Purification, characterization and partial amino acid sequencing of two new aspartic proteinases from fresh flowers of Cynara cardunculus L.
Two new aspartic proteinases have been isolated from stigmas of the cardoon Cynara cardunculus L. by a two-step purification procedure including extraction at low pH, gel filtration on Superdex 200, and ion-exchange chromatography on Mono Q. To follow the conventional nomenclature for aspartic proteinases, we have named these proteinases cardosin A and cardosin B. On SDS/PAGE, cardosin A migrated as two bands with apparent molecular masses of 31 000 Da and 15 000 Da whereas the chains of cardosin B migrated as bands of 34 000 Da and 14 000 Da. The partial amino acid sequences of the two cardosin revealed that they are similar but not identical, and that they differ from the previously reported cardoon proteinases named cynarases, which were assumed to be derived from a common precursor. Although the cardosins show some degree of similarity to each other, we could detect no immunological crossreactivity between them. Both cardosins were active at low pH and were inhibited by pepstatin, with Ki values of 3 nM for cardosin A and 1 nM for cardosin B, indicating that they belong to the class of aspartic proteinases. Significant differences between the two enzymes were also found for the Kcat/km values for the hydrolysis of two chromophoric synthetic peptides. The active-site ionization constants, pKe1 and pKe2, for cardosin A are 2.5 +/- 0.2 and 5.3+/- 0.2, whereas for cardosin B they are 3.73 +/- 0.09 and 6.7 +/- 0.1. The results herein described on the structural and kinetic properties of the cardosins indicate that they are the products of distinct genes which have probably arisen by gene duplication. A scheme for the proteolytic processing of the two enzymes is also proposed.
Veríssimo
P
P
Departamento de Bioquímica, Faculdade de Ciências e Tecnologia, Universidade de Coimbra, Portugal.
Faro
C
C
Moir
A J
AJ
Lin
Y
Y
Tang
J
J
Pires
E
E
D000595:Amino Acid Sequence; D016282:Aspartic Acid Endopeptidases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D015153:Blotting, Western; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D010944:Plants / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D016415:Sequence Alignment
D010940:Plant Proteins; D016282:Aspartic Acid Endopeptidases; C097455:aspartic proteinases, Cynara cardunculus