ABSTRACT
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.
Subject(s)
Antifungal Agents/chemistry , Apocynaceae/enzymology , Apocynaceae/microbiology , Cysteine Endopeptidases/chemistry , Plant Extracts/chemistry , Amino Acid Sequence , Antifungal Agents/isolation & purification , Antifungal Agents/metabolism , Apocynaceae/chemistry , Cysteine Endopeptidases/isolation & purification , Cysteine Endopeptidases/metabolism , Fusariosis/microbiology , Fusariosis/prevention & control , Fusarium/drug effects , Fusarium/growth & development , Molecular Sequence Data , Plant Diseases/microbiology , Plant Diseases/prevention & control , Plant Extracts/isolation & purification , Plant Extracts/metabolism , ProteolysisABSTRACT
OBJECTIVES AND DESIGN: Previous studies have described pro- and anti-inflammatory activities displayed by the latex from Calotropis procera. This report aims to clarify these observations and shows that such activities can be segregated from the whole latex. METHODS: The latex was divided into water-soluble fractions devoid of poly-isoprene by centrifugation and dialysis and both the activities were assayed by the peritonitis model in rats. The drugs dexamethasone, thalidomide, meclizine, indomethacin and celecoxib were used to modulate the inflammatory stimuli. RESULTS: Inflammation in rats was observed 2 h after intraperitoneal administration of the stimulus (DL fraction) in a dose dependent manner. This activity was inhibited by previous intravenous injection of dexamethasone, thalidomide and meclizine. Indomethacin and celecoxib did not reverse inflammation. These results suggest the involvement of histamine release and TNF-alpha mediated inflammation while prostaglandins seem not to be required. The anti-inflammatory fraction (NDL) inhibited inflammation triggered by proinflammatory fraction (DL) suggesting that NDL ought to follow a similar pathway of action to that of the anti-inflammatory drugs that were able to inhibit inflammation triggered by DL. CONCLUSIONS: Pro- and anti-inflammatory activities of the latex are displayed by compounds suitable to be fractionated on the basis of their molecular size.
