通过含有纯化乙酰胆碱受体的重组膜囊泡对阳离子转运进行定量分析。
Quantitation of cation transport by reconstituted membrane vesicles containing purified acetylcholine receptor.
作者信息
Wu W C, Moore H P, Raftery M A
出版信息
Proc Natl Acad Sci U S A. 1981 Feb;78(2):775-9. doi: 10.1073/pnas.78.2.775.
Abstract
A stopped-flow spectroscopic technique was used to study the kinetics of ion transport by reconstituted membrane preparations containing purified acetylcholine receptor. Influx of thallium (I) into membrane vesicles was monitored as a decrease, due to quenching by the thallous ion, in the fluorescence of an entrapped fluorophore. In a reproducible manner, the reconstituted receptor responded to cholinergic agonists by mediating rapid ion transport in the millisecond time range. The effect of agonists was blocked by receptor desensitization and by histrionicotoxin and was absent in membrane vesicles lacking receptor. Analysis of the fast kinetics of cation transport produced by saturating concentrations of agonists yielded an estimated rate of transport through a single reconstituted receptor channel. Comparison of this rate with those reported for in vivo preparations and for purified membranes shows that the reconstituted protein closely resembles the physiologically active receptor.
摘要
采用停流光谱技术研究了含有纯化乙酰胆碱受体的重组膜制剂的离子转运动力学。铊（I）流入膜囊泡的过程通过监测由于亚铊离子淬灭而导致的包裹荧光团荧光强度降低来进行。重组受体以可重复的方式对胆碱能激动剂作出反应，在毫秒时间范围内介导快速离子转运。激动剂的作用被受体脱敏和组氨酰毒素阻断，并且在缺乏受体的膜囊泡中不存在这种作用。对饱和浓度激动剂产生的阳离子转运动力学进行快速分析，得出了通过单个重组受体通道的估计转运速率。将该速率与体内制剂和纯化膜报道的速率进行比较表明，重组蛋白与生理活性受体非常相似。
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