1.9 and 2.6 mol phosphate/mol protein, respectively. Upon 2-h incubation with alkaline phosphatase, PHF lost two of the phosphate groups without apparent changes in the stability and morphology of PHF. The released phosphate originated from the N-terminal half of PHF-tau as determined by immunoblotting with antibodies to epitopes blocked by phosphorylation. Tau-1 and E-2, and by a prominent shift in the electrophoretic mobility of some fragments of PHF-tau. The shift in mobility was not observed with the C-terminal fragments of 25-26 kDa, which retained the epitope to Tau 46. The results suggest that the phosphorylation sites not affected by phosphatase may be located in the 25-26 kDa C-terminal region of PHF-tau and may play a role in structural stability of PHF.
我们对从正常人脑中分离出的tau蛋白、与双螺旋丝相关的tau蛋白（PHF-tau）以及不与PHF相关的阿尔茨海默病tau蛋白进行了磷酸盐分析。这些tau蛋白组分具有高纯度。正常tau蛋白和阿尔茨海默病tau蛋白通过热处理、酸提取以及钙调蛋白亲和层析（有无高效液相色谱）进行纯化。主要含有60、64和68 kDa的PHF-tau多肽及其降解片段的组分，要么作为细丝在蔗糖密度梯度上纯化（PHF），要么通过热处理和酸提取作为无定形蛋白纯化（PHF-tau）。发现PHF和PHF-tau含有6 - 8摩尔磷酸盐/摩尔蛋白，而正常tau蛋白和阿尔茨海默病tau蛋白分别含有1.9和2.6摩尔磷酸盐/摩尔蛋白。与碱性磷酸酶孵育2小时后，PHF失去了两个磷酸基团，而PHF的稳定性和形态没有明显变化。通过用针对被磷酸化阻断的表位的抗体进行免疫印迹测定，释放的磷酸盐源自PHF-tau的N端一半。Tau-1和E-2，以及PHF-tau某些片段的电泳迁移率有显著变化。在25 - 26 kDa的C端片段中未观察到迁移率变化，这些片段保留了与Tau 46的表位。结果表明，不受磷酸酶影响的磷酸化位点可能位于PHF-tau的25 - 26 kDa C端区域，并且可能在PHF的结构稳定性中起作用。
