title: A novel sucrose/H+ symport system and an intracellular sucrase in Leishmania donovani.
abstract: The flagellated form of pathogenic parasitic protozoa Leishmania, resides in the alimentary tract of its sandfly vector, where sucrose serves as a major nutrient source. In this study we report the presence of a sucrose transport system in Leishmania donovani promastigotes. The kinetics of sucrose uptake in promastigotes are biphasic in nature with both high affinity K(m) (K(m) of ∼ 75 μM) and low affinity K(m) (K(m)∼ 1.38 mM) components. By contrast the virulent amastigotes take up sucrose via a low affinity process with a K(m) of 2.5mM. The transport of sucrose into promastigotes leads to rapid intracellular acidification, as indicated by changes in the fluorescence of the pH indicator 2',7'-bis-(2-carboxyethyl)-5-(6) Carboxyfluorescein (BCECF). In experiments with right side-out plasma membrane vesicles derived from L. donovani promastigotes, an artificial pH gradient was able to drive the active accumulation of sucrose. These data are consistent with the operation of a H(+)-sucrose symporter. The symporter was shown to be independent of Na(+) and to be insensitive to cytochalasin B, to the flavonoid phloretin and to the Na(+)/K(+) ATPase inhibitor ouabain. However, the protonophore carbonylcyanide P- (trifluromethoxy) phenylhydrazone (FCCP) and a number of thiol reagents caused significant inhibition of sucrose uptake. Evidence was also obtained for the presence of a stable intracellular pool of the sucrose splitting enzyme, sucrase, in promastigote stage parasites. The results are consistent with the hypothesis that L. donovani promastigotes take up sucrose via a novel H(+)-sucrose symport system and that, on entering the cell, the sucrose is hydrolysed to its component monosaccharides by an intracellular sucrase, thereby providing an energy source for the parasites.
authors.affiliation: Division of Infectious Diseases and Immunology, Indian Institute of Chemical Biology, West Bengal, India.
authors.forename: Arpita
authors.initials: A
authors.lastname: Singh
authors.forename: Debjani
authors.initials: D
authors.lastname: Mandal
mesh_terms: D003593:Cytoplasm / Q000737:chemistry; D004791:Enzyme Inhibitors / Q000378:metabolism; D006859:Hydrogen / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D017136:Ion Transport; D007700:Kinetics; D007893:Leishmania donovani / Q000201:enzymology* / Q000378:metabolism*; D012964:Sodium / Q000378:metabolism; D013393:Sucrase / Q000378:metabolism*; D013395:Sucrose / Q000378:metabolism*; D027981:Symporters / Q000378:metabolism*
chemical_list: D004791:Enzyme Inhibitors; D027981:Symporters; D013395:Sucrose; D006859:Hydrogen; D012964:Sodium; D013393:Sucrase