FIGURE 5.

Effect of pH on the kinetics of glutamate transport by hASCT2 reconstituted in proteoliposomes. Purified hASCT2 was reconstituted in proteoliposomes prepared at the indicated pH and containing 10 mM glutamine, as described in materials and methods. In (A), the transport was started by adding indicated concentrations of [³H]glutamate and 50 mM Na-gluconate to proteoliposomes buffered at different pH: pH 6.0 (○), pH 6.5 (●), and pH 7 (●). The transport was measured in 15 min according to the stop inhibitor method. Data were plotted according to linear Lineweaver–Burk equation as reciprocal transport rate vs. reciprocal glutamate concentration. In (B), data from Figure 5A were replotted according to Lineweaver-Burk equation as reciprocal transport rate vs. reciprocal proton concentration, performed at different glutamate concentration: 0.5 mM (○), 0.6 mM (●), 0.8 mM (), 1.2 mM (), 2 mM (△), and 5 mM (). The derived pKa values were as follows: 7.0 ± 0.2, 6.8 ± 0.2, 6.9 ± 0.2, 6.8 ± 0.2, 6.8 ± 0.2, 7.0 ± 0.2 at 0.5, 0.6, 0.8, 1.2, 2, and 5 mM glutamate, respectively. Results are means ± SD from five independent experiments.