Skip to main content
NCBI home page
Infection and Immunity logoLink to Infection and Immunity
. 1981 May;32(2):575–582. doi: 10.1128/iai.32.2.575-582.1981

Kinetics of adenosinediphosphoribosylation of elongation factor 2 in cells exposed to diphtheria toxin.

M R Moynihan, A M Pappenheimer Jr
PMCID: PMC351486  PMID: 7251138

Abstract

When susceptible cells are exposed to diphtheria toxin (Mr, 62,000) the N-terminal 21,150-dalton A fragment of toxin reaches the cytoplasm, where it catalyzes the transfer of adenosinediphosphoribose from nicotinamide adenine dinucleotide to elongation factor 2 (EF2). Adenosinediphosphoribose-EF2 is inactive, so that protein synthesis is blocked. Using a simple, rapid assay for the amount of adenosinediphosphoribosylatable EF2 in unfractionated lysates of cultured cells we have followed the kinetics of inactivation of EF2 in CV-1 and BHK cells exposed to diphtheria toxin. With both cell lines a lag was observed between the addition of toxin to the cells and the adenosinediphosphoribosylation of EF2. The lag decreased with increasing toxin concentration until a limiting value of about 12 min was reached. The rate of adenosinediphosphoribosylation of EF2 after the lag was 10 to 20 times more rapid in CV-1 cells than in BHK cells exposed to the same toxin concentration. The concentration of fragment A active in the cytoplasm of toxin-treated cells was estimated from the rate of adenosinediphosphoribosylation observed. Comparison of these estimates with data from studies of binding of 125I-toxin to cells suggests that the fragment A of only a minor fraction of toxin molecules bound to cell surface receptors reaches the cytoplasm and participates in the inactivation of EF2. A model summarizing our current views on the process by which fragment A enters cells is presented.

