Glucose uptake occurs by facilitated diffusion in procyclic forms of Trypanosoma brucei

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 (K-m = 80 mu M, V-max = 4 nmol min(-1) 10(-8) cells), which showed substrate inhibition at glucose concentrations above 1.5 mM (K-i = 21 mM). Control experiments measuring deoxy-D-[H-3]Glc uptake under zero-trans conditions indicated that substrate inhibition occurred on the level of glycolysis. Temperature-dependent kinetics revealed a temperature quotient of Q(10) = 2.33 and an activation energy of E(a) = 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 mu M), the H+/ATPase inhibitor N,N'-dicyclohexylcarbodiimide (20 mu M), the ionophor monensin (1 mu M), 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-[C-14]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.