Skip to main content
PMC home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1989 Jan;86(2):554–557. doi: 10.1073/pnas.86.2.554

Endogenous ADP-ribosylation of elongation factor 2 in polyoma virus-transformed baby hamster kidney cells.

J L Fendrick 1, W J Iglewski 1
PMCID: PMC286510  PMID: 2536169

Abstract

Polyoma virus-transformed baby hamster kidney (pyBHK) cells were cultured in medium containing [32P]orthophosphate and 10% (vol/vol) fetal bovine serum. A 32P-labeled protein with an apparent molecular mass of 97 kDa was immunoprecipitated from cell lysates with antiserum to ADP-ribosylated elongation factor 2 (EF-2). The 32P labeling of the protein was enhanced by culturing cells in medium containing 2% serum instead of 10% serum. The 32P label was completely removed from the protein by treatment with snake venom phosphodiesterase and the digestion product was identified as [32P]AMP, indicating the protein was mono-ADP-ribosylated. HPLC analysis of tryptic peptides of the 32P-labeled 97-kDa protein and purified EF-2, which was ADP-ribosylated in vitro with diphtheria toxin fragment A and [32P]NAD, demonstrated an identical labeled peptide in the two proteins. The data strongly suggest that EF-2 was endogenously ADP-ribosylated in pyBHK cells. Maximum incorporation of radioactivity in EF-2 occurred by 12 hr and remained constant over the subsequent 12 hr. It was estimated that 30-35% of the EF-2 was ADP-ribosylated in cells cultured in medium containing 2% serum. When 32P-labeled cultures were incubated in medium containing unlabeled phosphate, the 32P label was lost from the EF-2 within 30 min.

Full text

PDF
555

Images in this article

555Image
on p.555
555Image
on p.555
556Image
on p.556
556Image
on p.556
556Image
on p.556

