Cystein peptidase and inhibitors present in the latex of Carica quercifolia (St. Hil.) Hieron. (Caricaceae)

TORRES M. J., MARTÍN M.I., LA BLUNDA J., LÓPEZ L.M.I. AND NATALUCCI C.

Pinamar

Congreso; X Congreso de la PABMB y XLI Reunión Anual de la SAIB; 2005

Sociedad Argentina de Investigaciones Bioquímicas y Biología Molecular

The latex of Carica quercifolia fruits was obtained doing superficial incisions in the fruit and receiving it in citric-citrate buffer 0.1 M pH 5.6 containing EDTA 5mM. The preparation was centrifuged at 11500g during 20 min. Gums and other insoluble materials were discarded, and a limpid supernatant was obtained (crude extract). This extract was characterized by SDS-PAGE and isoelectrofocusing-zimogram revealing the presence of several basic proteins with high proteolytic activity and also other acid fractions without proteolytic activity. The pH profile showed that the activity is manifested in a wide pH range (more than 80% of maximum activity among pH 6,9 and 9,1). The enzymatic activity was inhibited by E-64 and iodoacetic acid, but it was not affected for 1-10 phenantroline, pepstatine neither PMFS. Therefore, the new endopeptidases should be included in the cysteine group, as all the other proteases isolated from plants belonging to the Caricaceae family. Chromatographic purification of crude extract was achieved by FPLC using Sephacryl S-100 and then CM-Sepharose FF (pH 7,5). Three fractions with proteolyitic activity were isolated and a fourth with inhibitory activity of papain.