Active transport of L-proline in the protozoan parasite Trypanosoma brucei brucei.
The characteristics of L-proline transport in the procyclic form of Trypanosoma brucei were studied by using L-[14C]proline and a quick separation technique by centrifugation through an oil mixture. L-Proline uptake displayed typical Michaelis-Menten kinetics, with a Km of 19 microM and a maximum transport velocity of 17 nmol/min per 10(8) cells at 27 degrees C. The maximum concentration gradient factor obtained after 1 min of incubation was 270-fold in 0.02 mM proline. Cells permeabilized with 80 microM digitonin were still able to accumulate 14C label, but to a lower extent. The temperature-dependence of proline uptake gave an apparent activation energy of 74.9 kJ.mol-1. In competition studies with a 10-fold excess of structural analogues, L-alanine, L-cysteine and L-azetidine-2-carboxylate were found to inhibit L-proline uptake. Variation of pH or addition of the protonophore carbonyl cyanide m-chlorophenylhydrazone ('CCCP') did not affect proline transport, showing that it is not driven by a protonmotive force. The absence of Na+, with or without monensin, did not affect proline transport. The absence of K+ and the addition of the Na+,K(+)-ATPase inhibitor ouabain had no significant effect on proline uptake activity. The thiol-modifying reagent iodoacetate (10 mM) decreased proline uptake by half. KCN (1 mM) inhibited proline uptake to a lesser extent, and the degree of inhibition was proportional to the intracellular ATP concentration. Preliminary experiments on proline transport in plasma-membrane vesicles of the cells, using a filtration technique, showed an uptake of proline (0.67 nmol/mg of protein) by the vesicles, but only in the presence of intravesicular ATP. The results thus obtained suggest that the proline carrier system in T. brucei is ATP-driven and independent of Na+, K+ or H+ co-transport.
L'Hostis
C
C
Department of Biochemistry, University of Geneva, Switzerland.
Geindre
M
M
Deshusses
J
J
D000255:Adenosine Triphosphate / Q000378:metabolism; D000409:Alanine / Q000494:pharmacology; D000818:Animals; D001383:Azetidinecarboxylic Acid / Q000494:pharmacology; D001667:Binding, Competitive; D001693:Biological Transport, Active / Q000187:drug effects; D002463:Cell Membrane Permeability / Q000187:drug effects; D003545:Cysteine / Q000494:pharmacology; D004072:Digitonin / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D006909:Hydroxyproline / Q000494:pharmacology; D007461:Iodoacetates / Q000494:pharmacology; D019807:Iodoacetic Acid; D007700:Kinetics; D011190:Potassium Cyanide / Q000494:pharmacology; D011392:Proline / Q000378:metabolism*; D013696:Temperature; D013816:Thermodynamics; D014346:Trypanosoma brucei brucei / Q000378:metabolism*
D007461:Iodoacetates; D001383:Azetidinecarboxylic Acid; D000255:Adenosine Triphosphate; D011392:Proline; D003545:Cysteine; D004072:Digitonin; D011190:Potassium Cyanide; D000409:Alanine; D006909:Hydroxyproline; D019807:Iodoacetic Acid