lipid-a and potassium-hydroxide

The lipopolysaccharide (LPS) of strain 8081-c-R2, a spontaneous R-mutant of Yersinia enterocolitica serotype O:8, was isolated using extraction with phenol/chloroform/light petroleum. Its compositional analysis indicated the presence of D-GlcN, D-Glc, L-glycero-D-manno- and D-glycero-D-manno-heptose, 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo) and phosphate. From deacylated LPS obtained after successive treatment with hydrazine and potassium hydroxide, three oligosaccharides (1-3) were isolated using high-performance anion-exchange chromatography, the structures of which were determined by compositional analysis and one- and two-dimensional NMR spectroscopy as [carbohydrate structure see text] in which all sugars are pyranoses, and R and R' represent beta-D-Glc (in 1 and 2) and beta-D-GlcN (in 1 only), respectively. D-alpha-D-Hep is D-glycero-alpha-D-manno-heptose, L-alpha-D-Hep is L-glycero-alpha-D-manno-heptose, Kdo is 3-deoxy-D-manno-oct-2-ulosonic acid, and P is phosphate. The liberated lipid A was analyzed by compositional analyses and MALDI-TOF MS. Its beta-D-GlcN4P-(1-->6)-alpha-D-GlcN-1-->P backbone is mainly tetra-acylated with two amide- and one ester-linked (at O3 of the reducing GlcN) (R)-3-hydroxytetradecanoic acid residues, and one tetradecanoic acid that is attached to the 3-OH group of the amide-linked (R)-3-hydroxytetradecanoic acid of the nonreducing GlcN. Additionally, small amounts of tri- and hexa-acylated lipid A species occur.

Topics: Carbohydrate Sequence; Chromatography, Ion Exchange; Electrophoresis, Polyacrylamide Gel; Hydrazines; Hydroxides; Lipid A; Lipopolysaccharides; Magnetic Resonance Spectroscopy; Models, Chemical; Molecular Sequence Data; Phosphates; Plasmids; Potassium Compounds; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Yersinia enterocolitica

The lipopolysaccaride of Chlamydophila psittaci 6BC was isolated from tissue culture-grown elementary bodies using a modified phenol/water procedure followed by extraction with phenol/chloroform/light petroleum. Compositional analyses indicated the presence of 3-deoxy-Dmanno-oct-2-ulosonic acid, GlcN, organic bound phosphate and fatty acids in a molar ratio of approximately 3. 3 : 2 : 1.8 : 4.6. Deacylated lipopolysaccharide was obtained after successive microscale treatment with hydrazine and potassium hydroxide, and was then separated by high performance anion-exchange chromatography into two major fractions, the structures of which were determined by 600 MHz NMR spectroscopy as alpha-Kdo-(2-->8)-alpha-Kdo-(2-->4)-alpha-Kdo-(2-->6)-beta-D-GlcpN -(1 -->6)-alpha-D-GlcpN 1,4'-bisphosphate and alpha-Kdo-(2-->4)-[alpha-Kdo-(2-->8)]-alpha-Kdo-(2-->4)-alpha-Kdo-(2- ->6)-beta-D-GlcpN-(1-->6)-alpha-D-GlcpN 1,4'-bisphosphate. The distribution of fatty acids in lipid A was determined by compositional analyses and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry experiments on lipid A and de-O-acylated lipid A. It was shown that the carbohydrate backbone of lipid A is replaced by a complex mixture of fatty acids, including long-chain and branched (R)-configured 3-hydroxy fatty acids, the latter being exclusively present in an amide linkage.

Topics: Animals; Carbohydrate Conformation; Carbohydrate Sequence; Cell Line; Chlamydophila psittaci; Chloroform; Chromatography, Ion Exchange; Chromatography, Thin Layer; Hydrazines; Hydroxides; Lipid A; Lipopolysaccharides; Magnetic Resonance Spectroscopy; Mice; Molecular Sequence Data; Phenol; Potassium Compounds; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Sugar Acids; Time Factors; Water