A highly selective, high-affinity transporter for uracil in Trypanosoma brucei brucei: evidence for proton-dependent transport.
The presence of an uptake mechanism for uracil in procyclic forms of the protozoan parasite Trypanosoma brucei brucei was investigated. Uptake of [3H]uracil at 22 degrees C was rapid and saturable and appeared to be mediated by a single high-affinity transporter, designated U1, with an apparent Km of 0.46 +/- 0.09 microM and a Vmax of 0.65 +/- 0.08 pmol x (10(7) cells)(-1) x s(-1). [3H]Uracil uptake was not inhibited by a broad range of purine and pyrimidine nucleosides and nucleobases (concentrations up to 1 mM), with the exception of uridine, which acted as an apparent weak inhibitor (Ki value of 48 +/- 15 microM). Similarly, most chemical analogues of uracil, such as 5-chlorouracil, 3-deazauracil, and 2-thiouracil, had little or no affinity for the U1 carrier. Only 5-fluorouracil was found to be a relatively potent inhibitor of uracil uptake (Ki = 3.2 +/- 0.4 microM). Transport of uracil was independent of extracellular sodium and potassium gradients, as replacement of NaCl in the assay buffer by N-methyl-D-glucamine, KCl, LiCl, CsCl, or RbCl did not affect initial rates of transport. However, the proton ionophore carbonyl cyanide chlorophenylhydrazone inhibited up to 70% of [3H]uracil flux. These data show that uracil uptake in T. b. brucei procyclics is mediated by a single high-affinity transporter with high substrate selectivity and are consistent with a nucleobase-H+-symporter model for this carrier.
de Koning
H P
HP
Research School of Biosciences, University of Kent, Canterbury, United Kingdom.
Jarvis
S M
SM
D000818:Animals; D002352:Carrier Proteins / Q000737:chemistry*; D004305:Dose-Response Relationship, Drug; D007700:Kinetics; D009705:Nucleosides; D013997:Time Factors; D014346:Trypanosoma brucei brucei / Q000737:chemistry*; D014498:Uracil / Q000037:antagonists & inhibitors / Q000378:metabolism*
D002352:Carrier Proteins; D009705:Nucleosides; D014498:Uracil