title: Multiplicity of aspartic proteinases from Cynara cardunculus L.
abstract: Aspartic proteinases (AP) play major roles in physiologic and pathologic scenarios in a wide range of organisms from vertebrates to plants or viruses. The present work deals with the purification and characterisation of four new APs from the cardoon Cynara cardunculus L., bringing the number of APs that have been isolated, purified and biochemically characterised from this organism to nine. This is, to our knowledge, one of the highest number of APs purified from a single organism, consistent with a specific and important biological function of these protein within C. cardunculus. These enzymes, cardosins E, F, G and H, are dimeric, glycosylated, pepstatin-sensitive APs, active at acidic pH, with a maximum activity around pH 4.3. Their primary structures were partially determined by N- and C-terminal sequence analysis, peptide mass fingerprint analysis on a MALDI-TOF/TOF instrument and by LC-MS/MS analysis on a Q-TRAP instrument. All four enzymes are present on C. cardunculus L. pistils, along with cyprosins and cardosins A and B. Their micro-heterogeneity was detected by 2D-electrophoresis and mass spectrometry. The enzymes resemble cardosin A more than they resemble cardosin B or cyprosin, with cardosin E and cardosin G being more active than cardosin A, towards the synthetic peptide KPAEFF(NO(2))AL. The specificity of these enzymes was investigated and it is shown that cardosin E, although closely related to cardosin A, exhibits different specificity.
authors.affiliation: CESAM, Department of Biology, University of Aveiro, 3810-193, Aveiro, Portugal. acsarmento@ua.pt
authors.forename: Ana Cristina
authors.initials: AC
authors.lastname: Sarmento
authors.forename: Henrique
authors.initials: H
authors.lastname: Lopes
authors.forename: Cláudia S
authors.initials: CS
authors.lastname: Oliveira
authors.forename: Rui
authors.initials: R
authors.lastname: Vitorino
authors.forename: Bart
authors.initials: B
authors.lastname: Samyn
authors.forename: Kjell
authors.initials: K
authors.lastname: Sergeant
authors.forename: Griet
authors.initials: G
authors.lastname: Debyser
authors.forename: Jozef
authors.initials: J
authors.lastname: Van Beeumen
authors.forename: Pedro
authors.initials: P
authors.lastname: Domingues
authors.forename: Francisco
authors.initials: F
authors.lastname: Amado
authors.forename: Euclides
authors.initials: E
authors.lastname: Pires
authors.forename: M Rosário M
authors.initials: MR
authors.lastname: Domingues
authors.forename: Marlene T
authors.initials: MT
authors.lastname: Barros
mesh_terms: D016282:Aspartic Acid Endopeptidases / Q000378:metabolism*; D002853:Chromatography, Liquid; D031942:Cynara / Q000201:enzymology*; D015180:Electrophoresis, Gel, Two-Dimensional; D006863:Hydrogen-Ion Concentration; D010940:Plant Proteins / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D053719:Tandem Mass Spectrometry
chemical_list: D010940:Plant Proteins; D016282:Aspartic Acid Endopeptidases; C097455:aspartic proteinases, Cynara cardunculus