Braell W A
Proc Natl Acad Sci U S A. 1987 Mar;84(5):1137-41. doi: 10.1073/pnas.84.5.1137.
Cell-free fusion between endocytic vesicles has been obtained in a sensitive assay based on the avidin-biotin binding reaction. Chinese hamster ovary cells are allowed to endocytose either avidin-linked beta-galactosidase or biotinylated IgG. Postnuclear supernatant extracts prepared from these cells are incubated at 37 degrees C in the presence of an ATP-regenerating system. Fusion between vesicles from the two extracts permits the avidin-biotin association to occur, so that the amount of avidin-enzyme-biotinylated IgG complex produced is proportional to the amount of fusion between vesicles. The amount of complex formed is measured, after detergent lysis of the vesicles, by an ELISA technique, using a fluorogenic substrate for beta-galactosidase. The fusion process requires ATP hydrolysis and specific cytosolic proteins. The vesicles that fuse appear to be endocytic vesicles populated by the endocytosed proteins within 5 min of their internalization at 37 degrees C. Ionophores and weak bases do not inhibit fusion, suggesting that a pH gradient across the vesicle membrane is not crucial for the fusion process.
基于抗生物素蛋白-生物素结合反应的灵敏检测方法已实现了内吞小泡之间的无细胞融合。使中国仓鼠卵巢细胞内吞抗生物素蛋白连接的β-半乳糖苷酶或生物素化的免疫球蛋白G。从这些细胞制备的核后上清提取物在37℃下于ATP再生系统存在的条件下孵育。来自两种提取物的小泡之间的融合使抗生物素蛋白-生物素结合得以发生，因此产生的抗生物素蛋白-酶-生物素化免疫球蛋白G复合物的量与小泡之间的融合量成比例。在通过去污剂裂解小泡后，使用β-半乳糖苷酶的荧光底物，通过酶联免疫吸附测定技术测量形成的复合物的量。融合过程需要ATP水解和特定的胞质蛋白。融合的小泡似乎是在37℃内化5分钟内被内吞蛋白占据的内吞小泡。离子载体和弱碱不抑制融合，这表明跨小泡膜的pH梯度对融合过程并不关键。
