Skip to main content
SpringerLink
Log in

Purification and Biochemical Characterization of Methionine Aminopeptidase (MetAP) from Mycobacterium smegmatis mc2155

  • Published:
Applied Biochemistry and Biotechnology Aims and scope Submit manuscript

Abstract

The methionine aminopeptidase (MetAP) catalyzes the removal of amino terminal methionine from newly synthesized polypeptide. MetAP from Mycobacterium smegmatis mc2 155 was purified from the culture lysate in four sequential steps to obtain a final purification fold of 22. The purified enzyme exhibited a molecular weight of ≈ 37 kDa on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Activity staining was performed to detect the methionine aminopeptidase activity on native polyacrylamide gel. The enzyme was characterized biochemically, using l-methionine p-nitroanilide as substrate. The enzyme was found to have a temperature and pH optimum of 50 °C and 8.5, respectively, and was found to be stable at 50 °C with half-life more than 8 h. The enzyme activity was enhanced by Mg2+ and Co2+ and was inhibited by Fe2+ and Cu2+. The enzyme activity inhibited by EDTA is restored in presence of Mg2+ suggesting the possible role of Mg2+ as metal cofactor of the enzyme in vitro.

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

Fig. 1
Fig. 2
Fig. 3

Similar content being viewed by others

References

  1. Addlagatta, A., Quillin, M. L., Omotoso, O., Liu, J. O., & Matthews, B. W. (2005). Biochemistry, 44, 7166–7174.

    Article  CAS  Google Scholar 

  2. Vaughan, M. D., Sampson, P. B., & Honek, J. F. (2002). Current Medicinal Chemistry, 9, 385–409.

    CAS  Google Scholar 

  3. Flores, Y. M., Reyes, Y. N., Zavala, B. R., Rodriguez, C. H., & Tanaca, L. V. (2004). FEMS Microbiology Letters, 234, 247–253.

    Article  Google Scholar 

  4. Li, X., & Chang, Y. H. (1995). Proceedings of the National Academy of Sciences of the United States of America, 92, 12357–12361.

    Article  CAS  Google Scholar 

  5. Miller, C. G., Strauch, K. L., Kukral, A. M., Miller, J. L., Wingfield, P. T., Mazzei, G. J., et al. (1987). Proceedings of the National Academy of Sciences of the United States of America, 84, 2718–2722.

    Article  CAS  Google Scholar 

  6. Chang, S. Y. P., McGary, E. C., & Chang, S. (1989). Journal of Bacteriology, 171, 4071–4072.

    CAS  Google Scholar 

  7. Sassetti, C. M., Boyd, D. H., & Rubin, E. J. (2003). Molecular Microbiology, 48, 77–84.

    Article  CAS  Google Scholar 

  8. Lowther, W. T., & Matthews, B. W. (2000). Biochimica et Biophysica Acta, 1477, 157–167.

    CAS  Google Scholar 

  9. Parish, T., & Stoker, G. K. (1998). In T. Parish, & G.K. Stoker (Eds.), Methods in molecular biology, vol. 101. Mycobacteria Protocols pp. 1–10. Totowa, NJ: Humana.

    Google Scholar 

  10. Frottin, F., Martinez, A., Peynot, P., Mitra, S., Holz, R. C., Giglione, C., et al. (2006). Molecular and Cellular Proteomics, 5, 2336–2349.

    Article  CAS  Google Scholar 

  11. Evdokimov, A. G., Pokross, M., & Walter, R. L. (2007). Proteins Structure Function and Bioinformatics, 66, 538–546.

    Article  CAS  Google Scholar 

  12. Li, J. Y., Chen, L. L., Cui, Y. M., Luo, Q. L., Li, J., Nan, F. J., et al. (2003). Biochemical and Biophysical Research Communications, 307, 172–179.

    Article  CAS  Google Scholar 

  13. Gadagkar, R., & Gopinathan, K. P. (1980). Journal of Biosciences, 2, 337–348.

    Article  CAS  Google Scholar 

  14. Laemmli, U. K. (1970). Nature, 227, 680–685.

    Article  CAS  Google Scholar 

  15. Božić , N., & Vujčić , Z. (2005). Electrophoresis, 26, 2476–2480.

    Article  CAS  Google Scholar 

  16. Nampoothiri, K. M., Nagy, V., Kovacs, K., Szakacs, G., & Pandey, A. (2005). Letters in Applied Microbiology, 41, 498–504.

