Skip to main content
PMC home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1976 Jul;127(1):555–563. doi: 10.1128/jb.127.1.555-563.1976

Lipoprotein from the outer membrane of Escherichia coli: purification, paracrystallization, and some properties of its free form.

S Inoyye, K Takeishi, N Lee, M DeMartini, A Hirashima, M Inouye
PMCID: PMC233089  PMID: 58861

Abstract

In the envelope of Escherichia coli, is a lipoprotein of molecular weight 7,200 as a major envelope protein. This lipoprotein was previously shown to exist in two different forms in the outer membrane of E. coli: the free form and the boundform, which is covalently linked to the peptidoglycau. The free form of the lipoprotein has been purified and paracrystallized by adding acetone to a sodium dodecyl sulfate solution in the presence of magnesium ion. The paracrystals were needle shaped. An electron micrograph of the negatively stained paracrystals showed a highly ordered ultrastructure. The chemical structure of the free form was compared with that of the bound form by (i) the amino acid composition, (ii) the fatty acid composition, and (iii) the peptide analysis after cyanogen bromide cleavage. The alpha-helical content of the free form of the lipoprotein was measured from the circular dichroism spectrum of the lipoprotein in 0.01% sodium dodecyl sulfate and found to be 87%. Using the purified lipoprotein as antigen, antiserum against the free form of the lipoprotein was obtained. Immunoprecipitation of the lipoprotein with the antiserum was found to be very specific, since only the free form of the lipoprotein was found as a major peak when the antiserum was reacted with the whole envelope proteins solubilized in 0.2% sodium dodecyl sulfate, and the immunoprecipitate thus formed was analyzed by polyacrylamide gel electrophoresis.

