RESUMO
Aminopeptidase activity was detected in Encephalitozoon intestinalis using a fluorometric assay. The aminopeptidase was capable of hydrolysing different amino acids bound to 7-amino-4-trifluoromethyl coumarin, with maximal activity against the amino acid, leucine. Aminopeptidase activity was localized in E. intestinalis spores and in intracellular stages. Enzymatic activity was inhibited by the traditional aminopeptidase inhibitors, bestatin and its analogue, nitrobestatin. Inhibition with the chelating agents, EDTA and 1,10-phenanthroline, suggested that the enzyme activity belongs to the metalloaminopeptidase class. Subcellular fractionation demonstrated that maximal enzyme activity was localized in the cytosolic fraction. Direct fluorogenic substrate analysis by native polyacrylamide gel electrophoresis estimated a molecular weight of 70.8 kDa. Direct fluorogenic analysis by polyacrylamide ampholyte gel electrophoresis indicated an isoelectric point of 4.8. The enzyme was both heat (> 37 degrees C) and cold (< 0 degrees C) labile with an optimal activity at pH 7.2. This is the first report characterizing a cytosolic aminopeptidase in microsporidia.
Assuntos
Encephalitozoon/enzimologia , Leucina/análogos & derivados , Leucil Aminopeptidase/metabolismo , Animais , Quelantes/farmacologia , Cumarínicos/química , Ácido Edético/farmacologia , Fluorometria , Humanos , Ponto Isoelétrico , Leucina/farmacologia , Leucil Aminopeptidase/antagonistas & inibidores , Leucil Aminopeptidase/isolamento & purificação , Peso Molecular , Fenantrolinas/farmacologia , Inibidores de Proteases/farmacologia , Especificidade por SubstratoAssuntos
Aminopeptidases/antagonistas & inibidores , Encephalitozoon cuniculi/efeitos dos fármacos , Encefalitozoonose/tratamento farmacológico , Encefalitozoonose/mortalidade , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/uso terapêutico , Leucina/análogos & derivados , Peptídeos , Animais , Antibacterianos/farmacologia , Cicloexanos , Modelos Animais de Doenças , Encephalitozoon cuniculi/crescimento & desenvolvimento , Encefalitozoonose/parasitologia , Ácidos Graxos Insaturados/farmacologia , Feminino , Leucina/farmacologia , Leucina/uso terapêutico , Camundongos , Camundongos Nus , O-(Cloroacetilcarbamoil)fumagilol , Testes de Sensibilidade Parasitária/métodos , Sesquiterpenos/farmacologia , Sesquiterpenos/uso terapêuticoRESUMO
The efficacy of cyclosporin A (CsA) treatment against Schistosoma mansoni in mice was compared with treatments that included co-administration of one of two anti-sera (infected rabbit serum (IRS) obtained by repeated infection and a worm membrane antigen anti-serum (WSS) obtained by immunization with worm surface supernatants). These two sera recognized a number of worm antigens but differed in precise detail. Administration of CsA alone to mice harbouring mature infections of S. mansoni reduced worm burdens and preferentially targeted female worms. Sera administered alone had no effect on worm burdens. Co-administration of worm membrane antigen anti-serum (WSS) with CsA reduced worm burden significantly compared with drug treatment alone. Male worms were more susceptible to this combined treatment regime. Anti-infection serum (IRS) had a lesser stimulatory activity in combination with CsA which was not statistically different from the effects of CsA alone on worm burdens. The data suggest that CsA-induced surface damage to the parasite may reveal specific antigens that were previously unavailable for host attack.
