Therien H M, Mushynski W E
J Cell Biol. 1976 Dec;71(3):807-22. doi: 10.1083/jcb.71.3.807.
A new method has been developed for isolating synaptic junctional complexes (SJC) of high structural integrity. The major step in the isolation involves homogenization of a synaptosomal membrane (SM) fraction in a biphasic system consisting of Freon 113 and an aqueous phase containing 0.2% Triton X-100. Well-preserved SJCs, along with membrane vesicles, were recovered in the aqueous phase after low-speed centrifugation of the homogenate. The membranes were subsequently separated from the SJCs by centrifugation on a discontinuous sucrose density gradient. The purity and identity of subcellular fractions were monitored by thin sectioning electron microscopy, using specific and nonspecific staining methods. From the electron microscope studies we conclude that SJCs and their components occupy about 65% of the area covered by structures in this fraction. The assay of enzyme activities indicates that homogenization in Triton-Freon and subsequent steps of the isolation procedure affect the activities of Na, K-ATPase, cytochrome oxidase, and acid phosphatase to different extents, but do not cause total inactivation. Electrophoresis of the SJC-enriched fraction on sodium dodecyl sulfate-polyacrylamide gels has demonstrated that a polypeptide which co-migrates with tubulin is the major component in this fraction, and that a polypeptide co-migrating with actin is also present.
已开发出一种用于分离具有高度结构完整性的突触连接复合体（SJC）的新方法。分离的主要步骤包括在由氟利昂113和含有0.2% Triton X-100的水相组成的双相系统中对突触体膜（SM）部分进行匀浆。匀浆经低速离心后，保存良好的SJC以及膜泡在水相中回收。随后通过在不连续蔗糖密度梯度上离心将膜与SJC分离。使用特异性和非特异性染色方法，通过超薄切片电子显微镜监测亚细胞组分的纯度和特性。从电子显微镜研究中我们得出结论，SJC及其组分占该组分中结构覆盖面积的约65%。酶活性测定表明，在Triton-氟利昂中匀浆以及分离过程的后续步骤对Na、K-ATP酶、细胞色素氧化酶和酸性磷酸酶的活性有不同程度的影响，但不会导致完全失活。在十二烷基硫酸钠-聚丙烯酰胺凝胶上对富含SJC的组分进行电泳表明，与微管蛋白共迁移的一种多肽是该组分中的主要成分，并且还存在与肌动蛋白共迁移的一种多肽。
