Wolf-Watz H, Normark S, Bloom G D
J Bacteriol. 1973 Sep;115(3):1191-7. doi: 10.1128/jb.115.3.1191-1197.1973.
A rapid method for the isolation of large quantities of bacterial outer membrane is described. This cell envelope component was removed from plasmolyzed cells of Escherichia coli K-12 by lysozyme-ethylenediaminetetra-acetic acid treatment, aggregated by lowering the pH to 5.0, and recovered by centrifugation. Aggregates of membrane fragments were clearly identified in an electron microscope. A criterion of homogeneity of the preparation was obtained by isopycnic sucrose gradient centrifugation. A single band appeared at a density of 1.24 g/cc. The cytoplasmic membrane marker, succinate dehydrogenase activity, was 40 times lower in the outer membrane preparation than in complete cell envelope preparations. A rich activity was, however, found for the outer membrane marker, phospholipase A. The compositions of outer membranes from a transductant pair were compared. One transductant was a chain-forming, antibiotic-supersensitive envA strain, whereas the other contained the envA(+) allele. The envA strain showed a slightly modified protein pattern and a lower relative content of phosphatidylglycerol.
本文描述了一种快速分离大量细菌外膜的方法。通过溶菌酶 - 乙二胺四乙酸处理，从大肠杆菌K - 12的质壁分离细胞中去除这种细胞包膜成分，将pH值降至5.0使其聚集，然后通过离心回收。在电子显微镜下可清晰鉴定出膜碎片聚集体。通过等密度蔗糖梯度离心获得了制备物均匀性的标准。在密度为1.24 g/cc处出现一条带。外膜制备物中细胞质膜标记物琥珀酸脱氢酶活性比完整细胞包膜制备物低40倍。然而，在外膜标记物磷脂酶A方面发现了丰富的活性。比较了一对转导子的外膜组成。一个转导子是形成链状、对抗生素超敏感的envA菌株，而另一个含有envA(+)等位基因。envA菌株显示出略有改变的蛋白质模式和较低的磷脂酰甘油相对含量。
