Quenched flow technique with plasma membrane vesicles: acetylcholine receptor-mediated transmembrane ion flux.
Quenched flow technique with plasma membrane vesicles: acetylcholine receptor-mediated transmembrane ion flux.
复制标题
质膜囊泡猝灭流技术：乙酰胆碱受体介导的跨膜离子流。
DOI：
10.1016/0003-2697(81)90257-8
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发表时间：
1981
影响因子：
2.9
通讯作者：
Hess,GP
中科院分区：
文献类型：
--
作者：
Cash,DJ;Hess,GP
描述了适用于小体积膜囊泡悬浮液的具有宽范围孵育时间的通用淬灭流动机器的构造和使用。可以进行几次连续孵育，并且可以用不同的稀释液进行混合。该设计允许在实验过程中很容易改变的管道具有很大的通用性。乙酰胆碱受体介导的离子流入和流出的时间过程，随后与不同的配体浓度使用膜囊泡与反应时间范围从5毫秒到分钟。这些测量暴露了对不同配体的不同响应，以及离子通量和受体失活的不同配体依赖性。用双孵育和浓度跳跃技术研究了配体对受体的灭活作用和配体稀释后受体的恢复。特别是与使用膜囊泡在快速混合系统中的问题被认为是。用标记膜作为内标物研究囊泡的破裂通常很小，但可能很显著，这取决于管路布置和膜制备。观察到断裂时，断裂的发生具有很高的重现性。与囊泡系统的混合淬灭死时间明显大于与模型水解反应测得的。这种差异可能是由于囊泡的低扩散速率。
The construction and use of a versatile quenched flow machine suitable for use with small volumes of membrane vesicle suspension with a wide range of incubation times is described. Several sequential incubations can be made and the mixing can be performed with different dilutions. The design allows great versatility in plumbing which can be changed readily during an experiment. The time course of ion influx and efflux mediated by acetylcholine receptor was followed with varying ligand concentration using membrane vesicles with reaction times ranging from 5 ms to minutes. These measurements expose the varying response to different ligands, and the different ligand dependencies of ion flux and inactivation of receptor. Inactivation of the receptor by ligand and its recovery after dilution of ligand were studied by double incubation and concentration jump techniques. Problems particularly associated with the use of membrane vesicles in a rapid mixing system are considered. Breakage of the vesicles, studied with labeled membrane as an internal standard, was usually small but could be significant depending on the tubing arrangement and membrane preparation. Breakage, when observed, occurred very reproducibly. The mixing-quenching dead time with the vesicle system was apparently greater than that measured with a model hydrolysis reaction. This discrepancy may be attributable to the low diffusion rate of the vesicles.