title: Purine nucleobase transport in bloodstream forms of Trypanosoma brucei is mediated by two novel transporters.
abstract: The mechanism and inhibitor sensitivity of hypoxanthine transport by bloodstream forms of Trypanosoma brucei brucei was investigated. The dose response curve for the inhibition of hypoxanthine transport (1 microM) by guanosine was biphasic; approximately 90% of transport activity was inhibited with a Ki value of 10.8 +/- 1.8 microM, but 10% of the activity remained insensitive to concentrations as high as 2 mM. These two components of hypoxanthine transport are defined as guanosine-sensitive (H2) and guanosine-insensitive (H3). Hypoxanthine influx by both components was saturable, but there was a marked difference in their Km values (123 +/- 15 nM and 4.7 +/- 0.9 microM for H2 and H3, respectively) although the Vmax values (1.1 +/- 0.2 and 1.1 +/- 0.1 pmol (10[7] cells)[-1] s[-1], n = 3) were similar. Hypoxanthine uptake via the H2 carrier was inhibited by purine bases and analogues as well as by some pyrimidine bases and one nucleoside (guanosine), whereas the H3 transporter was sensitive only to inhibition by purine nucleobases. H2-mediated hypoxanthine uptake was inhibited by ionophores, ion exchangers and the potential H+-ATPase inhibitors, N,N'-dicyclohexylcarbodiimide (DCCD) and N-ethylmaleimide (NEM). Measurements of the intracellular pH and membrane potential of bloodstream trypanosomes in the presence and absence of these agents established a linear correlation between protonmotive force and rate of [3H]hypoxanthine (30 nM) uptake. We conclude that hypoxanthine transport in bloodstream forms of T. b. brucei occurs by two transport systems with different affinities and substrate specificities, one of which, H2, appears to function as a H+-/hypoxanthine symporter.
authors.affiliation: Research School of Biosciences, University of Kent at Canterbury, UK.
authors.forename: H P
authors.initials: HP
authors.lastname: de Koning
authors.forename: S M
authors.initials: SM
authors.lastname: Jarvis
mesh_terms: D000818:Animals; D001692:Biological Transport; D002352:Carrier Proteins / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006151:Guanosine / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D019271:Hypoxanthine / Q000378:metabolism* / Q000494:pharmacology; D007476:Ionophores / Q000494:pharmacology; D007700:Kinetics; D008564:Membrane Potentials; D018892:Proton-Motive Force; D006180:Proton-Translocating ATPases / Q000037:antagonists & inhibitors; D013379:Substrate Specificity; D014346:Trypanosoma brucei brucei / Q000378:metabolism*
chemical_list: D002352:Carrier Proteins; D004791:Enzyme Inhibitors; D007476:Ionophores; D006151:Guanosine; D019271:Hypoxanthine; D006180:Proton-Translocating ATPases