FIGURE 8.

Transport of proton by hASCT2 reconstituted in proteoliposomes. Purified hASCT2 was reconstituted in proteoliposomes containing 10 mM glutamine and 0.1 mM pyranine as described in materials and methods. The fluorescence measurement was started by adding 150 μL proteoliposomes to the transport buffer (up to 3 mL final volume) composed by Hepes Tris 20 mM pH 7 and 100 mM Na-gluconate. After ∼1 min, as indicated by the arrow, 5 mM glutamate (thick black line) or 1 mM glutamine (gray line) was added to the sample and fluorescence change was recorded. As a control, the same measurement was performed in the absence of Na-gluconate (black line); in the blank sample, 10 μM HgCl₂ was added at time 0 to the transport buffer (light-gray line). In the figure, sketch of the experimental set up (proteoliposomes); Pyr: pyranine. The fluorescence signal is indicated as arbitrary units (AU). Results are representative of three independent experiments.