Skip to main content
NCBI home page
Biochemical Journal logoLink to Biochemical Journal
. 1987 Dec 15;248(3):859–864. doi: 10.1042/bj2480859

ADP-ribosyltransferase from Helix pomatia. Purification and characterization.

H J Burtscher 1, H Klocker 1, R Schneider 1, B Auer 1, M Hirsch-Kauffmann 1, M Schweiger 1
PMCID: PMC1148628  PMID: 3124818

Abstract

ADP-ribosyltransferases from several higher eukaryotes have been purified and characterized, but little is known about ADP-ribosyltransferases in lower eukaryotes. We have purified an ADP-ribosyltransferase (EC 2.4.2.30) from Helix pomatia. The enzyme has an apparent Km of 26.7 microM. Optimal conditions for the enzyme reaction are 17.5 degrees C and pH 8. The time course is linear during the first 10 min of the reaction. The enzyme is capable of poly-ADP-ribosylation. The most highly purified preparation shows one major band at an Mr of 75,000 on electrophoresis in an SDS/polyacrylamide gel, with minor bands at Mr 115,000 and 155,000. Re-activation of SDS/polyacrylamide gels in situ shows the 75,000-Mr band to be enzymically active and additional active bands with Mr values of 115,000, 90,000 and 87,000 respectively. The 115,000-Mr and 75,000-Mr bands cross-react with a polyclonal affinity-purified antiserum against human ADP-ribosyltransferase. Like enzymes from higher eukaryotes, the activity from Helix pomatia is inhibited by thymidine, theophylline, theobromine nicotinamide, 3-methoxybenzamide and 3-aminobenzamide, and is dependent on histone and DNA.

Full text

PDF
859

Images in this article

861Fig. 1.
on p.861
862Fig. 2.
on p.862
862Fig. 3.
on p.862

Selected References

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

  1. Ansorge W. Fast and sensitive detection of protein and DNA bands by treatment with potassium permanganate. J Biochem Biophys Methods. 1985 May;11(1):13–20. doi: 10.1016/0165-022x(85)90037-5. [DOI] [PubMed] [Google Scholar]
  2. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  3. Burtscher H. J., Auer B., Klocker H., Schweiger M., Hirsch-Kauffmann M. Isolation of ADP-ribosyltransferase by affinity chromatography. Anal Biochem. 1986 Feb 1;152(2):285–290. doi: 10.1016/0003-2697(86)90410-0. [DOI] [PubMed] [Google Scholar]
  4. Carter S. G., Berger N. A. Purification and characterization of human lymphoid poly(adenosine diphosphate ribose) polymerase. Biochemistry. 1982 Oct 26;21(22):5475–5481. doi: 10.1021/bi00265a015. [DOI] [PubMed] [Google Scholar]
  5. Cleaver J. E., Morgan W. F. Poly(ADP-ribose) synthesis is involved in the toxic effects of alkylating agents but does not regulate DNA repair. Mutat Res. 1985 Jun-Jul;150(1-2):69–76. doi: 10.1016/0027-5107(85)90102-2. [DOI] [PubMed] [Google Scholar]
  6. Creissen D., Shall S. Regulation of DNA ligase activity by poly(ADP-ribose). Nature. 1982 Mar 18;296(5854):271–272. doi: 10.1038/296271a0. [DOI] [PubMed] [Google Scholar]
  7. DAVIS B. J. DISC ELECTROPHORESIS. II. METHOD AND APPLICATION TO HUMAN SERUM PROTEINS. Ann N Y Acad Sci. 1964 Dec 28;121:404–427. doi: 10.1111/j.1749-6632.1964.tb14213.x. [DOI] [PubMed] [Google Scholar]
  8. 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]
  9. Merril C. R., Goldman D., Sedman S. A., Ebert M. H. Ultrasensitive stain for proteins in polyacrylamide gels shows regional variation in cerebrospinal fluid proteins. Science. 1981 Mar 27;211(4489):1437–1438. doi: 10.1126/science.6162199. [DOI] [PubMed] [Google Scholar]
  10. Oka J., Ueda K., Hayaishi O. Snake venom phosphodiesterase: simple purification with Blue Sepharose and its application to poly(ADP-ribose) study. Biochem Biophys Res Commun. 1978 Feb 28;80(4):841–848. doi: 10.1016/0006-291x(78)91321-9. [DOI] [PubMed] [Google Scholar]
  11. Pekala P. H., Moss J. Poly ADP-ribosylation of protein. Curr Top Cell Regul. 1983;22:1–49. doi: 10.1016/b978-0-12-152822-5.50005-1. [DOI] [PubMed] [Google Scholar]
  12. Scovassi A. I., Stefanini M., Bertazzoni U. Catalytic activities of human poly(ADP-ribose) polymerase from normal and mutagenized cells detected after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. J Biol Chem. 1984 Sep 10;259(17):10973–10977. [PubMed] [Google Scholar]
  13. Tanuma S., Johnson G. S. ADP-ribosylation of nonhistone high mobility group proteins in intact cells. J Biol Chem. 1983 Apr 10;258(7):4067–4070. [PubMed] [Google Scholar]
  14. 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]
  15. Zahradka P., Ebisuzaki K. A shuttle mechanism for DNA-protein interactions. The regulation of poly(ADP-ribose) polymerase. Eur J Biochem. 1982 Oct;127(3):579–585. [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES