RESUMO
Parkia biglobosa (subfamily Mimosoideae), a typical tree from African savannas, possess a seed lectin that was purified by combination of ammonium sulfate precipitation and affinity chromatography on a Sephadex G-100 column. The P. biglobosa lectin (PBL) strongly agglutinated rabbit erythrocytes, an effect that was inhibited by d-mannose and d-glucose-derived sugars, especially α-methyl-d-mannopyranoside and N-acetyl-d-glucosamine. The hemagglutinating activity of PBL was maintained after incubation at a wide range of temperature and pH and also was independent of divalent cations. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, PBL exhibited an electrophoretic profile consisting of a single band with apparent molecular mass of 45 kDa. An analysis using electrospray ionization-mass spectrometry indicated that purified lectin possesses a molecular average mass of 47 562 ± 4 Da, and the analysis by gel filtration showed that PBL is a dimer in solution. The complete amino acid sequence of PBL, as determined using tandem mass spectrometry, consists of 443 amino acid residues. PBL is composed of a single non-glycosylated polypeptide chain of three tandemly arranged jacalin-related domains. Sequence heterogeneity was found in six positions, indicating that the PBL preparations contain highly homologous isolectins. PBL showed important antinociceptive activity associated to the inhibition of inflammatory process.
Assuntos
Analgésicos/isolamento & purificação , Anti-Inflamatórios não Esteroides/isolamento & purificação , Fabaceae/química , Dor/tratamento farmacológico , Peritonite/tratamento farmacológico , Lectinas de Plantas/isolamento & purificação , Ácido Acético , Sequência de Aminoácidos , Analgésicos/química , Analgésicos/farmacologia , Animais , Anti-Inflamatórios não Esteroides/química , Anti-Inflamatórios não Esteroides/farmacologia , Carragenina , Contagem de Células , Cromatografia de Afinidade , Testes de Hemaglutinação , Concentração de Íons de Hidrogênio , Camundongos , Dados de Sequência Molecular , Peso Molecular , Monócitos/efeitos dos fármacos , Monócitos/patologia , Neutrófilos/efeitos dos fármacos , Neutrófilos/patologia , Dor/induzido quimicamente , Dor/fisiopatologia , Peritonite/induzido quimicamente , Peritonite/patologia , Lectinas de Plantas/química , Lectinas de Plantas/farmacologia , Multimerização Proteica , Estrutura Terciária de Proteína , Coelhos , Sementes/química , TemperaturaRESUMO
A new galactose-specific lectin, named BBL, was purified from seeds of Bauhinia bauhinioides by precipitation with ammonium sulfate, followed by two steps of ion exchange chromatography. BBL haemagglutinated rabbit erythrocytes (native and treated with proteolytic enzymes) showing stability even after exposure to 60 °C for an hour. The lectin haemagglutinating activity was optimum between pH 8.0 and 9.0 and inhibited after incubation with D-galactose and its derivatives, especially α-methyl-D-galactopyranoside. The pure protein possessed a molecular mass of 31 kDa by SDS-PAGE and 28.310 Da by mass spectrometry. The lectin pro-inflammatory activity was also evaluated. The s.c. injection of BBL into rats induced a dose-dependent paw edema, an effect that occurred via carbohydrate site interaction and was significantly reduced by L-NAME, suggesting an important participation of nitric oxide in the late phase of the edema. These findings indicate that BBL can be used as a tool to better understand the mechanisms involved in inflammatory responses.
Assuntos
Bauhinia/química , Lectinas de Plantas/química , Lectinas de Plantas/isolamento & purificação , Animais , Edema/induzido quimicamente , Eritrócitos/efeitos dos fármacos , Galactose/análogos & derivados , Galactose/química , Hemaglutininas/efeitos dos fármacos , Hemaglutininas/imunologia , Masculino , NG-Nitroarginina Metil Éster/farmacologia , Óxido Nítrico/metabolismo , Lectinas de Plantas/farmacologia , Coelhos , Ratos , Ratos Wistar , Sementes/químicaRESUMO
A lectin from Canavalia maritima seeds (ConM) was purified and submitted to crystallization experiments. The best crystals were obtained using the vapour-diffusion method at a constant temperature of 293 K and grew in 7 d. A complete structural data set was collected to 2.1 A resolution using a synchrotron-radiation source. The ConM crystal belongs to the orthorhombic space group P2(1)2(1)2, with unit-cell parameters a = 67.15, b = 70.90, c = 97.37 A. A molecular-replacement search found a solution with a correlation coefficient of 69.2% and an R factor of 42.5%. Crystallographic refinement is under way.