The structure of postsynaptic densities isolated from dog cerebral cortex. I. Overall morphology and protein composition.
The structure of postsynaptic densities isolated from dog cerebral cortex. I. Overall morphology and protein composition.
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DOI：
10.1083/jcb.74.1.181
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发表时间：
1977-07
期刊：
影响因子：
--
通讯作者：
Siekevitz P
中科院分区：
文献类型：
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作者：
Cohen RS;Blomberg F;Berzins K;Siekevitz P
使用Triton X-100，通过Davis和Bloom（21，22）以及Cotman和Taylor（20）方法的短程序改良，从犬大脑皮层中分离出突触后密度（PSD）部分，包括一些粘附的突触下网状物物质。该馏分已被可视化的薄切片，复制品，负（磷钨酸）染色电子显微镜和其蛋白质分离的高分辨率SDS凝胶电泳。从形态学上看，该制剂似乎相当纯，几乎没有膜污染。密度由蛋白质组成，没有核酸，并且可检测到非常少的磷脂。该部分没有ATP酶或GT3酶活性，但它确实具有非常少量的细胞色素c氧化酶活性（比活性小于线粒体部分的0.5%）和少量的5 '-核苷酸酶活性（比活性在突触膜部分的6%至7%之间）。电子显微照片显示杯状结构长约400 nm，宽约40 nm，由直径13- 28 nm的表观颗粒组成。然而，正面视图，特别是复制品和PTA染色制剂的显微照片，显示了一个圆盘状结构，外径约400 nm，其中可见细丝从密度的中心部分延伸。高分辨率凝胶电泳研究表明，大约有15种主要蛋白质，可能还有10种或更多的次要蛋白质;主要蛋白质的摩尔质量为51，000，其次是45，000，40，000，31，000，26，000，还有几种是100，000。通过凝胶电泳的密度级分蛋白质与那些裂解的突触体膜部分含有一些粘附密度的比较表明，一些共迁移的蛋白质，但主要的膜部分蛋白质，分子量52,000，没有发现在密度级分。针对密度级分产生的抗体与溶解的突触膜蛋白的制剂反应。根据这两个标准，认为密度和突触膜具有一些共同的蛋白质。通过分别将（125）I标记的髓鞘、突触囊泡和线粒体级分蛋白与突触体混合，然后从混合物中分离密度级分，可以得出结论，主要的26，000 molwt密度级分蛋白是线粒体和密度共同的，密度的蛋白质都不是来自线粒体级分的污染物，次要的约150，000 molwt密度级分蛋白是线粒体和密度共同的，000条带是来自突触囊泡组分的污染物，17，000 mol wt条带的中度染色PSD组分蛋白是髓鞘的主要碱性蛋白污染的结果。基于标记酶测定和混合实验，认为密度级分在生物化学上是中等纯的，并且其蛋白质组成，除上述几个例外外，反映了其原位特征。
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.