Figure 3.

Effect of cholesterol on transport activity mediated by hASCT2. In (A), purified hASCT2 was reconstituted in proteoliposomes prepared without CHEMS or with 75 μg/mg phospholipids CHEMS as indicated and described in Materials and Methods. Transport assay was started adding 50 μM of [³H]glutamine and 50 mM Na-Gluconate to proteoliposomes containing 10 mM glutamine (black bar) or asparagine (dark gray bar) or threonine (light gray bar) or serine (white bar). Initial transport rates were calculated as k x limit (nmol/mg prot/min) from the first-order rate equation used to plot the time course data. Data are means ± SD of three independent experiments. In (B), HeLa cells were treated with 10 mM methyl-β-cyclodextrin (MCD) as described in Materials and Methods. Transport was started adding 10 μM [³H]glutamine in the presence or in the absence of 100 mM NaCl. Transport activity was calculated as the percent of residual activity with respect to the control condition (without MCD in the transport assay). ^*Significantly different from the control sample (proteoliposomes prepared without CHEMS in A or without MCD in B) as estimated by Student's t-test (P < 0.05).