Biochemistry (Moscow)

, Volume 75, Issue 5, pp 642–647 | Cite as

Biophysical characterization of a recombinant leucyl aminopeptidase from Bacillus kaustophilus

  • Meng-Chun Chi
  • Hui-Ping Chang
  • Gu-Gang Chang
  • Tzu-Fan Wang
  • Hsien-Bin Huang
  • Long-Liu LinEmail author


The biophysical properties of Bacillus kaustophilus leucyl aminopeptidase (BkLAP) were examined in terms of analytical ultracentrifugation, fluorescence spectroscopy, and circular dichroism. By using the analytical ultracentrifuge, we demonstrated that tetrameric BkLAP exists as the major form in solution at protein concentration of 1.5 mg/ml at pH 8.0. The native enzyme started to unfold beyond ∼1 M GdnHCl and reached an unfolded intermediate with [GdnHCl]1/2 at 1.8 M. Thermal unfolding of BkLAP was found to be highly irreversible and led to a marked formation of aggregates.

Key words

Bacillus kaustophilus leucyl aminopeptidase analytical ultracentrifuge thermal unfolding chemical denaturation 



B. kaustophilus LAP


circular dichroism


guanidine hydrochloride


leucyl aminopeptidase




nickel nitrilotriacetate




sodium dodecyl sulfate polyacrylamide gel electrophoresis


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  1. 1.
    Taylor, A. (1993) FASEB J., 7, 290–298.PubMedGoogle Scholar
  2. 2.
    Matsui, M., Fowler, J. H., and Walling, L. L. (2006) Biol. Chem., 387, 1535–1544.CrossRefPubMedGoogle Scholar
  3. 3.
    Burley, S. K., David, P. R., Taylor, A., and Lipscomb, W. (1990) Proc. Natl. Acad. Sci. USA, 87, 6878–6882.CrossRefPubMedGoogle Scholar
  4. 4.
    Strater, N., Sherrat, D. J., and Collons, S. D. (1999) EMBO J., 18, 4513–4522.CrossRefPubMedGoogle Scholar
  5. 5.
    Vogt, V. M. (1970) J. Biol. Chem., 245, 4760–4769.PubMedGoogle Scholar
  6. 6.
    Charlier, D., Kholti, A., Huysveld, N., Gigot, D., Maes, D., Thia-Toong, T. L., and Glansdorff, N. (2000) J. Mol. Biol., 302, 411–426.CrossRefPubMedGoogle Scholar
  7. 7.
    Chao, W. S., Gu, Y. Q., Pautot, V., Bray, E. A., and Walling, L. L. (1999) Plant Physiol., 120, 979–992.CrossRefPubMedGoogle Scholar
  8. 8.
    Beninga, J., Rock, K. L., and Goldberg, A. L. (1998) J. Biol. Chem., 273, 18534–18542.CrossRefGoogle Scholar
  9. 9.
    Daggett, M., and Fersht, A. R. (2003) Trends Biochem. Sci., 28, 18–25.CrossRefPubMedGoogle Scholar
  10. 10.
    Nolting, B., Golbik, R., and Fersht, A. R. (1995) Proc. Natl. Acad. Sci. USA, 92, 10668–10672.CrossRefPubMedGoogle Scholar
  11. 11.
    Plaxco, K. W., and Dobson, C. M. (1996) Curr. Opin. Struct. Biol., 6, 630–636.CrossRefPubMedGoogle Scholar
  12. 12.
    Panda, K. W., Gorovits, B. M., and Horowitz, P. M. (2000) J. Biol. Chem., 275, 63–70.CrossRefPubMedGoogle Scholar
  13. 13.
    Screerama, N., and Woody, K. W. (2004) Meth. Enzymol., 383, 318–351.CrossRefGoogle Scholar
  14. 14.
    Lin, L. L., Hsu, W. H., Wu, C. P., Chi, M. C., Chou, W. M., and Hu, H. Y. (2004) Extremophiles, 8, 79–87.CrossRefPubMedGoogle Scholar
  15. 15.
    Chi, M. C., Huang, H. B., Liu, J. S., Wang, W. C., Liang, W. C., and Lin, L. L. (2006) FEMS Microbiol. Lett., 260, 156–161.CrossRefPubMedGoogle Scholar
