Kedersha N L, Hill D F, Kronquist K E, Rome L H
J Cell Biol. 1986 Jul;103(1):287-97. doi: 10.1083/jcb.103.1.287.
Rat liver clathrin coated vesicles (CVs) were separated into several distinct subpopulations using non-sieving concentrations of agarose, which allowed the separation of species differing primarily in surface charge. Using preparative agarose electrophoresis (Kedersha, N. L., and L. H. Rome, 1986, Anal. Biochem., in press), the CVs were recovered and analyzed for differences in morphology, coat protein composition, and stripped vesicle protein composition. Coat proteins from different populations appeared identical on SDS PAGE, and triskelions stripped from the different populations showed the same mobility on the agarose gel, suggesting that the mobility differences observed in intact CVs were due to differences in the surface charge of underlying vesicles rather than to variations in their clathrin coats. Several non-coat polypeptides appeared to segregate exclusively with different populations as resolved by two-dimensional electrophoresis. Stripped CVs also exhibited considerable heterogeneity when analyzed by Western blotting: the fast-migrating population was enriched in the mannose 6-phosphate receptor, secretory acetylcholine esterase, and an Mr 195,000 glycoprotein. The slow-migrating population of CVs was enriched in the asialoglycoprotein receptor, and it appeared to contain all detectable concanavalin A-binding polypeptides as well as the bulk of detectable WGA-binding proteins. When CVs were prepared from 125I-asialoorosomucoid-perfused rat liver, ligand was found in the slow-migrating CVs, suggesting that these were endocytic in origin. Morphological differences were also observed: the fast-migrating population was enriched in smaller CVs, whereas the slow-migrating population exhibited an enrichment in larger CVs. As liver consists largely of hepatocytes, these subpopulations appear to originate from the same cell type and probably represent CVs of different intracellular origin and destination.
使用非筛分浓度的琼脂糖将大鼠肝脏网格蛋白包被囊泡（CVs）分离成几个不同的亚群，这使得主要在表面电荷上存在差异的不同种类得以分离。利用制备性琼脂糖电泳（凯德沙，N. L.，和L. H. 罗马，1986年，《分析生物化学》，即将发表），回收CVs并分析其形态、包被蛋白组成和脱包被囊泡蛋白组成的差异。不同亚群的包被蛋白在SDS - PAGE上看起来相同，并且从不同亚群中剥离的三脚蛋白在琼脂糖凝胶上显示出相同的迁移率，这表明在完整CVs中观察到的迁移率差异是由于下层囊泡表面电荷的差异，而不是其网格蛋白包被的变化。通过二维电泳分析，几种非包被多肽似乎仅与不同亚群分离。通过蛋白质免疫印迹分析时，脱包被的CVs也表现出相当大的异质性：快速迁移的亚群富含甘露糖6 - 磷酸受体、分泌型乙酰胆碱酯酶和一种分子量为195,000的糖蛋白。缓慢迁移的CVs亚群富含去唾液酸糖蛋白受体，并且似乎包含所有可检测到的伴刀豆球蛋白A结合多肽以及大部分可检测到的麦胚凝集素结合蛋白。当从用125I - 去唾液酸血清类黏蛋白灌注过的大鼠肝脏制备CVs时，在缓慢迁移CVs中发现了配体，这表明这些CVs起源于内吞作用。还观察到了形态学差异：快速迁移的亚群富含较小的CVs，而缓慢迁移的亚群在较大的CVs中表现出富集。由于肝脏主要由肝细胞组成，这些亚群似乎起源于同一细胞类型，并且可能代表不同细胞内起源和目的地的CVs。
