The component of Carica papaya seed toxic to A-aegypti and the identification of tegupain, the enzyme that generates it

As Aedes aegypti transmits the etiologic agents of both yellow and dengue fever; vector control is considered essential to minimise their incidence. the aim of this work was to identify the component of Carica papaya seed toxic to A. aegypti, and the identification of tegupain, the enzyme that generates it. Aqueous extracts (1%, w/v) of the seed tegument and cotyledon of C. papaya are not larvicidal isolately. However, a mixture of 17 mu g mL(-1) tegument extract and 27 mu g mL(-1) cotyledon extract caused 100% larval mortality in a bioassay. the mixture was no longer larvicidal after the tegument extract was pre-treated at 100 degrees C for 10 min. the enzyme tegupain efficiently hydrolysed the substrate Z-Phe-Arg-pNan (K-m 58.8 mu M, K-cat 28020 s(-1), K-cat/K-m 5 x 10(8) M-1 s(-1)), and its activity increased with 2 mM dithiothreitol (DTT), at 37 degrees C, pH 5.0. the chelating agent EDTA did not modify the enzyme activity. Inhibition of tegupain by cystatin (K-iapp 2.43 nM), E64 (3.64 nM, 83% inhibition), and the propeptide N-terminal sequence indicate that the toxic activity is due to a novel cysteine proteinase-like enzyme, rendered active upon the hydrolysis of a cotyledon component of C. papaya seeds. (c) 2013 Elsevier B.V. All rights reserved.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Universidade Federal de São Paulo, Dept Bioquim, BR-04044020 São Paulo, BrazilUniv Estadual Norte Fluminense, Biotechnol Lab, Campos Dos Goytacazes, RJ, BrazilUniversidade Federal de São Paulo, Dept Bioquim, BR-04044020 São Paulo, BrazilFAPESP: 2009/17058-6FAPESP: 2009/53766-5Web of Scienc

Biochemical characterization of a cysteine proteinase from Bauhinia forficata leaves and its kininogenase activity

In this work, the proteinase activity detect in the acetone precipitate (80%, v/v) of B. forficata leaves, trivially known as cow paw, and popularly used in folk medicine for treatment of diabetes mellitus, was purified by chromatography on Sephadex G-25, Canecystatin-Sepharose, and on Con A-Sepharose. the molecular weight 30 kDa was estimated by SDS-PAGE and zymography, and the N-terminal sequence and CD spectra indicated a relationship with the papain family of cysteine proteinases. Denominated baupain, the enzyme was activated by dithiotreitol and inhibited by E-64 and iodoacetamide, but not by benzamidine, TLCK. TPCK and EDTA. the 52 and S1 substrate specificity of baupain, assayed with two series of fluorescence resonance energy transfer (FRET) peptide substrates derived from Abz-KLRSSK-Q-EDDnp, indicates a preference for Phe and Tyr at P2 position over Leu found in papain. Baupain releases bradykinin from HMWK (human high molecular weight kininogen) though its proteolytic activity is blocked by the sequence motif QVVA of kininogen (K(iapp) = 1.9 x 10(-8) M). Canecystatin, from sugar cane, which also lodges the QVVA sequence, inhibits baupain (K(iapp) = 0.18 x 10(-9) M). (C) 2010 Elsevier B.V. All rights reserved.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Universidade Federal de São Paulo, Dept Bioquim, BR-04044020 São Paulo, BrazilUniversidade Federal de São Paulo, Dept Biophys, BR-04044020 São Paulo, BrazilUniv Estadual Oeste Parana, Ctr Engn & Ciencias Exatas, BR-85903000 Toledo, PR, BrazilUniversidade Federal de São Paulo, Dept Bioquim, BR-04044020 São Paulo, BrazilUniversidade Federal de São Paulo, Dept Biophys, BR-04044020 São Paulo, BrazilWeb of Scienc

Structural characterization of coagulant Moringa oleifera Lectin and its effect on hemostatic parameters

