Woodman P G, Warren G
Cell Biology Laboratory, Imperial Cancer Research Fund, London, England.
J Cell Biol. 1991 Mar;112(6):1133-41. doi: 10.1083/jcb.112.6.1133.
Brief internalization of [125I]transferrin was used to label coated endocytic vesicles, which were then purified using a combination of 2H2O and 2H2O/Ficoll density gradients. Purification was monitored using an assay measuring fusion of endocytic organelles, so as to isolate functional vesicles. Isolated vesicles had all the properties of clathrin-coated vesicles, being enriched for the major components of clathrin coats and uncoated by either 1 M Tris-HCl or an uncoating ATPase. Nearly half of the labeled vesicles were able to participate in subsequent fusion events, as measured by the cell-free assay. Fusion was specific, requiring energy and cytosol, and being sensitive to N-ethyl maleimide.
利用[125I]转铁蛋白的短暂内化作用来标记包被的内吞小泡，然后使用2H2O和2H2O/菲可密度梯度组合对其进行纯化。通过测量内吞细胞器融合的测定法来监测纯化过程，以便分离出功能性小泡。分离出的小泡具有网格蛋白包被小泡的所有特性，富含网格蛋白包被的主要成分，并且不会被1 M Tris-HCl或脱包被ATP酶脱包被。通过无细胞测定法测量，近一半的标记小泡能够参与随后的融合事件。融合具有特异性，需要能量和胞质溶胶，并且对N-乙基马来酰亚胺敏感。
