Advertisement

Identification of peptidases in Nicotiana tabacum leaf intercellular fluid

Authors

  • Mélanie Delannoy,

    1. Unité de Biochimie Physiologique, Institut des Sciences de la Vie, Université Catholique de Louvain, Louvain-la-Neuve, Belgium
    Search for more papers by this author
  • Georges Alves,

    1. Unité de Biochimie Physiologique, Institut des Sciences de la Vie, Université Catholique de Louvain, Louvain-la-Neuve, Belgium
    Search for more papers by this author
  • Didier Vertommen,

    1. HORM Unit, Christian de Duve Institute of Cellular Pathology, Université Catholique de Louvain, Louvain-la-Neuve, Belgium
    Search for more papers by this author
  • Julian Ma,

    1. St. Georg's Hospital Medical School, London UK
    Search for more papers by this author
  • Marc Boutry Dr.,

    Corresponding author
    1. Unité de Biochimie Physiologique, Institut des Sciences de la Vie, Université Catholique de Louvain, Louvain-la-Neuve, Belgium
    • Unité de Biochimie Physiologique, Institut des Sciences de la Vie, Université Catholique de Louvain, Croix du Sud 5-15, 1348 Louvain-la-Neuve, Belgium Fax: +32-10-473872
    Search for more papers by this author
  • Catherine Navarre

    1. Unité de Biochimie Physiologique, Institut des Sciences de la Vie, Université Catholique de Louvain, Louvain-la-Neuve, Belgium
    Search for more papers by this author

Abstract

Peptidases in the extracellular space might affect the integrity of recombinant proteins expressed in, and secreted from, plant cells. To identify extracellular peptidases, we recovered the leaf intercellular fluid from Nicotiana tabacum plants by an infiltration–centrifugation method. The activity of various peptidases was detected by an in vitro assay in the presence of specific inhibitors, using BSA and human serum γ-globulin as substrates. Peptidases were detected by 1- and 2-D zymography in a polyacrylamide gel containing gelatin as substrate. Proteolytic activity was observed over a wide range of molecular masses equal to, or higher than, 45 kDa. To identify the peptidases, the extracellular proteins were digested with trypsin and analyzed by LC and MS. Seventeen peptides showing identity or similarity to predicted plant aspartic, cysteine, and serine peptidases have been identified. The extracellular localization of a cysteine peptidase aleurain homolog was also shown.

Ancillary