Journal of Protein Chemistry

, Volume 20, Issue 6, pp 469–477 | Cite as

Purification and Characterization of a New Plant Endopeptidase Isolated from Latex of Asclepias fruticosa L. (Asclepiadaceae)

  • Sebastián A. Trejo
  • Laura M. I. López
  • Cecilia V. Cimino
  • Néstor O. Caffini
  • Claudia L. Natalucci


Asclepias fruticosa L. is a small shrub containing latex with proteolytic activity. The crude extract (latex diluted 1:250 and ultracentrifuged) contained 276 μg of protein/mL and the proteolytic activity reached 1.2 caseinolytic U/mL. This enzyme preparation was very stable even after 2 hours at 45°C, but was quickly inactivated after 5 minutes at 80°C. Chromatographic purification was achieved by FPLC using a cation exchanger (SP-Sepharose FF). Thus, a unique proteolitically active fraction could be isolated, being homogeneous by bidimensional electrophoresis and mass spectrometry (Mr = 23,652). The optimum pH range was achieved at 8.5–10.5. The enzyme activity was completely inhibited by specific cysteine peptidases inhibitors. Isoelectric focusing followed by zymogram showed the enzyme had a pI greater than 9.3. The N-terminus sequence (LPDSVDWREKGVVFPIRNQGK) shows a great deal of similarity to those of the other cysteine endopeptidases isolated from latices of Asclepiadaceae even when a high degree of homology could be observed with other plant cysteine endopeptidases.

