RESUMO
A novel peptide from the seeds of Trichosanthes kirilowii, trichokirin-S1, was purified by extraction of protein body, ammonia sulfate precipitation, Blue-gel affinity chromatography, FPLC Mono S ion exchange chromatography and Superose12 gel filtration chromatography. Its molecular weight was determined to be 11,426 by MALDI-TOF MS analysis. Its reaction mechanism to inactive ribosome was the same as that of the ribosome-inactivating protein trichosanthin, a rRNA N-glycosidase. The purified trichokirin-S1 showed a strong inhibitory activity on protein synthesis in cell-free rabbit reticulocyte lysate system, with IC(50) of 0.7 nmol/L. Therefore, trichokirin-S1 may be a promising and efficient toxin moiety of immunotoxins.
Assuntos
Proteínas de Plantas/isolamento & purificação , Sementes/química , Trichosanthes/química , Sequência de Aminoácidos , Animais , Sistema Livre de Células , Relação Dose-Resposta a Droga , Eletroforese em Gel de Poliacrilamida , Dados de Sequência Molecular , Peso Molecular , Extratos Vegetais/química , Extratos Vegetais/isolamento & purificação , Extratos Vegetais/farmacologia , Proteínas de Plantas/química , Proteínas de Plantas/farmacologia , Biossíntese de Proteínas/efeitos dos fármacos , Coelhos , Ratos , Ribossomos/efeitos dos fármacos , Ribossomos/metabolismo , Análise de Sequência de Proteína , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
A peptide, luffin P1, from seeds of Luffa cylindrica, was purified by ammonia sulfate precipitation, CM-52 ion exchange chromatography, Blue-gel affinity chromatography and FPLC Mono S ion exchange chromatography. Its molecular weight was 5226.5 as determined by MALDI-TOF-MS analysis. The sequence of N-terminal 11 amino acids of luffin P1 was identical with the partial N-terminal sequence (from G3 to R13) of 6.5K Arg/Glu rich peptide, which was also isolated from the seeds of Luffa cylindrica. Besides, luffin P1 had a very high homology with a trypsin inhibitor, named C2 peptide, from pumpkin seeds. Interestingly, the purified luffin P1 not only showed a strong inhibitory activity on protein synthesis in rabbit reticulocyte lysate cell-free translation system with IC(50) of 0.6 nmol/L, but also had trypsin inhibitory activity with IC(50) of 22 micromol/L.
Assuntos
Luffa/química , Proteínas de Plantas/isolamento & purificação , Sementes/química , Animais , Sequência de Bases , Cromatografia por Troca Iônica/métodos , Relação Dose-Resposta a Droga , Eletroforese em Gel de Poliacrilamida , Peso Molecular , Proteínas de Plantas/química , Proteínas de Plantas/farmacologia , Biossíntese de Proteínas/efeitos dos fármacos , Coelhos , Ratos , Análise de Sequência de Proteína , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Inibidores da Tripsina/farmacologiaRESUMO
A peptide designated Luffin P1 was purified from the seeds of Luffa cylindrica. Its molecular mass was determined to be 5226.1 Da by MALDI-TOF MS analysis. The purified Luffin P1 shows a strong inhibitory activity on protein synthesis in the cell-free rabbit reticulocyte lysate with IC(50) of 0.88 nM. Its reaction mechanism is the same as that of the ribosome-inactivating protein trichosanthin, which is an rRNA N-glycosidase. Besides, the results of gel filtration chromatography suggested the existence of polymers of Luffin P1 and polymerization of Luffin P1 enhanced its rRNA N-glycosidase activity. Luffin P1 was the smallest peptide yet reported that has translational inhibitory activity. The cDNA and deduced amino acid sequence of Luffin P1 has also been determined.