SPECIFICITY OF GLUCOSE-TRANSPORT IN TRYPANOSOMA-BRUCEI - EFFECTIVE INHIBITION BY PHLORETIN AND CYTOCHALASIN-B

Glucose transport in the bloodstream form of the protozoan parasite Trypanosoma brucei was characterized by enzymatically measuring the D-glucose uptake. Uptake kinetics showed a concentration-dependent saturable process, typical for a carrier-mediated transport system, with an apparent K(m) = 0.49 +/- 0.14 mM and V(max) = 252 +/- 43 nmol. min-1. mg cell protein-1 (equal to 2.25 x 10(8) trypanosomes). The specificity of glucose transport was investigated by inhibitor studies. Glucose uptake was shown to be sodium independent; neither the Na+/K+-ATPase inhibitor ouabain (1 mM) nor the ionophor monensin (1-mu-M) inhibited uptake. Transport was also unaffected by the H+-ATPase inhibitor N,N'-dicyclohexylcarbodiimide (DCCD; 20-mu-M) and the uncoupler carbonylcyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP; 1-mu-M). However, highly significant inhibition was obtained with both phloretin (82% at 0.13 mM; K(i) = 64-mu-M) and cytochalasin B (77% at 0.3 mM; K(i) = 0.44 mM), and partial inhibition with phlorizin (14% at 0.5 mM; K(i) = 3.0 mM). In each case, inhibition was noncompetitive, partially reversible (45%) for phloretin and completely reversible for cytochalasin B and phlorizin. Measurement of the temperature-dependent glucose uptake between 25-degrees-C and 37-degrees-C resulted in a temperature quotient of Q10 = 1.97 +/- 0.02 and an activation energy of E(a) = 52.12 +/- 1.00 kJ/mol for glucose uptake. We conclude that glucose uptake in T. brucei bloodstream forms occurs via a facilitated diffusion system, clearly distinguished from the human erythrocyte-type glucose transporter with about a 10-fold higher affinity for glucose and about a 1000-fold decreased sensitivity to the inhibitor cytochalasin B.