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
Article

Abstract

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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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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