Papel da lectina de folhas de Ipomoea asarifolia R. et Schult. na toxicidade a animais e seu envolvimento no mecanismo de defesa da planta

Abstract

Ipomoea asarifolia R. et Schult (common name: salsa) is a tropical plant of the Convolvulaceae family, is a shrubby, quickly growing toxic plant, widely distributed throughout Brazil. The present study was carried out towards the preparation of a lectin-enriched fraction (LEF) which could be used in biologic assays. To obtain LEF devoid of contaminants various purification strategies were tested. As a result of these approaches the excised leaves of Ipomoea asarifolia from plants growing in the field were washed with distilled water, mechanically wounded (6 mm - cuttings) and maintained in a chamber at 25±3 °C, in the dark, under near 100% relative humidity., for 72h. The leaf proteins were extracted (1:3, m/v) in 25 mM Tris-HC1, pH 7.5, containing 3% PVPP and 5 mM ascorbic acid, under mechanical stirring, for 2h, at 4 °C. The suspension was filtered through a nylon cloth and centrifuged at 10,000 g, for 30 min, at 4 °C. The crude extract obtained was precipitated to 30% ammonium sulfate, dialyzed, submitted to DEAE-cellulose chromatography, and after new dialysis, loaded on a Phenyl Sepharose 6-Fast Flow column. The fraction obtained was called lectin-enriched fraction (LEF). From this chromatographic step two proteins with hemaglutinating activity were identified in I. asarifolia leaves. A 44 kDa protein that has 65% amino-terminal identity (AVNLPAGHLSPGGVGNYVVTVGLCTP) with a DNA binding protein from chloroplast nucleoids of Nicotiana tabacum and a 20,1 kDa protein that has 72% amino-terminal identity (AGYTPVLDIGAEVLAAGEPY) with sporamin, a tuber protein of /. batatas. The 44 kDa protein seems to be constitutively expressed whereas the second one was apparently induced by leaf wounding. With respect to their immunological reactivity, it was found that the 44 kDa protein was also recognized by the antibody raised against sporamin. Nevertheless, the toxic effects of LEF observed after injection in the mouse intra orbital region, rat kidney perfusion and mouse vas deferens stimulation were attributed to the 44 kDa lectin, which was in much higher concentration in this fraction. The toxic lectin-containing LEF lost its haemagglutination activity after heating at 90 °C for 10 min and also after dialysis against 5 mM EDTA or EGTA. The LEF didn't present secondary metabolites. In addition, fluorescent studies showed that the lectin binds to the goat oocyte zona pellucida suggesting that it could be used as an integrity maker of this cell. Altogether the results of the present study strongly suggest that the lectin is involved in the defense mechanism and in the toxic effects of I. asarifolia.