Paraschos A, Gonzalez-Ros J M, Martinez-Carrion M
Biochim Biophys Acta. 1983 Sep 7;733(2):223-33. doi: 10.1016/0005-2736(83)90526-6.
A rapid, simple and reproducible method has been developed for the measurement of ion tracer flux with both native membrane vesicles and reconstituted lipid vesicle systems. Following the absorption of vesicles onto glass fiber filters, tracer flux is performed directly upon the deposited samples. In contrast to the more conventional vacuum and gel filtration techniques, absorption filtration exhibits comprehensive data retrieval whereby the removal of extravesicular ions, the retention of intravesicular ions and the amount of ions fluxed can be accurately analyzed. Both influx and efflux assays have been designed to measure the carbamylcholine-induced flux of 22Na+ which is characteristic of acetylcholine receptor-enriched membranes from Torpedo californica electroplax. The flux signal-to-background noise ratio is maximized in the efflux assay, since agonist activation is performed subsequent to the exhaustive removal of extravesicular tracer. An interesting feature of the influx assay is that the agonist-induced uptake of 22Na+ can be repeated with the original vesicles which additionally maximizes the flux signal. With either approach, the inactivation of ionophoric activity due to prolonged exposure to agonists ('desensitization') can be reversed upon removal of agonist without dilution of the deposited samples. Due to the large array of glass fiber filters and ion-exchange disks, the absorption filtration technique should be able to accommodate the transport and binding of soluble molecules to a variety of intact cells, membranes and reconstituted lipid vesicles.
已经开发出一种快速、简单且可重复的方法，用于测量天然膜囊泡和重组脂质囊泡系统中的离子示踪剂通量。在囊泡吸附到玻璃纤维滤器上之后，直接对沉积的样品进行示踪剂通量测定。与更传统的真空和凝胶过滤技术不同，吸附过滤具有全面的数据检索功能，据此可以准确分析胞外离子的去除、胞内离子的保留以及通量离子的量。流入和流出测定均设计用于测量氨基甲酰胆碱诱导的22Na+通量，这是来自加州电鳐电板富含乙酰胆碱受体的膜的特征。在流出测定中，通量信号与背景噪声之比最大化，因为激动剂激活是在彻底去除胞外示踪剂之后进行的。流入测定的一个有趣特征是，激动剂诱导的22Na+摄取可以用原始囊泡重复进行，这进一步最大化了通量信号。无论采用哪种方法，由于长时间暴露于激动剂导致的离子载体活性失活（“脱敏”），在去除激动剂时都可以逆转，而无需稀释沉积的样品。由于大量的玻璃纤维滤器和离子交换盘，吸附过滤技术应该能够适应可溶性分子与各种完整细胞、膜和重组脂质囊泡的转运和结合。
