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

• Published in: The Journal of biological chemistry Vol. 244; no. 9; pp. 2450 - 2458
• Main Authors: Quigley, J W, Cohen, S S
• Format: Journal Article
• Language: English
• Published: United States American Society for Biochemistry and Molecular Biology 10.05.1969
• Subjects: Amines - analysis; Amino Acids - analysis; Bacterial Proteins - analysis; Bacteriolysis; Binding Sites; Carbon Isotopes; Cell Membrane - analysis; Cell Wall - analysis; Centrifugation, Zonal; Coliphages; Deoxyribonucleases; DNA, Bacterial - analysis; Escherichia coli - cytology; Methods; Microscopy, Electron; Phospholipids - analysis; Phosphorus - analysis; Phosphorus Isotopes; Pimelic Acids - analysis; Protoplasts; RNA, Bacterial - analysis
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(19)78243-X

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.