Skip to main content
SpringerLink
Log in

Real-time determination of the activity of ATPase by use of a water-soluble polythiophene

  • Original Paper
  • Published:
Analytical and Bioanalytical Chemistry Aims and scope Submit manuscript

Abstract

This contribution introduces a fluorescence assay for real-time determination of the activity of p97/VCP, a 540-kDa homo-hexameric enzyme, belonging to the AAA-ATPase family. A fluorescent reporter “poly 1-(3-((4-methylthiophen-3-yl)oxy)propyl)quinuclidin-1-ium” (poly PTQ) is used to monitor the hydrolysis of ATP to ADP by p97/VCP. The proposed assay relies on the different strength of coordination of ATP and ADP to the polymer backbone. We used recovery of fluorescence intensity on addition of p97/VCP to a poly PTQ/ATP solution to determine the enzymatic activity. The kinetic data K m and V max were 0.30 mmol L−1 ATP and 0.134 nmol ATP min−1 μg−1 enzyme, respectively. The specificity of the assay was investigated by using an unhydrolyzable ATP analogue and sensitivity against p97 mutagenesis was further examined by detection of the activity of wild type and truncated p97/VCP. Our study demonstrates that determination of the real-time activity of p97/VCP is possible, because of the superior sensitivity and very fast optical response of poly PTQ.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Scheme 1
Fig. 1
Fig. 2
Fig. 3
Fig. 4

Similar content being viewed by others

References

  1. Chan KM, Delfert D, Junger KD (1986) Anal Biochem 157:375–380

    Article  CAS  Google Scholar 

  2. Wang Q, Song CC, Li CCH (2003) Biochem Biophys Res Commun 300:253–260

    Article  CAS  Google Scholar 

  3. Henkel RD, Vandeberg JL, Walsh RA (1988) Anal Biochem 169:312–318

    Article  CAS  Google Scholar 

  4. Matsunaga T, Kose E, Yasuda S, Ise H, Ikeda U, Ohmori S (2006) Biol Pharm Bull 29:560–564

    Article  CAS  Google Scholar 

  5. Hanocqquertier J, Baltus E, Schram E (1988) J Biolumin Chemilumin 2:17–24

    Article  CAS  Google Scholar 

  6. Sudo J, Terui J, Iwase H, Kakuno K (2000) J Chromatogr B 744:19–23

    Article  CAS  Google Scholar 

  7. Thomas SW, Joly GD, Swager TM (2007) Chem Rev 107:1339–1386

    Article  CAS  Google Scholar 

  8. Jiang H, Taranekar P, Reynolds JR, Schanze KS (2009) Angew Chem Int Ed 48:4300–4316

    Article  CAS  Google Scholar 

  9. Ho HA, Najari A, Leclerc M (2008) Acc Chem Res 41:168–178

    Article  CAS  Google Scholar 

  10. Liu B, Bazan GC (2004) Chem Mater 16:4467–4476

    Article  CAS  Google Scholar 

  11. Ho HA, Boissinot M, Bergeron MG, Corbeil G, Dore K, Boudreau D, Leclerc M (2002) Angew Chem Int Ed 41:1548–1551

    Article  CAS  Google Scholar 

  12. Ho HA, Leclerc M (2003) J Am Chem Soc 125:4412–4413

    Article  CAS  Google Scholar 

  13. Dore K, Dubus S, Ho HA, Levesque I, Brunette M, Corbeil G, Boissinot M, Boivin G, Bergeron MG, Boudreau D, Leclerc M (2004) J Am Chem Soc 126:4240–4244

