title: Glucose uptake occurs by facilitated diffusion in procyclic forms of Trypanosoma brucei.
abstract: The glucose transporter of Trypanosoma brucei procyclic forms was characterized and compared with its bloodstream form counterpart. Measuring the glucose consumption enzymatically, we determined a saturable uptake process of relatively high affinity (Km = 80 microM, Vmax = 4 nmol min-1 10(-8) cells), which showed substrate inhibition at glucose concentrations above 1.5 mM (Ki = 21 mM). Control experiments measuring deoxy-D-[3H]Glc uptake under zero-trans conditions indicated that substrate inhibition occurred on the level of glycolysis. Temperature-dependent kinetics revealed a temperature quotient of Q10 = 2.33 and an activation energy of Ea = 64 kJ mol-1. As shown by trans-stimulation experiments, glucose uptake was stereospecific for the D isomer, whereas L-glucose was not recognized. Inhibitor studies using either the uncoupler carbonylcyanide-4-(trifluoromethoxy)phenylhydrazone (5 microM), the H+/ATPase inhibitor N,N'-dicyclohexylcarbodiimide (20 microM), the ionophor monensin (1 microM), or the Na+/K+-ATPase inhibitor ouabain (1 mM) showed insignificant effects on transport efficiency. The procyclic glucose transporter was subsequently enriched in a plasma-membrane fraction and functionally reconstituted into proteoliposomes. Using Na+-free conditions in the absence of a proton gradient, the specific activity of D-[14C]glucose transport was determined as 2.9 nmol min-1 (mg protein)-1 at 0.2 mM glucose. From these cumulative results, we conclude that glucose uptake by the procyclic insect form of the parasite occurs by facilitated diffusion, similar to the hexose-transport system expressed in bloodstream forms. However, the markedly higher substrate affinity indicates a differential expression of different transporter isoforms throughout the lifecycle.
authors.affiliation: Physiologisch-chemisches Institut, Universität Tübingen, Germany.
authors.forename: U
authors.initials: U
authors.lastname: Wille
authors.forename: A
authors.initials: A
authors.lastname: Seyfang
authors.forename: M
authors.initials: M
authors.lastname: Duszenko
mesh_terms: D000818:Animals; D001692:Biological Transport; D003847:Deoxyglucose / Q000378:metabolism; D004058:Diffusion; D005947:Glucose / Q000037:antagonists & inhibitors / Q000378:metabolism*; D007700:Kinetics; D008081:Liposomes / Q000378:metabolism; D008565:Membrane Proteins / Q000302:isolation & purification / Q000378:metabolism; D009004:Monosaccharide Transport Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D015800:Protozoan Proteins / Q000378:metabolism; D013237:Stereoisomerism; D014346:Trypanosoma brucei brucei / Q000254:growth & development / Q000378:metabolism*
chemical_list: D008081:Liposomes; D008565:Membrane Proteins; D009004:Monosaccharide Transport Proteins; D015800:Protozoan Proteins; D003847:Deoxyglucose; D005947:Glucose