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 Km = 0.49 +/- 0.14 mM and Vmax = 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 microM) inhibited uptake. Transport was also unaffected by the H(+)-ATPase inhibitor N,N'-dicyclohexylcarbodiimide (DCCD; 20 microM) and the uncoupler carbonylcyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP; 1 microM). However, highly significant inhibition was obtained with both phloretin (82% at 0.13 mM; Ki = 64 microM) and cytochalasin B (77% at 0.3 mM; Ki = 0.44 mM), and partial inhibition with phlorizin (14% at 0.5 mM; Ki = 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 Ea = 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.
Physiologisch-chemisches Institut, Universität Tübingen, Federal Republic of Germany.
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Seyfang
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Duszenko
D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D003571:Cytochalasin B / Q000494:pharmacology*; D004024:Dicyclohexylcarbodiimide / Q000494:pharmacology; D005947:Glucose / Q000378:metabolism*; D007700:Kinetics; D008985:Monensin / Q000494:pharmacology; D010042:Ouabain / Q000494:pharmacology; D010693:Phloretin / Q000494:pharmacology*; D010695:Phlorhizin / Q000494:pharmacology; D013696:Temperature; D013816:Thermodynamics; D014346:Trypanosoma brucei brucei / Q000378:metabolism*
D003571:Cytochalasin B; D004024:Dicyclohexylcarbodiimide; D010042:Ouabain; D008985:Monensin; D010695:Phlorhizin; D005947:Glucose; D010693:Phloretin