Origin and development of laticifer and cytochemical localization of proteases from latex of Funastrum clausum (Jacq.) Schlechter (Asclepiadaceae)

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

Gramado, Brasil

Simposio; IX Simposio Latinoamericano de Farmacobotánica y III Reunión de la Sociedad Latinoamericana de Fitoquímica; 1999

Proteolytic enzymes are still very used in different pharmaceutical preparations (Caffini et al., 1988). Though proteases are frequently present in plant laticifers, not all the latex-containing species are good producers of proteolytic enzymes. Objective: to determine the origin and development of laticifer and cytochemical localization of proteases from latex of Funastrum clausum (Jacq.) Schlecht. The enzymes were detected in situ by histochemical assays made in transversal sections of fresh stems according to Fratello´s (1968) film-substrate method. Laticifer initials are differentiated in the embryo, arise an irregular ring of cells directly below the embryonic shoot apex. The nature of their growth habit and branching suggest that the tips of laticifer initials exhibit an intrusive form of growth. The cytochemical localization is evidenced through proteolytic activity as follows: graded occurrence of magenta, blue and white colors due to digestion of the colored film layers. Latex obtained by superficial incisions of  stems was gathered on 0.1 M phosphate buffer (pH 6.5) containing 5 mM EDTA and cysteine, and centrifuged at 16,000g for 30 minutes. This crude extract shows remarkable proteolytic activity when on casein: maximum activity was reached at pH 8.0-8.5 with 12 mM cysteine. The crud extract was purified by fractioned acetone precipitation followed by cationic exchange chromatography (CM Sepharose CL-6B; elution buffer citric acid-sodium phosphate pH 6.6; 0.0-0.6 M sodium chloride gradient). Two basic proteolytic active fractions were obtained, both homogeneous by SDS-PAGE (Laemmli, 1970).