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. 1973 Sep;70(9):2572–2576. doi: 10.1073/pnas.70.9.2572

Isolation and Characterization of Lipopolysaccharide Protein from Escherichia coli*

Ming-Chi Wu 1,2, Edward C Heath 1,2,†
PMCID: PMC427058  PMID: 4582189

Abstract

Extraction of the cell envelope of E. coli with 1% sodium dodecyl sulfate yielded a lipopolysaccharide protein that was purified to homogeneity by conventional techniques. Analysis of the pure protein indicated that it is a complex lipopolysaccharide protein with the following molar ratios of constitutents: 3-deoxyoctulosonate, 1.0; glucosamine, 1.3; neutral sugar (glucose + heptose), 1.0; organic phosphate, 2.3; and amino acid, 21. On a quantitative basis, all of the 3-deoxyoctulosonate present in the cell envelope preparation was solubilized in hot sodium dodecyl sulfate and subsequently accounted for in its entirely in the isolated protein component. After incubation of the cell-wall particulate fraction of cells grown on [3H]glucosamine with UDP-[14C]galactose under conditions designed to measure galactosyl transferase activity, the isolated lipopolysaccharide protein contained all of the [14C]galactose that was incorporated during the incubation. We concluded that the lipopolysaccharide of this organism occurs in the outer cellenvelope membrane exclusively in the form of lipopolysaccharide protein.

Keywords: cell wall, glycoprotein, membrane protein, endotoxin

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. AMES B. N., DUBIN D. T. The role of polyamines in the neutralization of bacteriophage deoxyribonucleic acid. J Biol Chem. 1960 Mar;235:769–775. [PubMed] [Google Scholar]
  2. Braun V., Rehn K. Chemical characterization, spatial distribution and function of a lipoprotein (murein-lipoprotein) of the E. coli cell wall. The specific effect of trypsin on the membrane structure. Eur J Biochem. 1969 Oct;10(3):426–438. doi: 10.1111/j.1432-1033.1969.tb00707.x. [DOI] [PubMed] [Google Scholar]
  3. ELBEIN A. D., HEATH E. C. THE BIOSYNTHESIS OF CELL WALL LIPOPOLYSACCHARIDE IN ESCHERICHIA COLI. I. THE BIOCHEMICAL PROPERTIES OF A URIDINE DIPHOSPHATE GALACTOSE 4-EPIMERASELESS MUTANT. J Biol Chem. 1965 May;240:1919–1925. [PubMed] [Google Scholar]
  4. Edstrom R. D., Heath E. C. The biosynthesis of cell wall lipopolysaccharide in Escherichia coli. VI. Enzymatic transfer of galactose, glucose, N-acetylglucosamine, and colitose into the polymer. J Biol Chem. 1967 Aug 25;242(16):3581–3588. [PubMed] [Google Scholar]
  5. Elson L. A., Morgan W. T. A colorimetric method for the determination of glucosamine and chondrosamine. Biochem J. 1933;27(6):1824–1828. doi: 10.1042/bj0271824. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. GRAHAM J. L., GRUNBAUM B. W. A rapid method for microelectrophoresis and quantitation of hemoglobins on cellulose acetate. Am J Clin Pathol. 1963 Jun;39:567–578. doi: 10.1093/ajcp/39.6.567. [DOI] [PubMed] [Google Scholar]
  7. Ghalambor M. A., Levine E. M., Heath E. C. The biosynthesis of cell wall lipopolysaccharide in Escherichia coli. 3. The isolation and characterization of 3-deoxyoctulosonic acid. J Biol Chem. 1966 Jul 10;241(13):3207–3215. [PubMed] [Google Scholar]
  8. Heath E. C. Complex polysaccharides. Annu Rev Biochem. 1971;40:29–56. doi: 10.1146/annurev.bi.40.070171.000333. [DOI] [PubMed] [Google Scholar]
  9. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  10. Okuda S., Weinbaum G. An envelope-specific glycoprotein from Escherichia coli B. Biochemistry. 1968 Aug;7(8):2819–2825. doi: 10.1021/bi00848a018. [DOI] [PubMed] [Google Scholar]
  11. Osborn M. J., Gander J. E., Parisi E., Carson J. Mechanism of assembly of the outer membrane of Salmonella typhimurium. Isolation and characterization of cytoplasmic and outer membrane. J Biol Chem. 1972 Jun 25;247(12):3962–3972. [PubMed] [Google Scholar]
  12. Osborn M. J., Gander J. E., Parisi E. Mechanism of assembly of the outer membrane of Salmonella typhimurium. Site of synthesis of lipopolysaccharide. J Biol Chem. 1972 Jun 25;247(12):3973–3986. [PubMed] [Google Scholar]
  13. Osborn M. J. Structure and biosynthesis of the bacterial cell wall. Annu Rev Biochem. 1969;38:501–538. doi: 10.1146/annurev.bi.38.070169.002441. [DOI] [PubMed] [Google Scholar]
  14. Ptashne M. ISOLATION OF THE lambda PHAGE REPRESSOR. Proc Natl Acad Sci U S A. 1967 Feb;57(2):306–313. doi: 10.1073/pnas.57.2.306. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. SEIFTER S., DAYTON S. The estimation of glycogen with the anthrone reagent. Arch Biochem. 1950 Jan;25(1):191–200. [PubMed] [Google Scholar]
  16. Schnaitman C. A. Effect of ethylenediaminetetraacetic acid, Triton X-100, and lysozyme on the morphology and chemical composition of isolate cell walls of Escherichia coli. J Bacteriol. 1971 Oct;108(1):553–563. doi: 10.1128/jb.108.1.553-563.1971. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Takayama K., MacLennan D. H., Tzagoloff A., Stoner C. D. Studies on the electron transfer system. LXVII. Polyacrylamide gel electrophoresis of the mitochondrial electron transfer complexes. Arch Biochem Biophys. 1966 Apr;114(1):223–230. doi: 10.1016/0003-9861(66)90324-9. [DOI] [PubMed] [Google Scholar]
  18. Wardi A. H., Allen W. S. Alcian blue staining of glycoproteins. Anal Biochem. 1972 Aug;48(2):621–623. doi: 10.1016/0003-2697(72)90118-2. [DOI] [PubMed] [Google Scholar]
  19. Weber K., Osborn M. The reliability of molecular weight determinations by dodecyl sulfate-polyacrylamide gel electrophoresis. J Biol Chem. 1969 Aug 25;244(16):4406–4412. [PubMed] [Google Scholar]
  20. Wober W., Alaupović P. Studies on the protein moiety of endotoxin from gram-negative bacteria. Characterization of the protein moiety isolated by phenol treatment of endotoxin from Serratia marcescens 08 and Escherichia coli 0 141:K85(B). Eur J Biochem. 1971 Apr;19(3):340–356. doi: 10.1111/j.1432-1033.1971.tb01323.x. [DOI] [PubMed] [Google Scholar]

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