    Article  CAS  Google Scholar 

  14. Ho HA, Leclerc M (2004) J Am Chem Soc 126:1384–1387

    Article  CAS  Google Scholar 

  15. Tang YL, Feng F, He F, Wang S, Li Y, Zhu DB (2006) J Am Chem Soc 128:14972–14976

    Article  CAS  Google Scholar 

  16. Yao ZY, Bai H, Li C, Shi GQ (2010) Chem Commun 46:5094–5096

    Article  CAS  Google Scholar 

  17. Yao ZY, Feng XL, Hong WJ, Li C, Shi GQ (2009) Chem Commun 31:4696–4698

    Article  Google Scholar 

  18. Yao ZY, Feng XL, Li C, Shi GQ (2009) Chem Commun 39:5886–5888

    Article  Google Scholar 

  19. Nilsson KPR, Rydberg J, Baltzer L, Inganäs O (2003) Proc Natl Acad Sci U S A 100:10170–10174

    Article  CAS  Google Scholar 

  20. Nilsson KPR, Rydberg J, Baltzer L, Inganäs O (2004) Proc Natl Acad Sci U S A 101:11197–11202

    Article  CAS  Google Scholar 

  21. Nilsson KPR, Inganäs O (2003) Nat Mater 2:419–410

    Article  CAS  Google Scholar 

  22. Aberem MB, Najari A, Ho HA, Gravel JF, Nobert P, Boudreau D, Leclerc M (2006) Adv Mater 18:2703–2707

    Article  CAS  Google Scholar 

  23. Li C, Numata M, Takeuchi M, Shinkai S (2005) Angew Chem Int Ed 44:6371–6374

    Article  CAS  Google Scholar 

  24. Chen LH, McBranch DW, Wang HL, Helgeson R, Wudl F, Whitten DG (1999) Proc Natl Acad Sci U S A 96:12287–12292

    Article  CAS  Google Scholar 

  25. Pinto MR, Schanze KS (2004) Proc Natl Acad Sci U S A 101:7505–7510

    Article  CAS  Google Scholar 

  26. Tang YL, Teng F, Yu MH, An LL, He F, Wang S, Li YL, Zhu DB, Bazan GC (2008) Adv Mater 20:703–705

    Article  CAS  Google Scholar 

  27. Liu Y, Schanze KS (2009) Anal Chem 81:231–239

    Article  Google Scholar 

  28. Wang YY, Zhang Y, Liu B (2010) Anal Chem 82:8604–8610

    Article  CAS  Google Scholar 

  29. Feng FD, Liu LB, Yang QO, Wang S (2010) Macromol Rapid Commun 31:1405–1421

    Article  CAS  Google Scholar 

  30. Zheng W, He L (2009) J Am Chem Soc 131:3432–3433

    Article  CAS  Google Scholar 

  31. Yildiz UH (2009) Fluorescent Ionene-Dye Nanoparticles by Electrostatic Self-Assembly. Mainz, (http://ubm.opus.hbz-nrw.de/volltexte/2009/1902/)

  32. Lakowicz JR (1999) Principles of Fluorescence Spectroscopy, 2nd edn. Kluwer Academic/Plenum Publisher, New York

    Google Scholar 

  33. Song CC, Wang Q, Li CCH (2003) J Biol Chem 278:3648–3655

    Article  CAS  Google Scholar 

  34. Bednarek SY, Park S, Rancour DM (2007) J Biol Chem 282:5217–5224

    Google Scholar 

  35. Hanson PI, Dalal S, Rosser MFN, Cry DM (2004) Mol Biol Cell 15:637–648

    Google Scholar 

  36. Latterich M, Halawani D, Leblanc AC, Rouiller I, Michnick SW, Servant MJ (2009) Mol Cell Biol 29:4484–4494

    Article  Google Scholar 

  37. DeLaBarre B, Christianson JC, Kopito RR, Brunger AT (2006) Molecular Cell 22:451–462

    Article  CAS  Google Scholar 

Download references

Acknowledgments

The School of Materials Science and Engineering and the provost office, NTU, are acknowledged for strategic funding to support the Centre for Biomimetic Sensor Science.

Author information

Authors and Affiliations

  1. Center for Biomimetic Sensor Science, Nanyang Technological University, 50 Nanyang Drive, Singapore, 637553, Singapore

    Umit Hakan Yildiz & Bo Liedberg

  2. Division of Chemical Biology, School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore, 637551, Singapore

    Chia Wei Sheng, Diyar Mailepessov, Diana Chia Xueqi & Susana Geifman Shochat

Authors
  1. Umit Hakan Yildiz
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Chia Wei Sheng
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Diyar Mailepessov
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Diana Chia Xueqi
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Susana Geifman Shochat
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Bo Liedberg
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Bo Liedberg.

Electronic supplementary material

Below is the link to the electronic supplementary material.

ESM 1

(PDF 441 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Yildiz, U.H., Sheng, C.W., Mailepessov, D. et al. Real-time determination of the activity of ATPase by use of a water-soluble polythiophene. Anal Bioanal Chem 404, 2369–2375 (2012). https://doi.org/10.1007/s00216-012-6341-8

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00216-012-6341-8

Keywords

Search

Navigation