Cheng T P, Wood J G
Brain Res. 1982 May 6;239(1):201-12. doi: 10.1016/0006-8993(82)90842-3.
Immunofixation of sodium lauryl sulphate (SDS)-acrylamide gels has been used to study the distribution of the major protein (clathrin) of coated vesicles in various compartments of synaptic terminals. Synaptosomal subcellular fractions were isolated and purified from pig brain homogenates by the procedure of Ueda et al. and lysed in 6 mM Tris-Cl buffer at pH 6.6, 7.8, and 8.1. The synaptosomal particulate and soluble fractions were separated by centrifugation. The synaptic junctional complex (SJC) and postsynaptic density (PSD) fractions were obtained by detergent treatment of the synaptic plasma membrane (SPM). The synaptosomal subcellular fractions and purified coated vesicle (PCV) fractions were subjected to SDS gel electrophoresis (7.5%). The resulting slabs were divided vertically into 4 segments which were stained with Coomassie blue dye, or immunofixed with preimmune and anti-clathrin serum, or affinity labeled with concanavalin A (Con A)-peroxidase. The Comassie blue stained gel indicated the presence of 180,000-molecular weight band in gels of most synaptosomal subcellular fractions. However, immunofixation of an identical gel revealed positive staining of the 180,000-molecular weight protein in PCV, synaptosomal (SF), SPM and synaptoplasmic (SS) fractions only. These findings not only support the contention that a pool of cytosolic-coated vesicle protein is localized at synaptic terminals, they also indicate that clathrin appears highly unlikely to contribute to the structural frameworks of the SJC and PSD of mature synapses.
十二烷基硫酸钠（SDS）-丙烯酰胺凝胶免疫固定法已被用于研究有被小泡的主要蛋白质（网格蛋白）在突触终末各部分的分布。按照上田等人的方法，从猪脑匀浆中分离并纯化突触体亚细胞组分，然后在pH值为6.6、7.8和8.1的6 mM Tris-Cl缓冲液中裂解。通过离心分离突触体颗粒组分和可溶性组分。通过用去污剂处理突触质膜（SPM）获得突触连接复合体（SJC）和突触后致密物（PSD）组分。将突触体亚细胞组分和纯化的有被小泡（PCV）组分进行SDS凝胶电泳（7.5%）。将所得凝胶板垂直分成4段，分别用考马斯亮蓝染色，或用免疫前血清和抗网格蛋白血清进行免疫固定，或用伴刀豆球蛋白A（Con A）-过氧化物酶进行亲和标记。考马斯亮蓝染色的凝胶显示，大多数突触体亚细胞组分的凝胶中有一条分子量为180,000的条带。然而，对同一块凝胶进行免疫固定后发现，只有在PCV、突触体（SF）、SPM和突触浆（SS）组分中，分子量为180,000的蛋白质呈现阳性染色。这些发现不仅支持了胞质有被小泡蛋白池定位于突触终末的观点，还表明网格蛋白极不可能参与成熟突触的SJC和PSD的结构框架。
