Enzymatic Profile of Proteases obtained from latices of Araujia angustifolia (Hook et Arn.) Decaisne and Araujia hortorum fourn. Fruits (Asclepiadaceae)

OBREGÓN, WALTER DAVID; CURCIARELLO RENATA; LIGGIERI ,CONSTANZA; TREJO, SEBASTIAN; CIMINO, CECILIA; PRIOLO, NORA; CAFFINI, NESTOR

Barcelona, España

Congreso; VIth European Symposium of the Protein Society; 2005

Sociedad Europea de Proteínas

Six Cysteine porteases were isolated from the latices of Araujia hortorum Fourn. And Araujia angustifolia (Hook et Arn.) Decaisne (Asclepiadaceae). Latex from fruits was collected on cold buffer (pH 6,4) containing cysteine and EDTA. Suspensions were first centrifuged at 10,000g and then the supernatant ultracentrifuged at 100,000g to obtain the Crude Extracts (CE). Proteases were detected in CE by IEF and zymogram.Purification of A. angustifolia CE by FPLC (SP-Sepharose, pH 7,4,NaCl linear gradient 0-0,5 M) yielded also three basic fractions (araujiain hI, hII y hIII). The optimum pH of all these proteases  is in the alkaline range.E-64 and iodoacetate inhibited irreversibly all the peptidases, whereas reversible inhibition was shown by HgCl2 treatment. On the other hand, Cys, DTT and beta-mercaptoetanol enhanced  activity in all cases. Molecular masses of the peptidases (SDS-PAGE and MS) are between 22 and 29 kDa. When assayed on N-α-CBZ-aminoacid-p-nitrophenyl esters araujiain hI, II,III and  araijiain a II had a higher preference for the glutamine derivative and araujiain aI and III for the alanine derivative. Kinetic  parameters were determined on PFLNA and on the  N-α-CBZ-p-nitrophenyl esters of Ala and Gln. The N-terminal sequences of these proteases  showed a high similarity between them and with other plant proteases such as papain, bromelain and ficin.