Biochemical characterization and sub-cellular localization of an aldo-keto reductase from Trypanosoma cruzi

GARAVAGLIA, PA; CANNATA, JB; MAUGERI, D; RUIZ, AM; GARCIA, GA

Mar del Plata, Buenos Aires, Argentina

Congreso; XLIII Reunión Anual Sociedad Argentina de Investigación Bioquímica y Biología Molecular; 2007

Sociedad Argentina de Investigación Bioquímica y Biología Molecular

A novel aldo-keto reductase (AKR) gene from T.cruzi, TcAKR, was cloned and expressed in E. coli. The recombinant TcAKR showed NADPH dependent reductase activity with two commonly used AKR substrates, 4-nitrobenzaldehyde (4-NBA) and 2-hydroxyacetone (2-DHA), with Km values of 0.67 mM and 30 mM, respectively and 0.026 mM for NADPH. The pH optimum for both substrates was 6.5. No activity was detected with NADH as a co-factor. The native TcAKR was also partially purified from epimastigotes of T cruzi by affinity chromatography on Cibacron Blue-Sepharose and showed a specific activity for 4-NBA of 0.27 U/mg protein. The sub-cellular localization of TcAKR in epimastigote and trypomastigote stages of T. cruzi was investigated by electronic microscopy using an anti-recombinant TcAKR serum. Immunogold staining showed a widespread localization throughout the parasite. Otherwise, the release profile of TcAKR from intact epimastigotes by titration with digitonin was similar to the cytosolic marker pyruvate kinase. Altogether, these results suggest TcAKR is mainly localized in cytosol.