CHARACTERIZATION OF A NUCLEOSIDE PROTON SYMPORTER IN PROCYCLIC TRYPANOSOMA-BRUCEI-BRUCEI/

Adenosine transport at 22 degrees C in procyclic forms of Trypanosoma brucei brucei was investigated using an oil-inhibitor stop procedure f or determining initial rates of adenosine uptake in suspended cells. A denosine influx was mediated by a single high affinity transporter (K- m 0.26 +/- 0.02 mu M, V-max 0.63 +/- 0.18 pmol/10(7) cells s(-1)). Pur ine nucleosides, with the exception of tubercidin (7-deazaadenosine), and dipyridamole inhibited adenosine influx (K-i 0.18-5.2 mu M). Purin e nucleobases and pyrimidine nucleosides and nucleobases had no effect on adenosine transport. This specificity of the transporter appears t o be similar to the previously described P1 adenosine transporter in b loodstream forms of trypanosomes. Uptake of adenosine was Na+-independ ent, but ionophores reducing the membrane potential and/or the transme mbrane proton gradient (monitored with the fluorescent probes bis-(1,3 -diethylthiobarbituric acid)-trimethine oxonol and 2',7'-bis(carboxyet hyl)-5,6-carboxyfluorescein acetoxymethyl ester, respectively) inhibit ed adenosine transport. Similarly, an increase in extracellular pH fro m 7.3 to 8.0 reduced adenosine influx by 30%. A linear correlation was demonstrated between the rate of adenosine transport and the protonmo tive force. Adenosine uptake was accompanied by a proton influx in bas e-loaded cells and was also shown to be electrogenic. These combined r esults indicate that transport of adenosine in T. brucei brucei procyc lics is protonmotive force-driven and strongly suggest that the adenos ine transporter functions as an HC symporter.