Identification of peptidases in Nicotiana tabacum leaf intercellular fluid.
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 gamma-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.
Delannoy
Mélanie
M
Unité de Biochimie Physiologique, Institut des Sciences de la Vie, Université Catholique de Louvain, Louvain-la-Neuve, Belgium.
Alves
Georges
G
Vertommen
Didier
D
Ma
Julian
J
Boutry
Marc
M
Navarre
Catherine
C
D002853:Chromatography, Liquid / Q000379:methods; D003545:Cysteine / Q000737:chemistry; D003546:Cysteine Endopeptidases / Q000737:chemistry; D016650:Fluorescein-5-isothiocyanate; D006801:Humans; D006863:Hydrogen-Ion Concentration; D013058:Mass Spectrometry / Q000379:methods; D010447:Peptide Hydrolases / Q000737:chemistry; D018515:Plant Leaves / Q000378:metabolism; D010940:Plant Proteins / Q000737:chemistry*; D040901:Proteomics / Q000379:methods*; D012680:Sensitivity and Specificity; D012710:Serum Albumin, Bovine / Q000737:chemistry; D014026:Nicotiana / Q000378:metabolism*; D005719:gamma-Globulins / Q000737:chemistry
D010940:Plant Proteins; D005719:gamma-Globulins; D012710:Serum Albumin, Bovine; D010447:Peptide Hydrolases; D003546:Cysteine Endopeptidases; C046726:aleurain; D016650:Fluorescein-5-isothiocyanate; D003545:Cysteine