Multiplicity of aspartic proteinases from Cynara cardunculus L.
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.
Sarmento
Ana Cristina
AC
CESAM, Department of Biology, University of Aveiro, 3810-193, Aveiro, Portugal. acsarmento@ua.pt
Lopes
Henrique
H
Oliveira
Cláudia S
CS
Vitorino
Rui
R
Samyn
Bart
B
Sergeant
Kjell
K
Debyser
Griet
G
Van Beeumen
Jozef
J
Domingues
Pedro
P
Amado
Francisco
F
Pires
Euclides
E
Domingues
M Rosário M
MR
Barros
Marlene T
MT
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
D010940:Plant Proteins; D016282:Aspartic Acid Endopeptidases; C097455:aspartic proteinases, Cynara cardunculus