Apparent high degree of asymmetry of protein arrangement in the Escherichia coli outer cell envelope membrane.

Ghosts from Escherichia coli have been oxidized with CuSO4-o-phenanthroline or ferricyanide-ferrocene. Upon oxidation they became resistant to boiling dodecyl sulfate. The resulting rod-shaped "oxidation containers" apparently held together by disulfide bridges, are practically pure protein. They are soluble in dodecyl sulfate when reduced and they contain a set of about 30 different polypeptide chains. The four major ghost membrane proteins are not represented among the "oxidation proteins." Comparison of data obtained from digestion of ghosts with trypsin or particle-bound trypsin showed that most of the "oxidation proteins" appear to be located at the outer surface of the ghost membrane which is derived from the outer cell envelope membrane. One of the major ghost membrane proteins, II, is partially digested by trypsin, and it is shown that its trypsin sensitive part is also exposed only at the outer surface of the ghost membrane. Native cells could be oxidized only with low yields of "oxidation containers." However, cell envelopes prepared without detergents or chelating agents, as well as cells depleted of phospholipid or treated with sucrose-Triton X-100, are completely accessible to oxidation. In each case, the same set of proteins as that present in "oxidation containers" from ghosts was found to be covalently linked. Treatment of cells with trypsin caused the loss of about five "oxidation proteins" and a complete loss of oxidizability of the ghosts derived from these cells. It therefore appears that arrangement and localization of the "oxidation proteins" are not greatly different in cells and in ghosts, i.e., that these proteins are also situated asymmetrically at the outer cell envelope membrane.