Identification and biochemical characterization of the plasma membrane glucose transporter of Leishmania donovani.
The plasma membrane glucose transporter of Leishmania donovani, an obligate intracellular protozoan parasite of humans, was specifically labeled, identified, and biochemically characterized. Cytochalasin B, a known inhibitor of D-glucose transport in mammalian cells, but not cytochalasin E inhibited the transport of 2-deoxy-D-glucose in the extracellular promastigote form of this organism. Hydroxysuccinimidyl-4-azido-benzoate was used to photochemically cross-link [3H] cytochalasin B to the glucose transporter in isolated surface membranes and plasma membrane vesicles of L. donovani promastigotes. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the covalently labeled glucose transporter migrated as a 20-30-kDa protein band. This band was eluted from SDS-PAGE gels and subsequently analyzed by isoelectric focusing. The latter revealed two major peaks focusing at pH 6.8 and 6.6 [3H]Cytochalasin B-labeled membrane activity was detergent-solubilized, bound to concanavalin A-agarose beads, and specifically eluted with alpha-methyl mannoside. Analysis of the eluted material by SDS-PAGE revealed a D-glucose-inhibitable cytochalasin B peak with an apparent Mr approximately 20,000. The cumulative results indicate that the glucose transporter of L. donovani promastigotes is a glycoprotein which contains mannose as its major carbohydrate constituent.
D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D003571:Cytochalasin B / Q000494:pharmacology; D003847:Deoxyglucose / Q000378:metabolism; D007700:Kinetics; D007893:Leishmania donovani / Q000378:metabolism*; D009004:Monosaccharide Transport Proteins / Q000378:metabolism*
D009004:Monosaccharide Transport Proteins; D003571:Cytochalasin B; D003847:Deoxyglucose