Lakey J H, Pattus F
European Molecular Biology Laboratory, Heidelberg, FRG.
Eur J Biochem. 1989 Dec 8;186(1-2):303-8. doi: 10.1111/j.1432-1033.1989.tb15209.x.
Because of conflicting results from differing techniques, the degree of voltage sensitivity of Escherichia coli porins in planar bilayers is still a matter of debate. In order to provide the first comparative study, OmpF porin was purified in three ways; firstly as native outer membrane vesicles, secondly as salt-extracted porin trimers in sodium dodecyl sulphate and thirdly as solubilised trimers extracted with octyl-polyoxyethylene (Octyl-POE). These methods represent the major approaches to porin isolation and purification. All three were reconstituted into Schindler-type bilayers. Detergent-solubilised OmpF was also reconstituted into Montal-Mueller- and Mueller-Rudin-type bilayers. In all cases voltage-dependent closing of OmpF was observed. Octyl-POE-extracted PhoE porin was similarly investigated in all three types of planar bilayer. Two membrane-formation techniques appeared genuinely to alter the voltage sensitivity of the porins they contained. Firstly, porins in membranes formed by the Montal-Mueller technique sometimes showed an increase in voltage sensitivity during the first 30 min after bilayer formation. Secondly, membranes formed by the Mueller-Rudin technique on thick polyethylene septa showed both poor solvent drainage and a significantly reduced porin voltage sensitivity.
由于不同技术得出的结果相互矛盾，大肠杆菌孔蛋白在平面双层膜中的电压敏感性程度仍是一个有争议的问题。为了进行首次比较研究，以三种方式纯化了OmpF孔蛋白；第一种是作为天然外膜囊泡，第二种是作为在十二烷基硫酸钠中盐提取的孔蛋白三聚体，第三种是作为用辛基聚氧乙烯（辛基 - POE）提取的溶解三聚体。这些方法代表了孔蛋白分离和纯化的主要途径。将所有三种都重构到辛德勒型双层膜中。用去污剂溶解的OmpF也重构到蒙塔尔 - 米勒型和米勒 - 鲁丁型双层膜中。在所有情况下都观察到了OmpF的电压依赖性关闭。对用辛基 - POE提取的PhoE孔蛋白在所有三种类型的平面双层膜中进行了类似的研究。两种成膜技术似乎确实改变了它们所包含的孔蛋白的电压敏感性。首先，通过蒙塔尔 - 米勒技术形成的膜中的孔蛋白在双层膜形成后的最初30分钟内有时会表现出电压敏感性增加。其次，在厚聚乙烯隔板上通过米勒 - 鲁丁技术形成的膜显示出溶剂排水不良以及孔蛋白电压敏感性显著降低。
