Leucil-aminopeptidase recombinante de L. interrogans
AUTOR(ES)
Hugo de Almeida Silva
DATA DE PUBLICAÇÃO
2008
RESUMO
Screening proteolytic activities in non-pathogenic and as well in pathogenic bacteria of the genus Leptospira, we identified the presence of aminopeptydolitic activity in the latter only. Aiming at clarification of this finding, this work led to the biochemical characterization of the enzyme which appears to be responsible for the leucyl-aminopeptydase activity present in pathogenic Leptospira serotypes. By the cloning og the pepA gene coding a putative cytosolic leucyl-aminopeptidase (LAP), we obtained the active recombinant enzyme, showing phylogenetic and biochemical features similar to those present in the M17 metallopeptidase enzymes family. Like other enzymes belonging to this family, recombinant LAP revealed catalytic activity upon L-Leu-AMC substrate, with optimal activity at pH 8.5, and a slight termophilic behavior, with its optimal temperature at 50 C. Besides, the enzyme presented typical Michaelis-Menten kynetics. It is irreversibly inhibited by EDTA, although other LAPs shows reactivation towards ionic cofactors. Apparently, the enzyme forms a 320 kDa oligomer, structure which the activity on the L-Leu-AMC substrate in eletrophoresis gel seems to be dependent. In adittion, the antiserum against α-HaLAP produced in isogenic mice showed its high antigenicty. This finding suggests that the enzyme could be employed to produce specific antibody, which could be useful in clarifying the differential expression of the peptidase in the course of severe infections. This work opens a new avenue that may lead to elucidation of novel mechanisms exclusively associated with pathogenic species within the genus Leptospira.
ASSUNTO(S)
leucilaminopeptidase (lap) leptospira interrogans caracterização bioquímica anatomia patologica e patologia clinica
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