Sugawara E, Nikaido H
Department of Molecular and Cell Biology, University of California, Berkeley 94720.
J Biol Chem. 1992 Feb 5;267(4):2507-11.
Escherichia coli outer membrane protein OmpA was purified to homogeneity, as a monomer, from a K12 derivative deficient in both OmpF and OmpC porins. When proteoliposomes reconstituted from the purified OmpA, phospholipids, and lithium dodecyl sulfate were tested for permeability to small molecules by osmotic swelling, it was found that OmpA produced apparently nonspecific diffusion channels that allowed the penetration of various solutes. The pore-forming activity was destroyed by the heat denaturation of the OmpA protein, and the use of an OmpA-deficient mutant showed that the activity was not caused by copurifying contaminants. The size of the OmpA channel, estimated by comparison of diffusion rates of solutes of different sizes, was rather similar to that of E. coli OmpF and OmpC porins, i.e. about 1 nm in diameter. The rate of penetration of L-arabinose caused by a given amount of OmpA protein, however, was about a hundredfold lower than the rate produced by the same amount of E. coli OmpF porin. The addition of large amounts of lithium dodecyl sulfate to the reconstitution mixture increased the permeability through the OmpA channel, apparently by facilitating the correct insertion of OmpA into the bilayer.
从缺乏OmpF和OmpC孔蛋白的K12衍生物中，将大肠杆菌外膜蛋白OmpA纯化至同质单体。当用纯化的OmpA、磷脂和十二烷基硫酸锂重构的蛋白脂质体通过渗透膨胀测试对小分子的通透性时，发现OmpA产生了明显非特异性的扩散通道，允许各种溶质渗透。OmpA蛋白的热变性破坏了其成孔活性，使用缺乏OmpA的突变体表明该活性不是由共纯化的污染物引起的。通过比较不同大小溶质的扩散速率估计，OmpA通道的大小与大肠杆菌OmpF和OmpC孔蛋白相当，即直径约为1纳米。然而，给定数量的OmpA蛋白引起的L-阿拉伯糖渗透速率比相同数量的大肠杆菌OmpF孔蛋白产生的速率低约一百倍。向重构混合物中加入大量十二烷基硫酸锂可增加通过OmpA通道的通透性，显然是通过促进OmpA正确插入双层膜来实现的。
