RESUMO

The gene of Leishmania mexicana enolase was cloned and overexpressed in Escherichia coli as an active enzyme; the protein was biochemically analyzed. This enolase shares with enolases from other trypanosomatids the presence of three atypical residues, each with a reactive side group, near the active site, already described for the enzyme from Trypanosoma brucei. The natural enzyme was purified, using a three-step procedure, from a cytosolic fraction of L. mexicana promastigotes. The kinetic properties of the purified recombinant enzyme were similar to those of the natural enzyme. Both the recombinant and natural enzyme were inhibited by inorganic pyrophosphate. Subcellular localization analysis after differential centrifugation showed that the enzyme activity is only associated with the cytosolic fraction. However, an apparently inactive form of enolase was detected by Western blots in the microsomal fraction. Digitonin treatment of parasites and immunofluorescence studies with permeabilized and non-permeabilized parasites showed that enolase is also associated with membranes and it was found at the external face of the plasma membrane.

Assuntos

Leishmania mexicana/enzimologia , Fosfopiruvato Hidratase/química , Fosfopiruvato Hidratase/genética , Sequência de Aminoácidos , Animais , Anticorpos Antiprotozoários/imunologia , Sequência de Bases , Western Blotting , Permeabilidade da Membrana Celular/efeitos dos fármacos , Clonagem Molecular , Digitonina/farmacologia , Eletroforese em Gel de Poliacrilamida , Imunofluorescência , Indicadores e Reagentes/farmacologia , Cinética , Leishmania mexicana/genética , Leishmania mexicana/imunologia , Camundongos , Dados de Sequência Molecular , Fosfopiruvato Hidratase/biossíntese , Fosfopiruvato Hidratase/isolamento & purificação , Coelhos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Alinhamento de Sequência