Full text

PDF
555

Images in this article

559Image
on p.559
560Image
on p.560

Selected References

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

  1. Bosch V., Braun V. Distribution of murein-lipoprotein between the cytoplasmic and outer membrane of Escherichia coli. FEBS Lett. 1973 Aug 15;34(2):307–310. doi: 10.1016/0014-5793(73)80818-x. [DOI] [PubMed] [Google Scholar]
  2. Braun V., Bosch V. Repetitive sequences in the murein-lipoprotein of the cell wall of Escherichia coli. Proc Natl Acad Sci U S A. 1972 Apr;69(4):970–974. doi: 10.1073/pnas.69.4.970. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Braun V., Bosch V. Sequence of the murein-lipoprotein and the attachment site of the lipid. Eur J Biochem. 1972 Jun 23;28(1):51–69. doi: 10.1111/j.1432-1033.1972.tb01883.x. [DOI] [PubMed] [Google Scholar]
  4. Braun V., Rehn K., Wolff H. Supramolecular structure of the rigid layer of the cell wall of Salmonella, Serratia, Proteus, and Pseudomonas fluorescens. Number of lipoprotein molecules in a membrane layer. Biochemistry. 1970 Dec 22;9(26):5041–5049. doi: 10.1021/bi00828a001. [DOI] [PubMed] [Google Scholar]
  5. Braun V., Sieglin U. The covalent murein-lipoprotein structure of the Escherichia coli cell wall. The attachment site of the lipoprotein on the murein. Eur J Biochem. 1970 Apr;13(2):336–346. doi: 10.1111/j.1432-1033.1970.tb00936.x. [DOI] [PubMed] [Google Scholar]
  6. Braun V., Wolff H. The murein-lipoprotein linkage in the cell wall of Escherichia coli. Eur J Biochem. 1970 Jun;14(2):387–391. doi: 10.1111/j.1432-1033.1970.tb00301.x. [DOI] [PubMed] [Google Scholar]
  7. DeMartini M., Inouye S., Inouye M. Ultrastructure of paracrystals of a lipoprotein from the outer membrane of Escherichia coli. J Bacteriol. 1976 Jul;127(1):564–571. doi: 10.1128/jb.127.1.564-571.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Goodwin T. W., Morton R. A. The spectrophotometric determination of tyrosine and tryptophan in proteins. Biochem J. 1946;40(5-6):628–632. doi: 10.1042/bj0400628. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Greenfield N., Fasman G. D. Computed circular dichroism spectra for the evaluation of protein conformation. Biochemistry. 1969 Oct;8(10):4108–4116. doi: 10.1021/bi00838a031. [DOI] [PubMed] [Google Scholar]
  10. HIRS C. H. The oxidation of ribonuclease with performic acid. J Biol Chem. 1956 Apr;219(2):611–621. [PubMed] [Google Scholar]
  11. Halegoua S., Hirashima A., Inouye M. Existence of a free form of a specific membrane lipoprotein in gram-negative bacteria. J Bacteriol. 1974 Dec;120(3):1204–1208. doi: 10.1128/jb.120.3.1204-1208.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Hantke K., Braun V. Covalent binding of lipid to protein. Diglyceride and amide-linked fatty acid at the N-terminal end of the murein-lipoprotein of the Escherichia coli outer membrane. Eur J Biochem. 1973 Apr;34(2):284–296. doi: 10.1111/j.1432-1033.1973.tb02757.x. [DOI] [PubMed] [Google Scholar]
  13. Hirashima A., Childs G., Inouye M. Differential inhibitory effects of antibiotics on the biosynthesis of envelope proteins of Escherichia coli. J Mol Biol. 1973 Sep 15;79(2):373–389. doi: 10.1016/0022-2836(73)90012-0. [DOI] [PubMed] [Google Scholar]
  14. Hirashima A., Inouye M. Specific biosynthesis of an envelope protein of Escherichia coli. Nature. 1973 Apr 6;242(5397):405–407. doi: 10.1038/242405a0. [DOI] [PubMed] [Google Scholar]
  15. Hirashima A., Wang S., Inouye M. Cell-free synthesis of a specific lipoprotein of the Escherichia coli outer membrane directed by purified messenger RNA. Proc Natl Acad Sci U S A. 1974 Oct;71(10):4149–4153. doi: 10.1073/pnas.71.10.4149. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Hirashima A., Wu H. C., Venkateswaran P. S., Inouye M. Two forms of a structural lipoprotein in the envelope of Escherichia coli. Further characterization of the free form. J Biol Chem. 1973 Aug 25;248(16):5654–5659. [PubMed] [Google Scholar]
  17. Inouye M. A three-dimensional molecular assembly model of a lipoprotein from the Escherichia coli outer membrane. Proc Natl Acad Sci U S A. 1974 Jun;71(6):2396–2400. doi: 10.1073/pnas.71.6.2396. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Inouye M. Internal standards for molecular weight determinations of proteins by polyacrylamide gel electrophoresis. Applications to envelope proteins of Escherichia coli. J Biol Chem. 1971 Aug 10;246(15):4834–4838. [PubMed] [Google Scholar]
  19. Inouye M., Pardee A. B. Changes of membrane proteins and their relation to deoxyribonucleic acid synthesis and cell division of Escherichia coli. J Biol Chem. 1970 Nov 10;245(21):5813–5819. [PubMed] [Google Scholar]
  20. Inouye M., Shaw J., Shen C. The assembly of a structural lipoprotein in the envelope of Escherichia coli. J Biol Chem. 1972 Dec 25;247(24):8154–8159. [PubMed] [Google Scholar]
  21. 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]
  22. Lee N., Inouye M. Outer membrane proteins of Escherichia coli: biosynthesis and assembly. FEBS Lett. 1974 Feb 15;39(2):167–170. doi: 10.1016/0014-5793(74)80043-8. [DOI] [PubMed] [Google Scholar]
  23. Scandella C. J., Kornberg A. A membrane-bound phospholipase A1 purified from Escherichia coli. Biochemistry. 1971 Nov 23;10(24):4447–4456. doi: 10.1021/bi00800a015. [DOI] [PubMed] [Google Scholar]

Articles from Journal of Bacteriology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES