Display options
Cite
Bhosle SM, Hunt A, Chaudhary J. A Modified Coupled Spectrophotometric Method to Detect 2-5 Oligoadenylate Synthetase Activity in Prostate Cell Lines. Biol Proced Online. 2016;18:9doi: 10.1186/s12575-016-0038-x.
Bhosle, S. M., Hunt, A., & Chaudhary, J. (2016). A Modified Coupled Spectrophotometric Method to Detect 2-5 Oligoadenylate Synthetase Activity in Prostate Cell Lines. Biological procedures online, 189. https://doi.org/10.1186/s12575-016-0038-x
Bhosle, Sushma M, et al. "A Modified Coupled Spectrophotometric Method to Detect 2-5 Oligoadenylate Synthetase Activity in Prostate Cell Lines." Biological procedures online vol. 18 (2016): 9. doi: https://doi.org/10.1186/s12575-016-0038-x
Bhosle SM, Hunt A, Chaudhary J. A Modified Coupled Spectrophotometric Method to Detect 2-5 Oligoadenylate Synthetase Activity in Prostate Cell Lines. Biol Proced Online. 2016 Mar 17;18:9. doi: 10.1186/s12575-016-0038-x. eCollection 2016. PMID: 26997919; PMCID: PMC4797170.
Copy Download .nbib Format: NLM
Share it on

Biol Proced Online. 2016 Mar 17;18:9. doi: 10.1186/s12575-016-0038-x. eCollection 2016.

A Modified Coupled Spectrophotometric Method to Detect 2-5 Oligoadenylate Synthetase Activity in Prostate Cell Lines.

Biological procedures online

Sushma M Bhosle, Aisha Hunt, Jaideep Chaudhary

Affiliations

  1. Department of Biological Sciences, Centre for Cancer Research and Therapeutic Development, Clark Atlanta University, Atlanta, GA 30314 USA.

PMID: 26997919 PMCID: PMC4797170 DOI: 10.1186/s12575-016-0038-x

Abstract

BACKGROUND: 2'-5' oligoadenylate synthetases (OAS) are interferon inducible enzymes that polymerizes ATP to 2'-5'-linked oligomers of adenylate (2-5As). As part of the innate immune response, these enzymes are activated by viral double stranded RNA or mRNAs with significant double stranded structure. The 2-5As in turn activate RNaseL that degrade single stranded RNAs. Three distinct forms of OAS exist in human cells (OAS1, 2 and 3) with each form having multiple spliced variants. The OAS enzymes and their spliced variants have different enzyme activities. OAS enzymes also play a significant role in regulating multiple cellular processes such as proliferation and apoptosis. Moreover, Single nucleotide polymorphisms that alter OAS activity are also associated with viral infection, diabetes and cancer. Thus detection of OAS enzyme activity with a simple spectrophotometric method in cells will be important in clinical research.

RESULTS: Here we propose a modified coupled spectrophotometric assay to detect 2-5 oligoadenylate synthetase (OAS) enzyme activity in prostate cell lines as a model system. The OAS enzyme from prostate cancer cell lysates was purified using Polyinosinic: polycytidylic acid (poly I:C) bound activated sepharose beads. The activated OAS enzyme eluted from Sepharose beads showed expression of p46 isoform of OAS1, generally considered the most abundant OAS isoform in elutes from DU14 cell line but not in other prostate cell line. In this assay the phosphates generated by the OAS enzymatic reaction is coupled with conversion of the substrate 2-amino-6-mercapto-7-methylpurine ribonucleoside (methylthioguanosine, a guanosine analogue; MESG) to a purine base product, 2-amino-6-mercapto-7-methylpurine and ribose1-phosphate via a catalyst purine nucleoside phosphorylase (phosphorylase) using a commercially available pyrophosphate kit. The absorbance of the purine base product is measured at 360 nm. The higher levels of phosphates detected in DU145 cell line indicates more activity of OAS in this prostate cancer cell line.

CONCLUSION: The modified simple method detected OAS enzyme activity with sensitivity and specificity, which could help in detection of OAS enzymes avoiding the laborious and radioactive methods.

Keywords: 2-5 oligoadenylate synthetase; Prostate cancer; Pyrophosphate assay

References

  1. Nucleic Acids Res. 1995 Aug 11;23(15):2886-92 - PubMed
  2. Diabetes. 2005 May;54(5):1588-91 - PubMed
  3. Cell. 1979 Jun;17(2):255-64 - PubMed
  4. Anal Biochem. 1996 Dec 1;243(1):41-5 - PubMed
  5. Virology. 1979 Apr 30;94(2):282-96 - PubMed
  6. Proc Natl Acad Sci U S A. 1978 Jan;75(1):256-60 - PubMed
  7. Biochimie. 2007 Jun-Jul;89(6-7):779-88 - PubMed
  8. Am J Hum Genet. 2005 Apr;76(4):623-33 - PubMed
  9. FEBS Lett. 1978 Nov 15;95(2):257-64 - PubMed
  10. Anal Biochem. 1976 Dec;76(2):423-30 - PubMed
  11. Genes Immun. 2003 Sep;4(6):411-9 - PubMed
  12. Nature. 1979 Mar 29;278(5703):471-3 - PubMed
  13. Proc Natl Acad Sci U S A. 1992 Jun 1;89(11):4884-7 - PubMed
  14. Proc Natl Acad Sci U S A. 2005 Oct 11;102(41):14533-8 - PubMed
  15. J Virol. 2010 Nov;84(22):11898-904 - PubMed
  16. Science. 1981 May 29;212(4498):1030-2 - PubMed
  17. J Biol Chem. 1979 Dec 10;254(23):12034-7 - PubMed
  18. Eur J Biochem. 1978 Mar;84(1):149-59 - PubMed
  19. J Biol Chem. 1987 Mar 15;262(8):3852-7 - PubMed
  20. Cell Growth Differ. 1996 Aug;7(8):969-78 - PubMed
  21. Nature. 1981 Jan 29;289(5796):414-7 - PubMed
  22. J Biol Chem. 1995 Feb 24;270(8):4133-7 - PubMed
  23. Anal Biochem. 1992 Nov 15;207 (1):90-3 - PubMed
  24. J Infect Dis. 2005 Nov 15;192(10 ):1741-8 - PubMed
  25. Oncogene. 2005 Aug 18;24(35):5492-501 - PubMed
  26. Nat Chem Biol. 2009 Mar;5(3):174-82 - PubMed
  27. Cancer. 2011 Dec 15;117(24):5509-18 - PubMed
  28. Biochem Biophys Res Commun. 2005 Apr 22;329(4):1234-9 - PubMed
  29. Liver Int. 2009 Oct;29(9):1413-21 - PubMed
  30. Nucleic Acids Res. 1980 Jul 25;8(14 ):3073-85 - PubMed
  31. Tissue Antigens. 2006 Nov;68(5):446-9 - PubMed
  32. Cell. 1978 Mar;13(3):565-72 - PubMed
  33. Cell. 1993 Mar 12;72(5):753-65 - PubMed
  34. J Biol Chem. 1981 Aug 10;256(15):7806-11 - PubMed
  35. Apoptosis. 2006 May;11(5):725-38 - PubMed
  36. Anal Biochem. 1995 Sep 1;230(1):55-61 - PubMed
  37. Anal Biochem. 2010 Sep 1;404(1):56-63 - PubMed
  38. Genomics. 2003 Nov;82(5):537-52 - PubMed

Publication Types

Grant support