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. 1987 May;6(5):1273–1280. doi: 10.1002/j.1460-2075.1987.tb02364.x

Identification of 69-kd and 100-kd forms of 2-5A synthetase in interferon-treated human cells by specific monoclonal antibodies.

A G Hovanessian, A G Laurent, J Chebath, J Galabru, N Robert, J Svab
PMCID: PMC553929  PMID: 2440675

Abstract

Recently, the existence of 40-kd and 46-kd 2-5A synthetases in interferon-treated cells has been confirmed by cloning and characterization of cDNA corresponding to these small size enzymes. By the use of specific monoclonal antibodies, we describe here two forms of high mol. wt 2-5A synthetases of 69 and 100 kd in human cells. The monoclonal antibodies immunoprecipitate either a 69- or a 100-kd 2-5A synthetase. These purified 2-5A synthetases in immune complex preparations are active, i.e. addition of poly(I).poly(C) and ATP results in the synthesis of 2-5A. Both 2-5A synthetases are composed of several subspecies with similar isoelectric points in the range of 7-8 but have different subcellular localizations: 100-kd synthetase is recovered from the microsomal pellet whereas 69-kd synthetase is found to be associated with cell membranes as well as with the microsomal pellet. Different types of interferon-treated human cells express both or either forms of these enzymes. The 69- and 100-kd 2-5A synthetases were also identified by electrophoretic transfer immunoblot analysis using rabbit polyclonal antibodies against a synthetic peptide common on both 46- and 40-kd 2-5A synthetases. These results indicate that small and large size isozymes share a common peptide sequence.

