RESUMO
A 24,118 Da (MALDI-TOF) cysteine peptidase (EC 3.4.22.16) was purified from the latex extract of Cryptostegia grandiflora by two chromatographic steps involving ion exchange and Reverse-phase HPLC. The purified protein (Cg24-I) exhibits a single band profile following reduced SDS-PAGE and a unique amino terminal sequence, 1VPASIDWREKGTVL14, that is similar to other plant cysteine peptidases. Cg24-I displayed optimal proteolytic activity at pH 8.0 with 25 mM phosphate buffer. The proteolytic activity was inhibited with 0.2 mM E-64 and 1 mM iodoacetamide and was stimulated with 1 mM DTT. Cg24-I fully inhibited spore germination of phytopathogenic fungi Fusarium solani at a dose of 28.1 µg/mL. Its toxicity involves the membrane permeabilization of spores as probed by propidium iodide uptake. These results show that latex peptidases are part of the plant's defensive strategy against phytopathogens and that they most likely act synergistically with other recognized defensive proteins.
Assuntos
Antifúngicos/química , Apocynaceae/enzimologia , Apocynaceae/microbiologia , Cisteína Endopeptidases/química , Extratos Vegetais/química , Sequência de Aminoácidos , Antifúngicos/isolamento & purificação , Antifúngicos/metabolismo , Apocynaceae/química , Cisteína Endopeptidases/isolamento & purificação , Cisteína Endopeptidases/metabolismo , Fusariose/microbiologia , Fusariose/prevenção & controle , Fusarium/efeitos dos fármacos , Fusarium/crescimento & desenvolvimento , Dados de Sequência Molecular , Doenças das Plantas/microbiologia , Doenças das Plantas/prevenção & controle , Extratos Vegetais/isolamento & purificação , Extratos Vegetais/metabolismo , ProteóliseRESUMO
Latex from Caricaceae contains a number of proteins believed to be part of a defense mechanism that protects these plants from wounding. Prior evidence suggests that some components in Carica papaya improve healing of ulcerous wounds in mammals. This study shows the chromatographic isolation of a protein fraction from C. candamarcensis that stimulates cell proliferation of mammalian cells by measuring MTT reduction and thymidine incorporation. The effect appears to be cell specific as L929, MDA-MB231 and BHK-21 cells are stimulated while no effect is seen on CHO cells. The maximal stimulatory effect reaches 2.2-fold 72 h after addition of the active fraction to L929, 1.8-fold in MDA-MB231 cells and 1.6-fold in BHK cells. Proteolytic inactivation of the active fraction suggests that a protein is responsible for the proliferative activity and its size is estimated between 10 and 25 kDa. A potential candidate for this function is a 23 kDa protein found in the fraction that reacts with human EGF antibody.