title: Osmotin purified from the latex of Calotropis procera: biochemical characterization, biological activity and role in plant defense.
abstract: 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 50mM 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 [ATFTIRNNCPYTIWAAAVPGGGRRLNSGGTWTINVAPGTA]. 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 μg mL⁻¹), Neurospora sp. (IC₅₀=57.5 μg mL⁻¹) and Colletotrichum gloeosporioides (IC₅₀=32.1 μg 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.
authors.affiliation: Departamento de Bioquímica e Biologia Molecular da, Universidade Federal do Ceará, Campus do Pici, Cx., Postal 6033, Fortaleza, Ceará, CEP 60451-970, Brazil. cleversondiniz@hotmail.com
authors.forename: Cleverson Diniz Teixeira
authors.initials: CD
authors.lastname: de Freitas
authors.forename: Fábio César Sousa
authors.initials: FC
authors.lastname: Nogueira
authors.forename: Ilka Maria
authors.initials: IM
authors.lastname: Vasconcelos
authors.forename: José Tadeu Abreu
authors.initials: JT
authors.lastname: Oliveira
authors.forename: Gilberto Barbosa
authors.initials: GB
authors.lastname: Domont
authors.forename: Márcio Viana
authors.initials: MV
authors.lastname: Ramos
mesh_terms: D000595:Amino Acid Sequence; D000935:Antifungal Agents / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism* / Q000494:pharmacology; D031107:Calotropis / Q000737:chemistry / Q000378:metabolism*; D020231:Colletotrichum / Q000187:drug effects; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D005670:Fusarium / Q000187:drug effects; D006023:Glycoproteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology; D007840:Latex / Q000737:chemistry / Q000378:metabolism*; D013058:Mass Spectrometry; D008970:Molecular Weight; D009491:Neurospora / Q000187:drug effects; D057865:Plant Immunity; D010940:Plant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism* / Q000494:pharmacology; D020033:Protein Isoforms; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D000935:Antifungal Agents; D006023:Glycoproteins; D007840:Latex; D010940:Plant Proteins; D020033:Protein Isoforms