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Selected References

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

  1. Baglioni C., Minks M. A., Maroney P. A. Interferon action may be mediated by activation of a nuclease by pppA2'p5'A2'p5'A. Nature. 1978 Jun 22;273(5664):684–687. doi: 10.1038/273684a0. [DOI] [PubMed] [Google Scholar]
  2. Benech P., Merlin G., Revel M., Chebath J. 3' end structure of the human (2'-5') oligo A synthetase gene: prediction of two distinct proteins with cell type-specific expression. Nucleic Acids Res. 1985 Feb 25;13(4):1267–1281. doi: 10.1093/nar/13.4.1267. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Benech P., Mory Y., Revel M., Chebath J. Structure of two forms of the interferon-induced (2'-5') oligo A synthetase of human cells based on cDNAs and gene sequences. EMBO J. 1985 Sep;4(9):2249–2256. doi: 10.1002/j.1460-2075.1985.tb03922.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Buffet-Janvresse C., Magard H., Robert N., Hovanessian A. G. Assay and the levels of 2-5A-synthetase in lymphocytes of patients with viral, bacterial and autoimmune diseases. Ann Immunol (Paris) 1983 Sep-Oct;134D(2):247–258. doi: 10.1016/s0771-050x(83)80090-4. [DOI] [PubMed] [Google Scholar]
  5. Burnette W. N. "Western blotting": electrophoretic transfer of proteins from sodium dodecyl sulfate--polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Anal Biochem. 1981 Apr;112(2):195–203. doi: 10.1016/0003-2697(81)90281-5. [DOI] [PubMed] [Google Scholar]
  6. Clemens M. J., Williams B. R. Inhibition of cell-free protein synthesis by pppA2'p5'A2'p5'A: a novel oligonucleotide synthesized by interferon-treated L cell extracts. Cell. 1978 Mar;13(3):565–572. doi: 10.1016/0092-8674(78)90329-x. [DOI] [PubMed] [Google Scholar]
  7. Dougherty J. P., Samanta H., Farrell P. J., Lengyel P. Interferon, double-stranded RNA, and RNA degradation. Isolation of homogeneous pppA(2'p5'A)n-1 synthetase from Ehrlich ascites tumor cells. J Biol Chem. 1980 May 10;255(9):3813–3816. [PubMed] [Google Scholar]
  8. Farrell P. J., Balkow K., Hunt T., Jackson R. J., Trachsel H. Phosphorylation of initiation factor elF-2 and the control of reticulocyte protein synthesis. Cell. 1977 May;11(1):187–200. doi: 10.1016/0092-8674(77)90330-0. [DOI] [PubMed] [Google Scholar]
  9. Galabru J., Robert N., Buffet-Janvresse C., Rivière Y., Hovanessian A. G. Continuous production of interferon in normal mice: effect of anti-interferon globulin, sex, age, strain and environment on the levels of 2-5A synthetase and p67K kinase. J Gen Virol. 1985 Apr;66(Pt 4):711–718. doi: 10.1099/0022-1317-66-4-711. [DOI] [PubMed] [Google Scholar]
  10. Hovanessian A. G., Kerr I. M. The (2'-5') oligoadenylate (pppA2'-5'A2'-5'A) synthetase and protein kinase(s) from interferon-treated cells. Eur J Biochem. 1979 Feb 1;93(3):515–526. doi: 10.1111/j.1432-1033.1979.tb12850.x. [DOI] [PubMed] [Google Scholar]
  11. Hovanessian A. G., Rivière Y., Krust B. Assay of interferon-mediated protein kinase activity from plasma and tissue extracts. Anal Biochem. 1983 Mar;129(2):349–356. doi: 10.1016/0003-2697(83)90561-4. [DOI] [PubMed] [Google Scholar]
  12. Hovanessian A. G., Wood J., Meurs E., Montagnier L. Increased nuclease activity in cells treated with pppA2'p5'A2'p5' A. Proc Natl Acad Sci U S A. 1979 Jul;76(7):3261–3265. doi: 10.1073/pnas.76.7.3261. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Ilson D. H., Torrence P. F., Vilcek J. Two molecular weight forms of human 2',5'-oligoadenylate synthetase have different activation requirements. J Interferon Res. 1986 Feb;6(1):5–12. doi: 10.1089/jir.1986.6.5. [DOI] [PubMed] [Google Scholar]
  14. Kerr I. M., Brown R. E., Hovanessian A. G. Nature of inhibitor of cell-free protein synthesis formed in response to interferon and double-stranded RNA. Nature. 1977 Aug 11;268(5620):540–542. doi: 10.1038/268540a0. [DOI] [PubMed] [Google Scholar]
  15. Kerr I. M., Brown R. E. pppA2'p5'A2'p5'A: an inhibitor of protein synthesis synthesized with an enzyme fraction from interferon-treated cells. Proc Natl Acad Sci U S A. 1978 Jan;75(1):256–260. doi: 10.1073/pnas.75.1.256. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Krust B., Galabru J., Hovanessian A. G. Further characterization of the protein kinase activity mediated by interferon in mouse and human cells. J Biol Chem. 1984 Jul 10;259(13):8494–8498. [PubMed] [Google Scholar]
  17. Krust B., Rivière Y., Hovanessian A. G. p67K kinase in different tissues and plasma of control and interferon-treated mice. Virology. 1982 Jul 15;120(1):240–246. doi: 10.1016/0042-6822(82)90022-8. [DOI] [PubMed] [Google Scholar]
