Extraction of Nucleic Acid-free Lipopolysaccharides from Gram-negative Bacteria

O’Neill, G. J.; Todd, J. P.

doi:10.1038/190344b0

Cited by 19 publications

(5 citation statements)

References 2 publications

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“…The assay for 2-keto-3-deoxy sugars described by Weissbach and Hurwitz (29) was performed, using Osborn's modification (16). LPS extracted as described previously (30) from C. fetus strain 23 by the method of O'Neill and Todd (15) was used as a standard, and the assumption was made that detection of this class of sugars in envelope fractions reflected the presence of an LPS component, presumably 2keto-3-deoxyoctonate.…”

Section: Methodsmentioning

confidence: 99%

Major outer membrane protein of Campylobacter fetus: physical and immunological characterization

McCoy¹,

Wiltberger²,

Winter³

1976

Infect Immun

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The outer membrane proteins of Campylobacter fetus have been isolated by extraction of cell envelopes both in Triton X-100 and ethylenediaminetetraacetate (EDTA) and in sodium dodecyl sulfate (SDS). Each method yielded a major protein component, which migrated identically in SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and proved comparable in electrophoretic and molecular weight characteristics to the analagous protein from Escherichia coli. In addition, the surface of SDS-extracted C. fetus cells displayed a subunit structure similar to that observed in E. coli. The major envelope protein isolated with SDS appeared antigenically identical with one of the proteins isolated with Triton-EDTA on the basis of immunodiffusion reactions with specific antisera. Antibodies directed to the major envelope protein were not reactive in agglutination, immobilization, bactericidal, or opsonization reactions. Strains of C. fetus belonging to each of the three 0 serotypes possessed major envelope proteins comparable in SDS-PAGE but distinguished antigenically in a fashion paralleling the 0 serotype classification.The cell envelope of gram-negative bacteria is a complex structure consisting of the outer membranous layer, the murein layer, and the cytoplasmic membrane. The outer membrane contains substantial amounts of protein, phospholipid, and lipopolysaccharide (LPS) and appears to be heterogeneous with respect to structural organization (14,17,28). In Enterobacteriaceae, a low-molecular-weight lipoprotein is covalently bound to the murein layer and extends into the outer membrane (2, 3).The results of comparative studies of cell envelopes of a variety of gram-negative organisms have also revealed the presence of a major protein band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (21). This protein, which represents approximately 70% of the total outer membrane protein, is an integral structural component and is difficult to solubilize without dissolution of the membrane (23). Based on the results of chromatographic separation and cyanogen bromide cleavage, Schnaitman (24) has reported that the 42,000molecular weight major envelope protein in Escherichia coli 0111 is a complex consisting of at least four distinct polypeptide species (proteins 1, 2, 3a, and 3b). In a recent investigation, Rosenbusch (18) reported that separation of a major envelope protein ("matrix protein") from the murein layer in Escherichia coli BE by SDS ' Present address:

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Section: Methodsmentioning

confidence: 99%

Major outer membrane protein of Campylobacter fetus: physical and immunological characterization

McCoy¹,

Wiltberger²,

Winter³

1976

Infect Immun

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“…(v) Trichloroacetic acid-Organisms were extracted with 5% trichloroacetic acid by homogenization in the cold for short periods and also by gentle stirring at 4 C for 3 hr (11). In addition, the cells, after extraction with trichloroacetic acid, were treated with hot aqueous phenol (12). (vi) Cetyltrimethyl ammonium bromide-Organisms were extracted under the conditions described by Nowotny et al (11) and Westphal and Jann (16).…”

Section: Comparison Of Other Extraction Proceduresmentioning

confidence: 99%

Comparative Studies on the Isolation of Membrane Lipoteichoic Acid fromLactobacillus fermenti

1973

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Preparations of membrane lipoteichoic acid containing different amounts of protein were isolated from intact organisms of Lactobacillus fermenti NCTC 6991 by various procedures chosen for their ability to disrupt the hydrophobic interaction of lipoteichoic acid with other membrane components. The highest yield of lipoteichoic acid was obtained with hot aqueous phenol, and this preparation contained very little protein. Partial removal of cell lipids with chloroform-methanol followed by extraction with water at 100 C gave a lipoteichoic acid-protein complex that was a very effective immunogen; immunogenicity was shown to relate to protein content, though the specificity of the antibodies was directed against the teichoic acid component.

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“…The following additional techniques were tried in the present work. Acetone-dried cells were treated with 0.25Mtrichloroacetic acid at 2-40C before treatment with aqueous phenol (O'Neill & Todd, 1961); the lipopolysaccharide contained 9-11 % nucleic acid and was analytically different from that obtained by the normal procedure. Crude lipopolysaccharide was treated with diethylene glycol for 2h at 37°C and then at room temperature for 24h (Morgan & Partridge, 1940), but preferential solubilization of lipopolysaccharide was not obtained.…”

Section: Preparation Oflipopolysaccharidementioning

confidence: 99%

Further studies of the chemical composition of the lipopolysaccharide of Pseudomonas aeruginosa

Chester

Gray

Wilkinson

1972

Biochemical Journal

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1. Qualitative and quantitative analytical results for the lipopolysaccharide from acetone-dried cells of Pseudomonas aeruginosa (N.C.T.C. 1999) are presented and possible contamination of the material with nucleic acid was further examined. 2. Additional sugars detected (only in large-scale hydrolysates) were mannose and arabinose; traces of spermidine and putrescine were also found. 3. The heptose component is l-glycero-d-mannoheptose. 4. The thiobarbituric acid-positive component is a 3-deoxy-2-octulonic acid, of which only 35-40% links lipid A to the polysaccharide. This linkage is not broken by hydrolysis with acetic acid up to 0.08m. 5. Liberation of lipid A required hydrolysis with 0.1m-hydrochloric acid, which substantially degraded the polysaccharide moiety. 6. Fractions obtained from the degraded polysaccharide by high-voltage electrophoresis were examined; in these, the alanine/galactosamine molar ratio is approx. 1. 7. Hydrazinolysis of whole lipopolysaccharide showed that at least 40% of the alanine is in amide linkage, possibly with galactosamine. 8. Lipid A, solubilized by alkaline methanolysis was fractionated; most of the phosphorus of the higher-molecular-weight fractions was released as P(i) by a phosphomonoesterase. 9. Hydrazinolysis of lipid A destroyed approx. 80% of the glucosamine, and glycosidically linked glucosamine oligosaccharides could not be isolated.

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Extraction of Nucleic Acid-free Lipopolysaccharides from Gram-negative Bacteria

Cited by 19 publications

References 2 publications

Major outer membrane protein of Campylobacter fetus: physical and immunological characterization

Major outer membrane protein of Campylobacter fetus: physical and immunological characterization

Comparative Studies on the Isolation of Membrane Lipoteichoic Acid fromLactobacillus fermenti

Further studies of the chemical composition of the lipopolysaccharide of Pseudomonas aeruginosa

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