The Isolation of Envelopes of Escherichia coli Spheroplasts in the Zonal Ultracentrifuge

Relatively large amounts of cell envelopes from Escherichia coli spheroplasts have been isolated in the zonal ultracentrifuge after deoxyribonuclease treatment of the disrupted spheroplasts. The Anderson B-IV zonal rotor was adapted to this isolation. After isopycnic zonal centrifugation through a 34 to 77% sucrose density gradient, the center of the zone containing the cell wall-cell membrane material positions at 58.9% (w/v) sucrose, corresponding to a density of 1.220 g per ml. Such material was further purified by a washing procedure. Electron micrographs indicate that very little material other than envelopes is present in this fraction. The preparation contained fewer than 0.1% intact spheroplasts. The cell envelope fraction contains 6.6 ± 0.3% of the protein, 0.5 ± 0.2% of the DNA, 2.7 ± 0.3% of the RNA, and 92 ± 2% of the phospholipid of the cell. Variable amounts of putrescine and spermidine are found in this fraction. The content of spermidine was particularly high, amounting to 4 to 10% of the cell spermidine. However, the spermidine on the envelopes was shown to be exchangeable during isolation and was not shown to have been a characteristic membrane component. Most of the receptor sites for bacteriophage T6 seem to have been retained on the isolated envelopes. These envelopes contain less than 4% of the diaminopimelic acid of the cell, indicating that most of the material of the rigid layer had been removed.