A convenient large-scale method for the isolation of membrane vesicles permeable to a specific inorganic ion: isolation and characterization of functional acetylcholine receptor-containing vesicles from the electric organ of Electrophorus electricus.
A convenient large-scale method for the isolation of membrane vesicles permeable to a specific inorganic ion: isolation and characterization of functional acetylcholine receptor-containing vesicles from the electric organ of Electrophorus electricus.
复制标题
一种方便的大规模分离可渗透特定无机离子的膜囊泡的方法：从电虫的电器官中分离和表征含有功能性乙酰胆碱受体的囊泡。
DOI：
10.1016/0003-2697(82)90236-6
复制
发表时间：
1982
影响因子：
2.9
通讯作者：
Hess,GP
中科院分区：
文献类型：
--
作者：
Sachs,AB;Lenchitz,B;Noble,RL;Hess,GP
开发了一种方便、大规模的分离可渗透特定无机离子的膜囊泡的方法。该方法的一般原理是将囊泡内的Na+交换为外部的Cs+。这种交换迅速发生的囊泡可以根据其密度与交换缓慢发生的囊泡分开（G. P. Hess和J. P. Andrews, 1977）。USA74, 482 - 486)。该方法已被用于开发一种适合于从电鳗中大规模分离含有功能性乙酰胆碱受体的囊泡的方法。新程序涉及一个不连续的蔗糖梯度，用于囊泡的初始纯化。这允许使用低速离心机，其容量比以前使用的贝克曼超离心机大30倍。然后使用自形成的csc - percoll梯度和低速离心分离含有乙酰胆碱受体的功能性囊泡。分离步骤导致接近理论上可能的包含功能受体的囊泡的四倍纯化。其产率高达12毫克膜蛋白/离心运行，比以前使用的蔗糖- cscl密度梯度（Hess和Andrews，见上文）的产率高约100倍。梯度是自形成的，离心30分钟后达到平衡。在12种不同生物膜制备的12个实验中，功能囊泡的内体积为2.0±0.3 μl/mg囊泡蛋白，受体浓度为1.2±0.02 μl （1.2 μmol/l内体积）。这些囊泡的电子显微照片显示平均囊泡半径为1600±300 Å。根据这些结果，计算出平均12个受体分子/膜泡。
A convenient, large-scale method for the isolation of membrane vesicles permeable to specific inorganic ions has been developed. The general principle of this method involves the exchange of Na+within the vesicles for external Cs+. Vesicles in which this exchange rapidly occurs can be separated on the basis of their density from vesicles in which the exchange occurs slowly (G. P. Hess and J. P. Andrews (1977) Proc. Nat. Acad. Sci. USA74, 482–486). This approach has been adapted to develop a method suitable for the large-scale isolation of vesicles that contain functional acetylcholine receptors from the Electrophorus electricus electroplax. The new procedure involves a discontinuous sucrose gradient for an initial purification of the vescles. This allows the use of a low-speed centrifuge, which has a capacity up to 30 times greater than the Beckman ultracentrifuge previously used. A self-forming CsCl-Percoll gradient and low-speed centrifugation are then used for the isolation of the functional acetylcholine receptor-containing vesicles. The isolation step leads close to the theoretically possible fourfold purification of the vesicles that contain functional receptors. The yield, up to 12 mg membrane protein/centrifugal run, is about 100-fold higher than the yield from the sucrose-CsCl density gradient previously (Hess and Andrews, see above) used. The gradients are self-forming and an equilibrium is reached after centrifugation for only 30 min. In 12 experiments with membrane preparations from 12 different ceis, the functional vesicles had an internal volume of 2.0 ± 0.3 μl/mg vesicle protein and a receptor concentration of 1.2 ± 0.02 μm (1.2 μmol/liter of internal volume). Electron micrographs of these vesicles show an average vesicle radius of 1600 ± 300 Å. From these results, an average of 12 receptor molecules/membrane vesicle is calculated.