Figure 8.

Effect of SH reagents on the transport activity of hASCT2 reconstituted in proteoliposomes. (A) Effect of DTE. Purified hASCT2 was reconstituted in proteoliposomes prepared without CHEMS (∘, •) or with 75 μg/mg phospholipids CHEMS (□, ■) as described in Materials and Methods. Transport was started by adding 50 μM [³H]glutamine and 50 mM Na-Gluconate in the absence (∘, □) or the presence (∙, ■) of 10 mM DTE, to proteoliposomes containing 10 mM glutamine. Transport was stopped as described in Materials and Methods at the indicated time points. Values were plotted according to the first-order rate equation. (B) Effect of HgCl₂ and NEM. Purified hASCT2 was reconstituted in proteoliposomes prepared without CHEMS (dark gray bars) or with 75 μg/mg phospholipids CHEMS (light gray bars) as described in Materials and Methods. Transport was started by adding 50 μM [³H]glutamine and 50 mM Na-Gluconate, in the presence of 20 μM HgCl₂ or 0.5 mM NEM, to proteoliposomes containing 10 mM glutamine. Transport was measured in 20 min. Transport activity was calculated as the percent of residual activity with respect to the control condition (without any addition in the transport assay). ^*Significantly different from the control sample (proteoliposomes prepared without CHEMS) as estimated by Student's t-test (P < 0.05). In (A,B) data are means ± SD of three independent experiments.