Full text

PDF
575

Images in this article

579Image
on p.579

Selected References

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

  1. Arlinghaus R., Shaeffer J., Bishop J., Schweet R. Purification of the transfer enzymes from reticulocytes and properties of the transfer reaction. Arch Biochem Biophys. 1968 May;125(2):604–613. doi: 10.1016/0003-9861(68)90619-x. [DOI] [PubMed] [Google Scholar]
  2. Benjamin R. C., Gill D. M. Poly(ADP-ribose) synthesis in vitro programmed by damaged DNA. A comparison of DNA molecules containing different types of strand breaks. J Biol Chem. 1980 Nov 10;255(21):10502–10508. [PubMed] [Google Scholar]
  3. Boquet P. Interaction of diphtheria toxin fragments A, B and protein crm 45 with liposomes. Eur J Biochem. 1979 Oct 15;100(2):483–489. doi: 10.1111/j.1432-1033.1979.tb04192.x. [DOI] [PubMed] [Google Scholar]
  4. Boquet P., Pappenheimer A. M., Jr Interaction of diphtheria toxin with mammalian cell membranes. J Biol Chem. 1976 Sep 25;251(18):5770–5778. [PubMed] [Google Scholar]
  5. Dorland R. B., Middlebrook J. L., Leppla S. H. Receptor-mediated internalization and degradation of diphtheria toxin by monkey kidney cells. J Biol Chem. 1979 Nov 25;254(22):11337–11342. [PubMed] [Google Scholar]
  6. Draper R. K., Chin D., Stubbs L., Simon M. I. Studies of the diphtheria toxin receptor on Chinese hamster cells. J Supramol Struct. 1978;9(1):47–55. doi: 10.1002/jss.400090106. [DOI] [PubMed] [Google Scholar]
  7. Draper R. K., Simon M. I. The entry of diphtheria toxin into the mammalian cell cytoplasm: evidence for lysosomal involvement. J Cell Biol. 1980 Dec;87(3 Pt 1):849–854. doi: 10.1083/jcb.87.3.849. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Gill D. M., Dinius L. L. The elongation factor 2 content of mammalian cells. Assay method and relation to ribosome number. J Biol Chem. 1973 Jan 25;248(2):654–658. [PubMed] [Google Scholar]
  9. Hayaishi O., Ueda K. Poly(ADP-ribose) and ADP-ribosylation of proteins. Annu Rev Biochem. 1977;46:95–116. doi: 10.1146/annurev.bi.46.070177.000523. [DOI] [PubMed] [Google Scholar]
  10. Helenius A., Kartenbeck J., Simons K., Fries E. On the entry of Semliki forest virus into BHK-21 cells. J Cell Biol. 1980 Feb;84(2):404–420. doi: 10.1083/jcb.84.2.404. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Ito S., Shizuta Y., Hayaishi O. Purification and characterization of poly(ADP-ribose) synthetase from calf thymus. J Biol Chem. 1979 May 10;254(9):3647–3651. [PubMed] [Google Scholar]
  12. Ittelson T. R., Gill D. M. Diphtheria toxin: specific competition for cell receptors. Nature. 1973 Mar 30;242(5396):330–332. doi: 10.1038/242330b0. [DOI] [PubMed] [Google Scholar]
  13. Ivins B., Saelinger C. B., Bonventre P. F., Woscinski C. Chemical modulation of diphtheria toxin action on cultured mammalian cells. Infect Immun. 1975 Apr;11(4):665–674. doi: 10.1128/iai.11.4.665-674.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Kim K., Groman N. B. In vitro inhibition of diphtheria toxin action by ammonium salts and amines. J Bacteriol. 1965 Dec;90(6):1552–1556. doi: 10.1128/jb.90.6.1552-1556.1965. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Leppla S., Dorland R. B., Middlebrook J. L. Inhibition of diphtheria toxin degradation and cytotoxic action by chloroquine. J Biol Chem. 1980 Mar 25;255(6):2247–2250. [PubMed] [Google Scholar]
  16. Middlebrook J. L., Dorland R. B., Leppla S. H. Association of diphtheria toxin with Vero cells. Demonstration of a receptor. J Biol Chem. 1978 Oct 25;253(20):7325–7330. [PubMed] [Google Scholar]
  17. Middlebrook J. L., Dorland R. B. Response of cultured mammalian cells to the exotoxins of Pseudomonas aeruginosa and Corynebacterium diphtheriae: differential cytotoxicity. Can J Microbiol. 1977 Feb;23(2):183–189. doi: 10.1139/m77-026. [DOI] [PubMed] [Google Scholar]
  18. Moehring J. M., Moehring T. J., Danley D. E. Posttranslational modification of elongation factor 2 in diphtheria-toxin-resistant mutants of CHO-K1 cells. Proc Natl Acad Sci U S A. 1980 Feb;77(2):1010–1014. doi: 10.1073/pnas.77.2.1010. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Moehring T. J., Moehring J. M. Interaction of diphtheria toxin and its active subunit, fragment A, with toxin-sensitive and toxin-resistant cells. Infect Immun. 1976 May;13(5):1426–1432. doi: 10.1128/iai.13.5.1426-1432.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. POPE C. G. Observations on the diphtheria toxin-antitoxin reaction. Br J Exp Pathol. 1957 Apr;38(2):207–216. [PMC free article] [PubMed] [Google Scholar]
  21. Pappenheimer A. M., Jr Diphtheria toxin. Annu Rev Biochem. 1977;46:69–94. doi: 10.1146/annurev.bi.46.070177.000441. [DOI] [PubMed] [Google Scholar]
  22. Pappenheimer A. M., Jr, Uchida T., Harper A. A. An immunological study of the diphtheria toxin molecule. Immunochemistry. 1972 Sep;9(9):891–906. doi: 10.1016/0019-2791(72)90163-2. [DOI] [PubMed] [Google Scholar]
  23. Robinson E. A., Henriksen O., Maxwell E. S. Elongation factor 2. Amino acid sequence at the site of adenosine diphosphate ribosylation. J Biol Chem. 1974 Aug 25;249(16):5088–5093. [PubMed] [Google Scholar]
  24. Sandvig K., Olsnes S. Diphtheria toxin entry into cells is facilitated by low pH. J Cell Biol. 1980 Dec;87(3 Pt 1):828–832. doi: 10.1083/jcb.87.3.828. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Uchida T., Pappenheimer A. M., Jr, Greany R. Diphtheria toxin and related proteins. I. Isolation and properties of mutant proteins serologically related to diphtheria toxin. J Biol Chem. 1973 Jun 10;248(11):3838–3844. [PubMed] [Google Scholar]
  26. Van Ness B. G., Howard J. B., Bodley J. W. ADP-ribosylation of elongation factor 2 by diphtheria toxin. NMR spectra and proposed structures of ribosyl-diphthamide and its hydrolysis products. J Biol Chem. 1980 Nov 25;255(22):10710–10716. [PubMed] [Google Scholar]
  27. White J., Kartenbeck J., Helenius A. Fusion of Semliki forest virus with the plasma membrane can be induced by low pH. J Cell Biol. 1980 Oct;87(1):264–272. doi: 10.1083/jcb.87.1.264. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Wibo M., Poole B. Protein degradation in cultured cells. II. The uptake of chloroquine by rat fibroblasts and the inhibition of cellular protein degradation and cathepsin B1. J Cell Biol. 1974 Nov;63(2 Pt 1):430–440. doi: 10.1083/jcb.63.2.430. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Willingham M. C., Pastan I. The receptosome: an intermediate organelle of receptor mediated endocytosis in cultured fibroblasts. Cell. 1980 Aug;21(1):67–77. doi: 10.1016/0092-8674(80)90115-4. [DOI] [PubMed] [Google Scholar]
  30. Willingham M. C., Yamada S. S. A mechanism for the destruction of pinosomes in cultured fibroblasts. Piranhalysis. J Cell Biol. 1978 Aug;78(2):480–487. doi: 10.1083/jcb.78.2.480. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Yamaizumi M., Mekada E., Uchida T., Okada Y. One molecule of diphtheria toxin fragment A introduced into a cell can kill the cell. Cell. 1978 Sep;15(1):245–250. doi: 10.1016/0092-8674(78)90099-5. [DOI] [PubMed] [Google Scholar]
  32. Youle R. J., Neville D. M., Jr Receptor-mediated transport of the hybrid protein ricin-diphtheria toxin fragment A with subsequent ADP-ribosylation of intracellular elongation factor II. J Biol Chem. 1979 Nov 10;254(21):11089–11096. [PubMed] [Google Scholar]
  33. de Duve C., de Barsy T., Poole B., Trouet A., Tulkens P., Van Hoof F. Commentary. Lysosomotropic agents. Biochem Pharmacol. 1974 Sep 15;23(18):2495–2531. doi: 10.1016/0006-2952(74)90174-9. [DOI] [PubMed] [Google Scholar]

Articles from Infection and Immunity are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES