Morré D J, Lawrence J, Safranski K, Hammond T, Morré D M
Department of Medicinal Chemistry, Purdue University, West Lafayette, IN 47907.
J Chromatogr A. 1994 May 6;668(1):201-13. doi: 10.1016/0021-9673(94)80110-X.
In practice it has been possible to separate membrane particles of different origins but of similar chemical composition by preparative free-flow electrophoresis. Examples include the vacuolar (tonoplast) and plasma membranes of plants and membranes derived from the cis and trans regions of the rat liver Golgi apparatus. Yet, when analyzed for intrinsic molecules that might contribute to significant differences in surface charge, the separated membranes were surprisingly similar. As more information was generated, it became apparent that the membranes with greatest electrophoretic mobility (i.e. lysosomes, rightside-out tonoplast vesicles and membranes from the trans region of the Golgi apparatus), where those membranes with an inherent ability to acidify their interiors. By so doing, the vesicles generate a membrane potential, negative outside, which might serve as a basis for enhanced electrophoretic mobility. To test the hypothesis, tonoplast membranes were incubated with ATP to drive proton import or with monensin to dissipate the ATP-supported proton gradient. With ATP, mobility was enhanced. Also, when ATP-treated vesicles were analyzed in the presence of monensin, the ATP effect on mobility was reversed. Similarly with Golgi apparatus, mobility of the most electrophoretically mobile portions of the separation was enhanced by ATP and the ATP effect was reversed with monensin. A trans origin of the vesicles was verified by assay of the trans Golgi apparatus marker, thiamine pyrophosphatase. Finally, incubation with ATP (and reversal by monensin) was employed as an aid to the free-flow electrophoretic separation of kidney endosomes from complex mixtures. These lysosomal derivatives also are capable of acidification of their interiors in an ATP-dependent process and of generating, at the same time, a negative (outside) membrane potential. The findings provide both an experimental basis to enhance membrane separations by preparative free-flow electrophoresis and, at the same time, a theoretical basis to help explain why certain membranes of very similar overall chemical composition may be separated by electrophoretic methods.
在实践中，通过制备型自由流动电泳已能够分离出不同来源但化学组成相似的膜颗粒。例子包括植物的液泡（液泡膜）和质膜，以及源自大鼠肝脏高尔基体顺式和反式区域的膜。然而，当分析可能导致表面电荷显著差异的内在分子时，分离出的膜却惊人地相似。随着更多信息的产生，很明显，具有最大电泳迁移率的膜（即溶酶体、外翻的液泡膜囊泡和来自高尔基体反式区域的膜），是那些具有使内部酸化的固有能力的膜。通过这样做，这些囊泡产生一个膜电位，外部为负，这可能是增强电泳迁移率的基础。为了验证这一假设，将液泡膜与ATP一起孵育以驱动质子内流，或与莫能菌素一起孵育以消除ATP支持的质子梯度。加入ATP后，迁移率增强。此外，当在莫能菌素存在的情况下分析经ATP处理的囊泡时，ATP对迁移率的影响被逆转。高尔基体也是如此，分离物中电泳迁移率最高部分的迁移率因ATP而增强，且ATP的作用被莫能菌素逆转。通过检测反式高尔基体标记物硫胺焦磷酸酶，证实了囊泡的反式来源。最后，将与ATP孵育（并被莫能菌素逆转）用于从复杂混合物中自由流动电泳分离肾内体的辅助手段。这些溶酶体衍生物也能够在依赖ATP的过程中使内部酸化，并同时产生一个负的（外部）膜电位。这些发现既为通过制备型自由流动电泳增强膜分离提供了实验基础，同时也为解释为什么某些总体化学组成非常相似的膜可以通过电泳方法分离提供了理论基础。
