Journal
PLANT PHYSIOLOGY AND BIOCHEMISTRY
Volume 49, Issue 7, Pages 738-743Publisher
ELSEVIER FRANCE-EDITIONS SCIENTIFIQUES MEDICALES ELSEVIER
DOI: 10.1016/j.plaphy.2011.01.027
Keywords
Amino acid sequence; Antifungal activity; Fusarium solani; Laticifer proteins; Mass spectrometry; Thaumatin-like protein
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Funding
- FUNCAP
- CNPq (Universal, RENORBIO and Brazil/India cooperation)
- IFS
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A protein, similar to osmotin- and thaumatin-like proteins, was purified from Calotropis procera (Ait.) R.Br latex. The isolation procedure required two cation exchange chromatography steps on 50 mM Na-acetate buffer (pH 5.0) CM-Sepharose Fast Flow and 25 mM Na-phosphate buffer (pH 6.0) Resource-S, respectively. The protein purity was confirmed by an unique N-terminal sequence [ATFTIRNNCPYTI-WAAAVPGGGRRLNSGGTWTINVAPGTA]. The osmotin (CpOsm) appeared as a single band (20,100 Da) in sodium dodecyl sulfate-polyacrylamide gel electrophoresis and as two spots in two-dimensional electrophoresis (pI 8.9 and 9.1). Both polypeptides were further identified by mass spectrometry as two osmotin isoforms with molecular masses of 22,340 and 22,536 Da. The CpOsm exerted antifungal activity against Fusarium solani (IC50 = 67.0 mu g mL(-1)), Neurospora sp. (IC50 = 57.5 mu g mL(-1)) and Colletotrichum gloeosporioides (IC50= 32.1 mu g mL(-1)). However, this activity was lost when the protein was previously treated with a reducing agent (DTT, Dithiothreitol) suggesting the presence of disulfide bounds stabilizing the protein. The occurrence of osmotin in latex substantiates the defensive role of these fluids. (C) 2011 Elsevier Masson SAS. All rights reserved.
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