  16. 16.
    Chi, M. C., Liu, J. S., Wang, W. C., Lin, L. L., and Huang, H. B. (2008) Biochimie, 90, 811–819.CrossRefPubMedGoogle Scholar
  17. 17.
    Chi, M. C., Ong, P. L, Hsu, W. H., Chen, Y. H., Huang, H. B., and Lin, L. L. (2008) Int. J. Biol. Macromol., 43, 481–487.CrossRefPubMedGoogle Scholar
  18. 18.
    Laemmli, U. K. (1970) Nature, 227, 680–685.CrossRefPubMedGoogle Scholar
  19. 19.
    Brown, P. H., and Schuck, P. (2006) Biophys. J., 90, 4651–4661.CrossRefPubMedGoogle Scholar
  20. 20.
    Lunn, F. A., MacLeod, T. J., and Bearne, S. L. (2008) Biochem. J., 412, 113–121.CrossRefPubMedGoogle Scholar
  21. 21.
    Loveridge, E. J., Rodriguez, R. J., Swanwick, R. S., and Allemann, R. K. (2009) Biochemistry, 48, 5822–5933.CrossRefGoogle Scholar
  22. 22.
    Nakagawa, Y., Saburi, W., Takada, M., Hatada, Y., and Horikoshi, K. (2008) Biochim. Biophys. Acta, 1784, 2004–2011.PubMedGoogle Scholar
  23. 23.
    Gu, Y. Q., Pautot, V., Holzer, F. M., and Walling, L. L. (1996) Plant Physiol., 110, 1257–1266.PubMedGoogle Scholar
  24. 24.
    Gu, Y. Q., Holzer, F. M., and Walling, L. L. (1999) Eur. J. Biochem., 263, 726–735.CrossRefPubMedGoogle Scholar
  25. 25.
    Herrera-Camacho, I., Rosas-Murrieta, N. H., Rolo-Domingguez, A., Millan, L., Reyes-Leyva, J., Santos-Lopez, G., and Suarez-Rendueles, P. (2007) FEBS J., 274, 6228–6240.CrossRefPubMedGoogle Scholar
  26. 26.
    Gallagher, S. R. (2001) in Curr. Protoc. Protein Sci., Chap. 6, Unit 6.5, John Wiley & Sons, New York.Google Scholar
  27. 27.
    Shriver, J. W., and Edmondson, S. P. (2009) Meth. Mol. Biol., 490, 57–82.Google Scholar
  28. 28.
    Gruber, C. W., Cemazar, M., Mechler, A., Martin, L. L., and Crail, D. J. (2009) Peptide Sci., 92, 35–43.Google Scholar
  29. 29.
    Dengra-Pozo, J., Martinez-Rodriguez, S., Contreras, L. M., Prieto, J., Andujar-Sanchez, M., Clemente-Jimenez, J. M., Las Heras-Vazquez, F. J., Rodriguez-Vico, F., and Neira, J. L. (2009) Biopolymers, 91, 757–772.CrossRefGoogle Scholar
  30. 30.
    Freire, E., van Osdol, W. W., Mayorga, O. L., and Sanchez-Ruiz, J. M. (1990) Annu. Rev. Biophys. Biophys. Chem., 19, 159–188.CrossRefPubMedGoogle Scholar
  31. 31.
    Sanchez-Ruiz, J. M. (1992) Biophys. J., 61, 921–935.CrossRefPubMedGoogle Scholar
  32. 32.
    Tello-Solis, S. R., and Hernandez-Arana, A. (1995) Biochem. J., 311, 969–974.PubMedGoogle Scholar

Copyright information

© Pleiades Publishing, Ltd. 2010

Authors and Affiliations

  • Meng-Chun Chi
    • 1
  • Hui-Ping Chang
    • 1
  • Gu-Gang Chang
    • 2
  • Tzu-Fan Wang
    • 3
  • Hsien-Bin Huang
    • 3
  • Long-Liu Lin
    • 1
    Email author
  1. 1.Department of Applied ChemistryNational Chiayi UniversityChiayiTaiwan
  2. 2.Department of Life Sciences and Institute of Genome ScienceNational Yang-Ming UniversityTaipeiTaiwan
  3. 3.Department of Life Sciences and Institute of Molecular BiologyNational Chung Cheng UniversityChiayiTaiwan

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