Lectins are carbohydrate recognition proteins. cMoL, a coagulant Moringa oleifera Lectin, was isolated from seeds of the plant. Structural studies revealed a heat-stable and pH resistant protein with 101 amino acids, 11.67 theoretical pI and 81% similarity with a M. oleifera flocculent protein. Secondary structure content was estimated as 46% alpha-helix, 12% beta-sheets, 17% beta-turns and 25% unordered structures belonging to the alpha/beta tertiary structure class. cMoL significantly prolonged the time required for blood coagulation, activated partial thromboplastin (aPTF) and prothrombin times (PT), but was not so effective in prolonging aPTT in asialofetuin presence. cMoL acted as an anticoagulant protein on in vitro blood coagulation parameters and at least on aPTT, the lectin interacted through the carbohydrate recognition domain. (C) 2013 Elsevier B.V. All rights reserved.Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundacao de Amparo a Ciencia e Tecnologia do Estado de Pernambuco (FACEPE)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Univ Fed Pernambuco, Dept Bioquim, BR-50670901 Recife, PE, BrazilUniversidade Federal de São Paulo, Dept Bioquim, BR-04044020 São Paulo, BrazilUniv Estadual Oeste Parana, Ctr Engn & Ciencias Exatas, BR-85903000 Toledo, PR, BrazilLMU, Inst Clin Neuroimmunol, Munich, GermanyMax Planck Inst Biochem, Dept Prot Analyt, D-82152 Martinsried, GermanyUniversidade Federal de São Paulo, Dept Bioquim, BR-04044020 São Paulo, BrazilWeb of Scienc

Purification, primary structure and potential functions of a novel lectin from Bauhinia forficata seeds

A new lectin. Bfl. was purified from Bauhinia forficata seeds by ammonium sulfate fractionation. DEAE-Sephadex ion exchange chromatography, Sepharose-4B and chitin affinity chromatographies and Superdex 75 size exclusion chromatography. the molecular homogeneity and purity of BfL were assessed by reversed-phase H PLC. BfL appeared as a single band of approximately 27.0 kDa on SDS-PAGE under non-reducing and reducing conditions, and its molecular weight was determined to be 27,850 Da by LC/ESI-MS. Bit is a glycoprotein with a carbohydrate content of 6.24% determined by the phenol-sulfuric acid method. Fetuin, asialofetuin, thyroglobulin and azocasein inhibited the hemagglutinating activity of BfL, whereas saccharides did not. Bfl hemagglutinating activity was stable at 100 degrees C for 30 min, pH-dependent, with the highest activity at pH 6.0, and metal-independent. the primary structure of BfL shows similarity with other lectins from the genus Bauhinia. Deconvolution of the BfL circular dichroism (CD) spectrum indicated the presence of alpha-helix and beta structures. BfL increases coagulation time, but this effect is not related to human plasma kallikrein or human factor Xa inhibition. Bfl also inhibits ADP- and epinephrine-induced platelet aggregation in a dose-dependent manner and is the only currently described lectin from Bauhinia that exhibits anticoagulant and antiplatelet aggregating properties. (c) 2012 Elsevier B.V. All rights reserved.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Universidade Federal de São Paulo, Dept Bioquim, BR-04044020 São Paulo, BrazilUniversidade Federal de São Paulo, Dept Biofis, BR-04044020 São Paulo, BrazilUniv Estadual Oeste Parana, Ctr Engn & Ciencias Exatas, BR-85903000 Toledo, PR, BrazilUniv Fed Pernambuco, Dept Bioquim, BR-50670901 Recife, PE, BrazilInst Clin Neuroimmunol LMU, Munich, GermanyMax Planck Inst Biochem, Dept Prot Analyt, D-82152 Martinsried, GermanyUniversidade Federal de São Paulo, Dept Bioquim, BR-04044020 São Paulo, BrazilUniversidade Federal de São Paulo, Dept Biofis, BR-04044020 São Paulo, BrazilFAPESP: 07/58929-4FAPESP: 09/53766-5Web of Scienc