Groves J T, Wülfing C, Boxer S G
Department of Chemistry, Stanford University, California 94305-5080, USA.
Biophys J. 1996 Nov;71(5):2716-23. doi: 10.1016/S0006-3495(96)79462-6.
Electric fields have been used to manipulate and concentrate glycan-phosphatidyl inositol (GPI)-tethered proteins in planar supported bilayers. Naturally GPI-linked CD48, along with engineered forms of I-Ek and B7-2, in which their transmembrane domains have been genetically replaced with the GPI linkage, were studied. The proteins were labeled with fluorescently tagged antibodies, allowing the electric field-induced behavior to be followed by epifluorescence microscopy. All three protein complexes were observed to migrate toward the cathode with the B7-2 and CD48, each tethered to the membrane by a single GPI linker, moving significantly faster than the I-Ek, which has two GPI linkers. Patterns scratched into the membrane function as barriers to lateral diffusion and were used to isolate the proteins into highly concentrated corrals. All field-induced concentration profiles were completely reversible, indicating that the supported bilayer provides a stable, fluid environment in which GPI-tethered proteins can be manipulated. The ability to electrically control the spatial distribution of membrane-tethered proteins provides new opportunities for the study of biological membranes and the development of membrane-based devices.
电场已被用于在平面支撑双层膜中操纵和浓缩糖基磷脂酰肌醇（GPI）锚定蛋白。研究了天然GPI连接的CD48，以及I-Ek和B7-2的工程形式，其中它们的跨膜结构域已被基因替换为GPI连接。用荧光标记的抗体标记蛋白质，通过落射荧光显微镜观察电场诱导的行为。观察到所有三种蛋白质复合物都向阴极迁移，其中B7-2和CD48各自通过单个GPI接头连接到膜上，移动速度明显快于具有两个GPI接头的I-Ek。刻在膜上的图案起到横向扩散的屏障作用，用于将蛋白质隔离到高度浓缩的围栏中。所有电场诱导的浓度分布都是完全可逆的，这表明支撑双层膜提供了一个稳定的流体环境，其中GPI锚定蛋白可以被操纵。电控制膜锚定蛋白空间分布的能力为生物膜研究和基于膜的装置开发提供了新机会。
