Specificity of glucose transport in Trypanosoma brucei

Abstract

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 × 10⁸ 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 μM) inhibited uptake. Transport was also unaffected by the H⁺-ATPase inhibitor N,N′-dicyclohexylcarbodiimide (DCCD; 20 μM) and the uncoupler carbonylcyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP; 1 μM). However, highly significant inhibition was obtained with both phloretin (82% at 0.13 mM; K_i= 64 μM) 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°C and 37°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.