Cohen R S, Blomberg F, Berzins K, Siekevitz P
J Cell Biol. 1977 Jul;74(1):181-203. doi: 10.1083/jcb.74.1.181.
A postsynaptic density (PSD) fraction, including some adherent subsynaptic web material, has been isolated from dog cerebral cortex by a short-procedure modification of methods of Davis and Bloom (21, 22) and Cotman and Taylor (20), using Triton X-100. The fraction has been visualized by thin-section, replica, and negative (phosphotungstic acid) staining electron microscopy and its proteins separated by high-resoltuion SDS gel electrophoresis. Morphologically, the preparation seems to be quite pure, with very little membrane contamination. The density is composed of protein, no nuclei acids, and very little phospholipids being detectable. The fraction had no ATPase or GTPase activity, but it did have a very small amount of cytochrome c oxidase activity (of a specific activity less than 0.5 percent that of a mitochondrial fraction) and a small amount of 5'- nucleotidase activity (of a specific activity between 6 and 7 percent that of a synaptic membrane fraction). Electron micrographs reveal cup-shaped structures approximately 400nm long and approximately 40nm wide, made up of apparent particles 13-28nm in diameter. However, en face views, and particularly micrographs of replicas and PTA-stained preparations, reveal a disk-shaped structure, outside diameter approximately 400 nm, in which filaments are seen to extend from the central part of the density. High resolution gel electrophoresis studies indicated some 15 major proteins and perhaps 10 or more minor ones; the predominant protein had a mol wt of 51,000, followed by ones at 45,000, 40,000, 31,000, 26,000, and several at 100,000. A comparison by gel electrophoresis of density fraction proteins with those of a lysed synaptosomal membrane fraction containing some adherent densities indicated some comigrating proteins, but the major membrane fraction protein, mol wt 52,000, was not found in the density fraction. Antibodies raised against the density fraction reacted with a preparation of solubilized synaptic membrane proteins. By both these criteria, it was considered that the density and the synaptic membrane have some proteins in common. By separately mixing (125)I-labeled myelin, synaptic vesicle, and mitochondrial fraction proteins with synaptosomes, and then isolating the density fraction from the mixture, it was concluded that a major 26,000 mol wt density fraction protein was common to both mitochondria and density, that none of the proteins of the density were contaminants from the mitochondrial fraction, that a minor approximately 150,000 band was a contaminant from the synaptic vesicle fraction, and that the moderately staining PSD fraction protein of 17,000 mol wt band was the result of contamination by the major basic protein of myelin. On the basis of the marker enzymatic assays and the mixing experiments, it is considered that the density fraction is moderately pure biochemically, and that its protein composition, aside from a few exceptions noted above, reflects its in situ character.
通过对戴维斯和布鲁姆（21, 22）以及科特曼和泰勒（20）的方法进行简短程序修改，并使用 Triton X - 100，从犬大脑皮层中分离出了一个突触后致密物（PSD）组分，其中包括一些附着的亚突触网物质。该组分通过超薄切片、复型以及负染（磷钨酸）电子显微镜进行了观察，其蛋白质通过高分辨率 SDS 凝胶电泳进行了分离。从形态学上看，该制备物似乎相当纯净，几乎没有膜污染。该致密物由蛋白质组成，未检测到核酸，且磷脂含量极少。该组分没有 ATP 酶或 GTP 酶活性，但确实具有极少量的细胞色素 c 氧化酶活性（比活性低于线粒体组分的 0.5%）和少量的 5'-核苷酸酶活性（比活性介于突触膜组分的 6%至 7%之间）。电子显微镜照片显示出杯状结构，长约 400nm，宽约 40nm，由直径为 13 - 28nm 的明显颗粒组成。然而，从正面观察，特别是复型和磷钨酸染色制备物的显微照片显示出一种盘状结构，外径约 400nm，在其中可见细丝从致密物的中心部分延伸出来。高分辨率凝胶电泳研究表明有大约 15 种主要蛋白质，可能还有 10 种或更多次要蛋白质；主要蛋白质的分子量为 51,000，其次是 45,000、40,000、31,000、26,000 的蛋白质，还有几种分子量为 100,000 的蛋白质。通过凝胶电泳将致密物组分的蛋白质与含有一些附着致密物的裂解突触体膜组分的蛋白质进行比较，发现了一些共迁移的蛋白质，但在致密物组分中未发现分子量为 52,000 的主要膜组分蛋白质。针对致密物组分产生的抗体与溶解的突触膜蛋白质制备物发生反应。基于这两个标准，认为致密物和突触膜有一些共同的蛋白质。通过将（125）I 标记的髓磷脂、突触小泡和线粒体组分蛋白质分别与突触体混合，然后从混合物中分离出致密物组分，得出结论：一种主要的分子量为 26,000 的致密物组分蛋白质在线粒体和致密物中都存在，致密物的蛋白质中没有来自线粒体组分的污染物，一条约 150,000 的次要条带是来自突触小泡组分的污染物，分子量为 17,000 条带的中等染色 PSD 组分蛋白质是髓磷脂主要碱性蛋白质污染的结果。基于标记酶测定和混合实验，认为致密物组分在生化方面具有适度的纯度，并且除了上述少数例外情况外，其蛋白质组成反映了其原位特征。