  18. Laurent A. G., Gruest J., Krust B., Montagnier L. Characterization of monoclonal antibody specific for human alpha interferon. Hybridoma. 1982;1(3):313–322. doi: 10.1089/hyb.1.1982.1.313. [DOI] [PubMed] [Google Scholar]
  19. Merlin G., Chebath J., Benech P., Metz R., Revel M. Molecular cloning and sequence of partial cDNA for interferon-induced (2'-5')oligo(A) synthetase mRNA from human cells. Proc Natl Acad Sci U S A. 1983 Aug;80(16):4904–4908. doi: 10.1073/pnas.80.16.4904. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Nilsen T. W., Wood D. L., Baglioni C. Presence of 2',5'-oligo(A) and of enzymes that synthesize, bind, and degrade 2',5'-oligo(A) in HeLa cell nuclei. J Biol Chem. 1982 Feb 25;257(4):1602–1605. [PubMed] [Google Scholar]
  21. O'Farrell P. H. High resolution two-dimensional electrophoresis of proteins. J Biol Chem. 1975 May 25;250(10):4007–4021. [PMC free article] [PubMed] [Google Scholar]
  22. Revel M., Kimchi A., Friedman M., Wolf D., Merlin G., Panet A., Rapoport S., Lapidot Y. Cell-regulatory functions of interferon induced enzymes: antimitogenic effect of (2'-5')oligo-A, growth-related variations in (2'-5')oligo-A synthetase, and isolation of its mRNA. Tex Rep Biol Med. 1981;41:452–462. [PubMed] [Google Scholar]
  23. Safer B. 2B or not 2B: regulation of the catalytic utilization of eIF-2. Cell. 1983 May;33(1):7–8. doi: 10.1016/0092-8674(83)90326-4. [DOI] [PubMed] [Google Scholar]
  24. Saunders M. E., Gewert D. R., Tugwell M. E., McMahon M., Williams B. R. Human 2-5A synthetase: characterization of a novel cDNA and corresponding gene structure. EMBO J. 1985 Jul;4(7):1761–1768. doi: 10.1002/j.1460-2075.1985.tb03848.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Schweiger A., Kostka G. Synthesis of 2',5'-oligoadenylate by rat liver nuclear matrix protein. Biochem Biophys Res Commun. 1984 May 16;120(3):996–1001. doi: 10.1016/s0006-291x(84)80205-3. [DOI] [PubMed] [Google Scholar]
  26. Shimizu N., Sokawa Y. (2'-5')Oligoadenylate synthetase in pig spleen: isolation and characterization. J Biochem. 1983 Nov;94(5):1421–1428. [PubMed] [Google Scholar]
  27. Shimizu N., Sokawa Y., Sokawa J. Immunological cross-reactivity between large and small (2'-5')oligoadenylate synthetases from pig cells. J Biochem. 1984 Jun;95(6):1827–1830. doi: 10.1093/oxfordjournals.jbchem.a134797. [DOI] [PubMed] [Google Scholar]
  28. Shulman L. M., Barker P. E., Hart J. T., Messer Peters P. G., Ruddle F. H. Assignment of low-molecular-weight human (2', 5')A synthetase to chromosome 11. Somat Cell Mol Genet. 1984 May;10(3):247–257. doi: 10.1007/BF01535247. [DOI] [PubMed] [Google Scholar]
  29. Smekens-Etienne M., Goldstein J., Ooms H. A., Dumont J. E. Variation of (2'-5')oligo(adenylate) synthetase activity during rat-liver regeneration. Eur J Biochem. 1983 Feb 1;130(2):269–273. doi: 10.1111/j.1432-1033.1983.tb07146.x. [DOI] [PubMed] [Google Scholar]
  30. Sokawa J., Shimizu N., Sokawa Y. Presence of (2'-5')oligoadenylate synthetase in avian erythrocytes. J Biochem. 1984 Jul;96(1):215–222. doi: 10.1093/oxfordjournals.jbchem.a134815. [DOI] [PubMed] [Google Scholar]
  31. St Laurent G., Yoshie O., Floyd-Smith G., Samanta H., Sehgal P. B., Lengyel P. Interferon action: two (2'-5')(A)n synthetases specified by distinct mRNAs in Ehrlich ascites tumor cells treated with interferon. Cell. 1983 May;33(1):95–102. doi: 10.1016/0092-8674(83)90338-0. [DOI] [PubMed] [Google Scholar]
  32. Wathelet M., Moutschen S., Cravador A., DeWit L., Defilippi P., Huez G., Content J. Full-length sequence and expression of the 42 kDa 2-5A synthetase induced by human interferon. FEBS Lett. 1986 Feb 3;196(1):113–120. doi: 10.1016/0014-5793(86)80224-1. [DOI] [PubMed] [Google Scholar]
  33. Wells J. A., Swyryd E. A., Stark G. R. An improved method for purifying 2',5'-oligoadenylate synthetases. J Biol Chem. 1984 Jan 25;259(2):1363–1370. [PubMed] [Google Scholar]
  34. Williams B. R., Saunders M. E., Willard H. F. Interferon-regulated human 2-5A synthetase gene maps to chromosome 12. Somat Cell Mol Genet. 1986 Jul;12(4):403–408. doi: 10.1007/BF01570735. [DOI] [PubMed] [Google Scholar]
  35. Yang K., Samanta H., Dougherty J., Jayaram B., Broeze R., Lengyel P. Interferons, double-stranded RNA, and RNA degradation. Isolation and characterization of homogeneous human (2'-5')(a)n synthetase. J Biol Chem. 1981 Sep 10;256(17):9324–9328. [PubMed] [Google Scholar]

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