Barkla Bronwyn J, Vera-Estrella Rosario, Pantoja Omar
Instituto de Biotecnología, Universidad Nacional Autónoma de México, A.P. 510-3, Colonia Miraval, Cuernavaca, Morelos, México.
Anal Chem. 2007 Jul 15;79(14):5181-7. doi: 10.1021/ac070159v. Epub 2007 Jun 13.
Free flow zonal electrophoresis (FFZE) is a versatile technique that allows for the separation of cells, organelles, membranes, and proteins based on net surface charge during laminar flow through a thin aqueous layer. We have been optimizing the FFZE technique to enhance separation of plant vacuolar membranes (tonoplast) from other endomembranes to pursue a directed proteomics approach to identify novel tonoplast transporters. Addition of ATP to a mixture of endomembranes selectively enhanced electrophoretic mobility of acidic vesicular compartments during FFZE toward the positive electrode. This has been attributed to activation of the V-ATPase generating a more negative membrane potential outside the vesicles, resulting in enhanced migration of acidic vesicles, including tonoplast, to the anode (Morré, D. J.; Lawrence, J.; Safranski, K.; Hammond, T.; Morré, D. M. J. Chromatogr., A 1994, 668, 201-213). We confirm that ATP does induce a redistribution of membranes during FFZE of microsomal membranes isolated from several plant species, including Arabidopsis thaliana, Thellungiella halophila, Mesembryanthemum crystallinum, and Ananas comosus. However, we demonstrate, using V-ATPase-specific inhibitors, nonhydrolyzable ATP analogs, and ionophores to dissipate membrane potential, that the ATP-dependent migrational shift of membranes under FFZE is not due to activation of the V-ATPase. Addition of EDTA to chelate Mg2+, leading to the production of the tetravalent anionic form of ATP, resulted in a further enhancement of membrane migration toward the anode, and manipulation of cell surface charge by addition of polycations also influenced the ATP-dependent migration of membranes. We propose that ATP enhances the mobility of endomembranes by screening positive surface charges on the membrane surface.
自由流动区带电泳（FFZE）是一种通用技术，可在层流通过薄水层时，根据净表面电荷对细胞、细胞器、膜和蛋白质进行分离。我们一直在优化FFZE技术，以增强植物液泡膜（液泡膜）与其他内膜的分离，从而采用定向蛋白质组学方法来鉴定新型液泡膜转运蛋白。在FFZE过程中，向内膜混合物中添加ATP可选择性增强酸性囊泡区室向正极的电泳迁移率。这归因于V-ATPase的激活，在囊泡外部产生更负的膜电位，导致包括液泡膜在内的酸性囊泡向阳极迁移增强（莫雷，D. J.；劳伦斯，J.；萨弗兰斯基，K.；哈蒙德，T.；莫雷，D. M.《色谱杂志》，A辑，1994年，668卷，201 - 213页）。我们证实，ATP确实会在从包括拟南芥、盐芥、冰叶日中花和菠萝等多种植物物种中分离的微粒体膜的FFZE过程中诱导膜的重新分布。然而，我们使用V-ATPase特异性抑制剂、不可水解的ATP类似物和离子载体来消除膜电位，证明FFZE下膜的ATP依赖性迁移变化并非由于V-ATPase的激活。添加EDTA螯合Mg2 +，导致产生四价阴离子形式的ATP，进一步增强了膜向阳极的迁移，通过添加聚阳离子来操纵细胞表面电荷也影响了膜的ATP依赖性迁移。我们提出，ATP通过屏蔽膜表面的正表面电荷来增强内膜的迁移率。
