PROTEASES IN PLANTS: CYTOCHEMICAL LOCALIZATION AND ISOLATION FROM LATEX

MORCELLE DEL VALLE, S. R.; PRIOLO, N. S.; CORTADI, A.; CAFFINI, N. O.

Merlo, San Luis, Argentina

Congreso; XVII Reunión Científica anual de la Sociedad de Biología de Cuyo; 1999

Sociedad de Biología de Cuyo

PROTEASES IN PLANTS: CYTOCHEMICAL LOCALIZATION AND ISOLATION FROM LATEX   Morcelle del Valle, S., Priolo, N., Cortadi, A.*, Caffini, N.     LIPROVE, Fac. de Cs. Exac., Univ Nac La Plata 1900, Buenos Aires; *Cát Bot, Fac Cs Bioqím Farm Univ Nac Rosario. E-mail: morcelle@biol.unlp.edu.ar   Some latices contain porteases that degradate proteins during development, promote latex coagulation and protect against potencial predators. This work reports the cytochemical localization and further isolation and characterization of proteases present in the latex of Funastrum clausum Schlechter (Asclepiadaceae). Proteases were histochemically detected in situ in fresh stems. In longitudinal sections (Denker´s method) cryostat sections were incubated for 10 min at 50ºC on gelatine film and then stained with 0.5% toluidine blue. In transversal sections (Fratello´s method) sections were placed directly on to unexposed, processed color film and then incubated (10 min at 50ºC). Latex obtained from stems was placed in 55 mM citric-phosphate buffer (pH 6.4) with EDTA and cysteine and centrifuged at 16,000 g for 30 min. Proteolytic assays were made on 1% casein in Tris-HCl buffer (0.1 M, pH 8.8) with 12 mM cysteine. The crude extract was purified by cationic exchange chromatography. Protease localization in stem laticifers was evidenced by successive apparition of magenta, blue and white colors due to digestion of film layers. Enzyme preparations showed highest caseinolytic activity at pH 8-10. Inhibition assays with E-64 suggest that the catalytic mechanism might involve –SH groups. Purification allowed separation of 3 proteolytic fractions with pI>9.3 and similar molecular masses (SDS-PAGE).