INVESTIGADORES
MORCELLE DEL VALLE Susana Raquel
congresos y reuniones científicas
Título:
Cysteine endopeptidases from the latex of Araujia hortorum Fourn.: isolation, purification and characterization of the three protelytic fractions
Autor/es:
OBREGÓN, W.D.; ARRIBERE, M.C.; MORCELLE DEL VALLE, S.; PRIOLO, N.; CAFFINI, N.
Lugar:
Portoroz, Slovenia
Reunión:
Conferencia; International Conference on Cysteine Proteinases and their inhibitors: The new Millenium; 2000
Resumen:
Three endopeptidases (araujiain h I, araujiain h II, araujiain h III) were purified to mass spectroscopy homogeneity from the latex of Araujia hortorum Fourn. (Asclepiadaceae) fruits by ultracentrifugation and ion exchange chromatography. The enzymes have a molecular mass of 24,031; 23,718 and 23,545 (mass spectrometry) respectively. Isoelectric point of araujiain h I and araujiain h III are higher than 9.3, whereas araujiain h II has a pI of 8.9. Proteolytic activity was assayed on casein as substrate, and the three endopeptidases showed the major caseinolytic activity in the pH range of 8.0-9.0 in the presence of 12 mM cysteine. Inhibition assays were carried out with iodoacetate and E-64 as protease inhibitors: the proteolytic fractions were irreversibly inhibited, suggesting they belong to the cysteine proteases family. Esterolytic activity was performed with N-a-CBZ-amino acid-p-nitrophenyl esters. The three enzymes showed highest preference for the glutamine derivative followed by the aspartic acid one, but with other derivative, they showed different behaviour. Aminoacid sequence of the three proteases was performed, araujiain h I has the N-terminal blocked therefore, the sequence of an internal peptide (i.p.) obtained by hydrolysis with protease V( was determined. On the other hand, N-terminal of araujiain h II and araujiain h III are not blocked. The sequences found were: Araujiain h I (i.p.):                AFTYVAKNGITSRDKYPYRGQQGQCYQLQKVVRISGYQSV Araujiain h II (N-end):                      VPDSIDWREKDAVLPIRNQGQXGSIWAFXAIASVE Araujiain h III (N-end):                     LPESVDWRKKNLVFPVRNQGQXGSXXAFSAVAXYGT These results show high percentage of homology with other plant cysteine proteinases.