RESUMO
The Saccaromices cerevisiae D-serine dehydratase is a pyridoxal 5'-phosphate dependent enzyme that requires zinc for its function. It catalyses the conversion of D-serine into pyruvate and ammonia with the K(m) and k(cat) values of 0.39 mM and 13.1 s(-1) respectively. In this work, a new methodology for monitoring D-serine is presented. Our results show that this enzyme could be successfully used as a biological probe for detection of D-serine via fluorescence spectroscopy.
Assuntos
Técnicas Biossensoriais/métodos , Hidroliases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Absorção , Fluoresceína/química , Corantes Fluorescentes/química , Hidroliases/química , Modelos Moleculares , Fosfato de Piridoxal/metabolismo , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Serina/análise , Serina/química , Serina/metabolismo , Espectrometria de Fluorescência/métodosAssuntos
Bacillus subtilis/fisiologia , Proteínas de Bactérias/genética , Esporos Bacterianos , Proteínas de Bactérias/química , Sequência de Bases , Primers do DNA , Dimerização , Proteínas de Fluorescência Verde/genética , Plasmídeos , Proteínas Recombinantes/química , Proteínas Recombinantes/genéticaRESUMO
A T helper epitope was expressed in three innovative delivery vehicles recently developed in our laboratories and based respectively, on the filamentous bacteriophage fd, the E2 protein from the PDH complex of Bacillus stearothermophilus and the protein CotC of Bacillus subtilis spores. Studies of antigenicity and immunogenicity were performed by using a specific T cell hybridoma and by priming mononuclear cells isolated from the venous blood of human donors. The results indicate that the E2 system is the best suited for inducing a specific immune response towards a CD4 T cell epitope. Importantly, TCR clonal analysis demonstrated the persistence over years of a previously described antigen specific clonotype and its presence correlates with the immunogenic strength of the antigen delivery system.
