Proteases from the latex of Funastrum clausum (Asclepiadaceae).

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

Oaxaca, México

Congreso; 42nd. Annual Meeting of the American Society of Pharmacognosy; 2001

Proteases were detected in stem laticifers of Funastrum clausum (Jacq.) Schlechter (Asclepiadaceae) in situ by histochemical assays made in transversal sections according to Fratello´s method. Latex of stems was gathered on 55 mM citric-phosphate buffer (pH 6.4) with EDTA and cysteine and crude extract was obtained after ultracentrifugation (100,000´g). Caseinolytic activity was maximun at pH 8-9 in the presence of 12 mM cysteine. Inhibition assays were carried out in presence of E-64, resulting in a complete inhibition of the proteolytic activity, suggesting the proteases present in it belong to the cysteine family. Endoesterolytic activity of the crude extract, determined on N-a-CBZ-amino acid-p-nitrophenyl esters, exhibited highest preference for the Ala derivative, followed by those of Tyr and Gln. Assays in monofasic and bifasic systems of different proportions of water and organic solvents showed a remarkable stability in those containing methanol, ethanol and buthanol. Proteases present in the crude extract were purified by cationic exchange cromatography in FPLC (SP-Sepharose, elution buffer citric acid-phosphate pH 6.6, 0-1 M NaCl gradient). The protease purified had a molecular mass of 28 kDa (SDS PAGE) and pI higher than 9.3 (isoelectric focusing).