Selected References

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

  1. Brown B. A., Bodley J. W. Primary structure at the site in beef and wheat elongation factor 2 of ADP-ribosylation by diphtheria toxin. FEBS Lett. 1979 Jul 15;103(2):253–255. doi: 10.1016/0014-5793(79)81339-3. [DOI] [PubMed] [Google Scholar]
  2. Collier R. J., Cole H. A. Diphtheria toxin subunit active in vitro. Science. 1969 Jun 6;164(3884):1179–1181. doi: 10.1126/science.164.3884.1179. [DOI] [PubMed] [Google Scholar]
  3. Collier R. J. Diphtheria toxin: mode of action and structure. Bacteriol Rev. 1975 Mar;39(1):54–85. doi: 10.1128/br.39.1.54-85.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Hassell J. A., Engelhardt D. L. The regulation of protein synthesis in animal cells by serum factors. Biochemistry. 1976 Apr 6;15(7):1375–1381. doi: 10.1021/bi00652a004. [DOI] [PubMed] [Google Scholar]
  5. Hassell J. A., Engelhardt D. L. Translational inhibition in extracts from serum-deprived animal cells. Biochim Biophys Acta. 1973 Nov 14;324(4):545–553. doi: 10.1016/0005-2787(73)90213-x. [DOI] [PubMed] [Google Scholar]
  6. Henriksen O., Smulson M. E. Function and properties of aminoacyl transferase II. II. Subcellular distribution during asynchronous and synchronous growth. Arch Biochem Biophys. 1972 May;150(1):175–182. doi: 10.1016/0003-9861(72)90024-0. [DOI] [PubMed] [Google Scholar]
  7. Hershko A., Mamont P., Shields R., Tomkins G. M. "Pleiotypic response". Nat New Biol. 1971 Aug;232(33):206–211. [PubMed] [Google Scholar]
  8. Honjo T., Nishizuka Y., Hayaishi O. Diphtheria toxin-dependent adenosine diphosphate ribosylation of aminoacyl transferase II and inhibition of protein synthesis. J Biol Chem. 1968 Jun 25;243(12):3553–3555. [PubMed] [Google Scholar]
  9. Iglewski B. H., Kabat D. NAD-dependent inhibition of protein synthesis by Pseudomonas aeruginosa toxin,. Proc Natl Acad Sci U S A. 1975 Jun;72(6):2284–2288. doi: 10.1073/pnas.72.6.2284. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Iglewski W. J., Lee H., Muller P. ADP-ribosyltransferase from beef liver which ADP-ribosylates elongation factor-2. FEBS Lett. 1984 Jul 23;173(1):113–118. doi: 10.1016/0014-5793(84)81028-5. [DOI] [PubMed] [Google Scholar]
  11. Iglewski W. J., Lee H. Purification and properties of an altered form of elongation factor 2 from mutant cells resistant to intoxication by diphtheria toxin. Eur J Biochem. 1983 Aug 1;134(2):237–240. doi: 10.1111/j.1432-1033.1983.tb07556.x. [DOI] [PubMed] [Google Scholar]
  12. Iglewski W. J., Lee H. Purification and properties of an altered form of elongation factor 2 from mutant cells resistant to intoxication by diphtheria toxin. Eur J Biochem. 1983 Aug 1;134(2):237–240. doi: 10.1111/j.1432-1033.1983.tb07556.x. [DOI] [PubMed] [Google Scholar]
  13. Kaminskas E. Serum-mediated stimulation of protein synthesis in Ehrlich ascites tumor cells. J Biol Chem. 1972 Sep 10;247(17):5470–5476. [PubMed] [Google Scholar]
  14. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  15. Lee H., Iglewski W. J. Cellular ADP-ribosyltransferase with the same mechanism of action as diphtheria toxin and Pseudomonas toxin A. Proc Natl Acad Sci U S A. 1984 May;81(9):2703–2707. doi: 10.1073/pnas.81.9.2703. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Moldave K. Eukaryotic protein synthesis. Annu Rev Biochem. 1985;54:1109–1149. doi: 10.1146/annurev.bi.54.070185.005333. [DOI] [PubMed] [Google Scholar]
  17. Oppenheimer N. J., Bodley J. W. Diphtheria toxin. Site and configuration of ADP-ribosylation of diphthamide in elongation factor 2. J Biol Chem. 1981 Aug 25;256(16):8579–8581. [PubMed] [Google Scholar]
  18. Oppermann H., Levinson A. D., Varmus H. E., Levintow L., Bishop J. M. Uninfected vertebrate cells contain a protein that is closely related to the product of the avian sarcoma virus transforming gene (src). Proc Natl Acad Sci U S A. 1979 Apr;76(4):1804–1808. doi: 10.1073/pnas.76.4.1804. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. 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]
  20. Payne D. M., Jacobson E. L., Moss J., Jacobson M. K. Modification of proteins by mono(ADP-ribosylation) in vivo. Biochemistry. 1985 Dec 17;24(26):7540–7549. doi: 10.1021/bi00347a006. [DOI] [PubMed] [Google Scholar]
  21. 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]
  22. Sayhan O., Ozdemirli M., Nurten R., Bermek E. On the nature of cellular ADP-ribosyltransferase from rat liver specific for elongation factor 2. Biochem Biophys Res Commun. 1986 Sep 30;139(3):1210–1214. doi: 10.1016/s0006-291x(86)80306-0. [DOI] [PubMed] [Google Scholar]
  23. Sitikov A. S., Davydova E. K., Ovchinnikov L. P. Endogenous ADP-ribosylation of elongation factor 2 in polyribosome fraction of rabbit reticulocytes. FEBS Lett. 1984 Oct 15;176(1):261–263. doi: 10.1016/0014-5793(84)80953-9. [DOI] [PubMed] [Google Scholar]
  24. Soeiro R., Amos H. Arrested protein synthesis in polysomes of cultured chick embryo cells. Science. 1966 Nov 4;154(3749):662–665. doi: 10.1126/science.154.3749.662. [DOI] [PubMed] [Google Scholar]
  25. Sokol P. A., Iglewski B. H., Hager T. A., Sadoff J. C., Cross A. S., McManus A., Farber B. F., Iglewski W. J. Production of exoenzyme S by clinical isolates of Pseudomonas aeruginosa. Infect Immun. 1981 Oct;34(1):147–153. doi: 10.1128/iai.34.1.147-153.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Ueda K., Hayaishi O. ADP-ribosylation. Annu Rev Biochem. 1985;54:73–100. doi: 10.1146/annurev.bi.54.070185.000445. [DOI] [PubMed] [Google Scholar]
  27. Van Ness B. G., Barrowclough B., Bodley J. W. Recognition of elongation factor 2 by diphtheria toxin is not solely defined by the presence of diphthamide. FEBS Lett. 1980 Oct 20;120(1):4–6. doi: 10.1016/0014-5793(80)81032-5. [DOI] [PubMed] [Google Scholar]
  28. 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]
  29. Vasil M. L., Kabat D., Iglewski B. H. Structure-activity relationships of an exotoxin of Pseudomonas aeruginosa. Infect Immun. 1977 Apr;16(1):353–361. doi: 10.1128/iai.16.1.353-361.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES