Forbush B
Anal Biochem. 1984 Aug 1;140(2):495-505. doi: 10.1016/0003-2697(84)90200-8.
A new method for the measurement of rapid isotopic release from a membrane compartment is described. Membrane vesicles loaded with isotope, or broken membranes with bound radioactive ligand, are filtered onto the surface of a cellulose ester filter; the rate of the loss of isotope from the membrane compartment is followed continuously by collecting fluid which is passed through the filters under high pressure. A change in release rate is initiated by changing the solution or by delivering a flash of light to a photosensitive sample. The approach has been used to study rapid 22Na efflux from membrane vesicles rich in the ouabain-sensitive Na pump, and to examine dissociation of 32P and 86Rb from membrane-bound Na,K-ATPase. Since the rate of efflux is measured, and not the total counts remaining on the filter, the technique has high sensitivity. A complete time course is obtained using only a few micrograms of membrane protein. The apparatus described is simple, inexpensive, and easily constructed; with the present device, time resolution is approximately 10 ms.
本文描述了一种测量膜隔室中快速同位素释放的新方法。将装载有同位素的膜囊泡或带有结合放射性配体的破碎膜过滤到纤维素酯滤膜表面；通过收集在高压下通过滤膜的液体，连续跟踪膜隔室中同位素的损失速率。通过改变溶液或向光敏样品发出闪光来引发释放速率的变化。该方法已用于研究富含哇巴因敏感钠泵的膜囊泡中快速的22Na外流，并用于检测32P和86Rb从膜结合的钠钾ATP酶上的解离。由于测量的是外流速率，而不是滤膜上剩余的总计数，因此该技术具有高灵敏度。仅使用几微克的膜蛋白就能获得完整的时间进程。所描述的仪器简单、便宜且易于构建；使用当前设备，时间分辨率约为10毫秒。