Asclepias fruticosa Asclepiadaceae latex milkweed plant endopeptidases 


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  1. Abraham, K. I. and Joshi, P. N. (1979a). Biochim.Biophys.Acta 568, 111–119.Google Scholar
  2. Abraham, K. I. and Joshi, P. N. (1979b). Biochim.Biophys.Acta 568, 120–126.Google Scholar
  3. Altschul, S. F., Madden, T. L., Schaffer, A. A., Zhang, J., Zhang, Z., Miller, W., and Lipman, D. J. (1997). Nucleic Acids Res. 25, 3389–3402.Google Scholar
  4. Arribére, M. C., Cortadi, A. A., Gattuso, M. A., Bettiol, M. P., Priolo, N. S., and Caffini, N. O. (1998). Phytochem.Anal. 9, 267–273.Google Scholar
  5. Barragán, B. E., Hernández-Arana, A., Oliver, M., Castaneda-Agulló, M., and Del Castillo, L. M. (1985). Rev.Latinoamer.Quim.16, 158–160.Google Scholar
  6. Barrett, A. J. and Kirschke, H. (1981). Meth.Enzymol. 80, 535–561.Google Scholar
  7. Bose, S. M. and Madhavakrishna, W. (1958). Enzymologia 19, 186–200.Google Scholar
  8. Bradford, M. M. (1976). Anal.Biochem. 72, 248–254.Google Scholar
  9. Brockbank, W. J. and Lynn, K. R. (1979). Biochim.Biophys.Acta 578, 13–22.Google Scholar
  10. Choi, K. H., Laursen, R. A., and Allen, K. N. (1999). Biochemistry 38, 11624–11633.Google Scholar
  11. Cohen, L. W., Coghlan, V. M., and Dihel, L. C. (1986). Gene 48, 219–227.Google Scholar
  12. Dubois, T., Kleinschmidt, T., Schnek, A. G., Looze, Y., and Braunitzer, G. (1988). Biol.Chem.Hoppe-Seyler 369, 741–754.Google Scholar
  13. Endress, M. E. and Bruyns, P. V. (2000). Bot.Rev. 66, 1–56.Google Scholar
  14. Firsthoefel, N. R., Cushman, M. A. F., Ostrem, J. A., and Cushman, J. C. (1998). Plant Sci. 136, 195–206.Google Scholar
  15. Gan, S. and Amasino, R. M. (1995). Science 270, 1986–1988.Google Scholar
  16. Good, N. E. and Izawa, S. (1972). Meth.Enzymol. 24, 53–68.Google Scholar
  17. Greenberg, D. M. and Winnick, T. (1940). J.Biol.Chem. 135, 761–787.Google Scholar
  18. Griffiths, C. M., Hosken, S. E., Oliver, D., Chojecki, J., and Thomas, H. (1997). Plant Mol.Biol. 34, 815–821.Google Scholar
  19. Guerrero, F. D., Jones, J. T., and Mullet, J. E. (1990). Plant Mol.Biol. 15, 11–26.Google Scholar
  20. Jaziri, M., Kleinschmidt, T., Walraevens, V., Schnek, A. G., and Looze, Y. (1994). Biol.Chem.Hoppe-Seyler 375, 379–385.Google Scholar
  21. Jones, M. L., Larsen, P. B., and Woodson, W. R. (1995). Plant.Mol. Biol. 28, 505–512.Google Scholar
  22. Laemmli, U. K. (1970). Nature 227, 680–685.Google Scholar
  23. López, L. M. I. (1995). “Aislamiento, purificación y caracterización de proteasas del látex de Maclura pomifera (Raf.) Schneid. (Moraceae).” Ph. D. thesis, Faculty of Exact Sciences, La Plata University, Argentina.Google Scholar
  24. López, L. M. I., Sequeiros, C., Natalucci, C. L., Brullo, A., Maras, B., Barra, D., and Caffini, N. (2000). Protein Express.Purif. 18, 133–140.Google Scholar
  25. Lynn, K. R., Brockbank, W. J., and Clevette, N. A. (1980a). Biochim. Biophys.Acta 612, 119–125.Google Scholar
  26. Lynn, K. R., Yaguchi, M., and Roy, C. (1980b). Biochim.Biophys. Acta 624, 579–580.Google Scholar
  27. Madhavakrishna, W. and Bose, S. M. (1960). Enzymologia 22, 251–261.Google Scholar
  28. Noh, Y. S. and Amasino, R. M. (1999). Plant Mol.Biol. 41, 195–206.Google Scholar
  29. Obregón, W. D., Arribére, M. C., Morcelle del Valle, S., Liggieri, C., Caffini, N. O., and Priolo, N. S. (2001). J.Protein Chem. 20, 317–325.Google Scholar
  30. Pal, G. and Sinha, N. K. (1980). Arch.Biochem.Biophys. 202, 321–329.Google Scholar
  31. Priolo, N., Morcelle del Valle, S., Arribére, M. C., López, L. M. I., and Caffini, N. O. (2000). J.Protein Chem. 19, 39–49.Google Scholar
  32. Priolo, N., Arribére, M. C., Caffini, N., Barberis, S., Nieto Vázquez, R., and Luco, J. (2001). J.Mol.Catal.B: Enzymatic 635, 1–13.Google Scholar
  33. Ritonja, A., Buttle, D. J., Rawlings, N. D., Turk, V., and Barrett, A. J. (1989). FEBS Lett. 258, 109–112.Google Scholar
  34. Rodriquez-Romero, A. and Hernández-Arana, A. (1998). In Handbook of Proteolytic Enzymes (Barrett, A. J., Rawlings, N. D., and Woessner, J. F., eds.), Academic Press, London, pp. 576–578.Google Scholar
  35. Rowan, A. D. and Buttle, D. J. (1994). Meth.Enzymol. 244, 555–568.Google Scholar
  36. Salvesen, G. and Nagase, H. (1989). In Proteolytic enzymes: A practical approach (Beynon, R. J. and Bond, J. S., eds.), Oxford, United Kingdom: IRL Press, pp. 83–104.Google Scholar
  37. Schaffer, M. A. and Fischer, R. L. (1988). Plant Physiol. 87, 431–436.Google Scholar
  38. Sengupta, A., Bhattacharya, D., Pal, G., and Sinha, N. K. (1984). Arch. Biochem.Biophys. 232, 17–25.Google Scholar
  39. Tablero, M., Arreguín, R., Arreguín, B., Soriano, M., Sánchez, R. I., Rodriguez-Romero, A., and Hernández-Arana, A. (1991). Plant Sci. 74, 7–15.Google Scholar
  40. Tournaire, C., Kushnir, S., Bauw, G., Inze, D., Teyssendier de la Serve, B., and Renaudin, J. P. (1996). Plant Physiol. 111, 159–168.Google Scholar
  41. Tranbarger, T. J. and Misra, S. (1996). Gene 172, 221–226.Google Scholar
  42. Ueda, T., Seo, S., Ohashi, Y., and Hashimoto, J. (2000). Plant Mol. Biol. 44, 649–657.Google Scholar
  43. Utsumi, S., Damodaran, S., and Kinsella, J. E. (1984). J.Agric.Food Chem. 32, 1406–1412.Google Scholar
  44. Vairo Cavalli, S. E., Cortadi, A., Arribére, M. C., Conforti, P., Caffini, N. O., and Priolo, N. S. (2001). Biol.Chem.Hoppe-Seyler 382, 879–883.Google Scholar
  45. Watanabe, H., Abe, K., Emori, Y., Hosoyama, H., and Arai, S. (1991). J.Biol.Chem. 266, 16897–16902.Google Scholar
  46. Watson, D. C., Yaguchi, M., and Lynn, K. R. (1990). Biochem.J. 266, 75–81.Google Scholar
  47. Westergaar, J. L., Hackbarth, C., Treuhaft, M. W., and Roberts, R. C. (1980). J.Immunol.Meth. 34, 167–175.Google Scholar
  48. Winnick, T., Davies, A. R., and Greenberg, D. M. (1940). J.Gen. Physiol. 23, 275–308.Google Scholar
  49. Ye, Z. H. and Varner, J. E. (1996). Plant Mol.Biol. 30, 1233–1246.Google Scholar
  50. Yu, W. J. and Greenwood, J. S. (1994). Exp.Bot. 45, 261–268.Google Scholar

Copyright information

© Plenum Publishing Corporation 2001

Authors and Affiliations

  • Sebastián A. Trejo
    • 1
  • Laura M. I. López
    • 1
  • Cecilia V. Cimino
    • 1
  • Néstor O. Caffini
    • 1
  • Claudia L. Natalucci
    • 1
  1. 1.Laboratorio de Investigación de Proteínas Vegetales (LIPROVE), Departamento de Ciencias Biológicas, Facultad de Ciencias ExactasUniversidad Nacional de La PlataArgentina

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