Purification and Characterization of a Novel Intracellular Sucrase Enzyme ofLeishmania donovaniPromastigotes

Abstract

The promastigote stage ofLeishmaniaresides in the sand fly gut, enriched with sugar molecules. Recently we reported thatLeishmania donovanipossesses a sucrose uptake system and a stable pool of intracellular sucrose metabolizing enzyme. In the present study, we purified the intracellular sucrase nearly to its homogeneity and compared it with the purified extracellular sucrase. The estimated size of intracellular sucrase is ~112 kDa by gel filtration chromatography, native PAGE, and substrate staining. However, in SDS-PAGE, the protein is resolved at ~56 kDa, indicating the possibility of a homodimer in its native state. The kinetics of purified intracellular sucrase shows its higher substrate affinity with aKmof 1.61 mM than the extracellular form having aKmof 4.4 mM. The highly specific activity of intracellular sucrase towards sucrose is optimal at pH 6.0 and at 30°C. In this report the purification and characterization of intracellular sucrase provide evidence that sucrase enzyme exists at least in two different forms inLeishmania donovanipromastigotes. This intracellular sucrase may support further intracellular utilization of transported sucrose.