Action on bovine alpha s1-casein of cardosins A and B, aspartic proteinases from the flowers of the cardoon Cynara cardunculus L.
The cleavage of purified bovine alpha s1-casein separately by cardosin A and cardosin B, two distinct milk-clotting aspartic proteinases (APs) present in the stigmas of the plant Cynara cardunculus L., was studied. Casein digestion peptides were separated either by SDS-PAGE or by reverse-phase HPLC, and their N-terminal amino acid sequences were subsequently determined by automated Edman degradation, thus identifying the cleavage sites. Results showed that both enzymes exert a similar but distinct action on bovine alpha s1-casein. In common they have the preference for the bond Phe23-Phe24, and the cleavage of Trp164-Tyr165 and Phe153-Tyr154. Cardosin A also cleaves the bond Tyr165-Tyr166, whereas Cardosin B cleaves an extra type of bond, Phe150-Arg151, revealing a slightly broader specificity. A model for the action of both enzymes on bovine alpha s1-casein is proposed and discussed. In comparison with the reported action of chymosin on bovine alpha s1-casein, both cardosins proved to have a broader specificity towards this particular substrate due to a higher ability to cleave bonds between residues with large hydrophobic side-chains.
Ramalho-Santos
M
M
Departamento de Bioquímica, Faculdade de Ciências e Tecnologia, Universidade de Coimbra, Portugal.
Veríssimo
P
P
Faro
C
C
Pires
E
E
D000818:Animals; D001120:Arginine / Q000378:metabolism; D016282:Aspartic Acid Endopeptidases / Q000302:isolation & purification / Q000378:metabolism*; D002364:Caseins / Q000378:metabolism*; D002417:Cattle; D008970:Molecular Weight; D010446:Peptide Fragments / Q000032:analysis / Q000737:chemistry; D010649:Phenylalanine / Q000378:metabolism; D010940:Plant Proteins / Q000302:isolation & purification / Q000378:metabolism*; D010944:Plants / Q000201:enzymology*; D017421:Sequence Analysis; D013379:Substrate Specificity; D014364:Tryptophan / Q000378:metabolism; D014443:Tyrosine / Q000378:metabolism
D002364:Caseins; D010446:Peptide Fragments; D010940:Plant Proteins; D014443:Tyrosine; D010649:Phenylalanine; D014364:Tryptophan; D001120:Arginine; D016282:Aspartic Acid Endopeptidases; C097455:aspartic proteinases, Cynara cardunculus