    Article  CAS  Google Scholar 

  17. Karadzic, I., Izrael, L., Gojgic-Cvijovic, G., & Vujcic, Z. (2002). Journal of Bioscience and Bioengineering, 94, 309–314.

    Article  CAS  Google Scholar 

  18. Reese, E. T., & Maguire, A. (1969). Applied and Environmental Microbiology, 17, 242–245.

    CAS  Google Scholar 

  19. Etienne, G., Laval, F., Villeneuve, C., Dinadayala, P., Abouwarda, A., Zerbib, D., et al. (2005). Microbiology, 151, 2075–2086.

    Article  CAS  Google Scholar 

  20. Saxena, R., & Chakraborti, P. K. (2005). Biochemical and Biophysical Research Communications, 332, 418–425.

    CAS  Google Scholar 

  21. Nagy, V., Nampoothiri, K. M., Pandey, A., Rahulan, R., & Szakacs, G. (2008). Journal of Applied Microbiology, 104, 380–387.

    CAS  Google Scholar 

  22. Boufous, H., & Vadeboncoeur, C. (2003). Biochimie, 85, 993–997.

    Article  CAS  Google Scholar 

  23. Kwon, C., Park, J., & Cho, J. M. (1996). Journal of Industrial Microbiology & Biotechnology, 17, 30–35.

    CAS  Google Scholar 

  24. Chung, J. M., Chung, I. Y., & Lee, Y. S. (2002). Journal of Biochemistry and Molecular Biology, 35, 228–235.

    CAS  Google Scholar 

  25. Chang, Y. H., Teichert, U., & Smith, J. A. (1990). Journal of Biological Chemistry, 265, 19892–19897.

    CAS  Google Scholar 

  26. Kendall, R. L., & Bradshaw, R. A. (1992). Journal of Biological Chemistry, 267, 20667–20673.

    CAS  Google Scholar 

  27. Tsunasawa, S., Izu, Y., Miyagi, M., & Kato, I. (1997). Journal of Biochemistry, 122, 843–850.

    CAS  Google Scholar 

  28. Wang, J., Sheppard, G. S., Lou, P., Kawai, M., Park, C., Egan, D. A., et al. (2003). Biochemistry, 42, 5035–5042.

    Article  CAS  Google Scholar 

  29. Papir, G., Spungin-bialik, A., Ben-Meir, D., Fudim, E., & Gilboa, R. (1998). European Journal of Biochemistry, 258, 313–319.

    Article  CAS  Google Scholar 

  30. Walker, K. W., & Bradshaw, R. A. (1998). Protein Science, 7, 2684–2687.

    Article  CAS  Google Scholar 

  31. Chen, D. Z., Patel, D. V., Hackbarth, C. J., Wang, W., Dreyer, G., Young, D. C., et al. (2000). Biochemistry, 39, 1256–1262.

    Article  CAS  Google Scholar 

  32. Migita, K., & Nishimura, T. (2006). Bioscience, Biotechnology, and Biochemistry, 70, 1110–1117.

    Article  CAS  Google Scholar 

  33. Lee, G. D., Chun, S. S., Kho, Y. H., & Chun, H. K. (1998). Journal of Applied Microbiology, 84, 561–566.

    Article  Google Scholar 

Download references

Acknowledgement

The corresponding author is thankful to DBT, New Delhi for the financial support

Author information

Authors and Affiliations

  1. Biotechnology Division, National Institute for Interdisciplinary Science and Technology (CSIR), Trivandrum, 695019, Kerala, India

    Sai Shyam Narayanan, Ajeena Ramanujan, Shyam Krishna & Kesavan Madhavan Nampoothiri

Authors
  1. Sai Shyam Narayanan
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Ajeena Ramanujan
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Shyam Krishna
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Kesavan Madhavan Nampoothiri
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Kesavan Madhavan Nampoothiri.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Narayanan, S.S., Ramanujan, A., Krishna, S. et al. Purification and Biochemical Characterization of Methionine Aminopeptidase (MetAP) from Mycobacterium smegmatis mc2155. Appl Biochem Biotechnol 151, 512–521 (2008). https://doi.org/10.1007/s12010-008-8227-y

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12010-008-8227-y

Keywords

Search

Navigation