Trans effects on the transport of [¹⁴C]isoleucine and [¹⁴C] leucine in isolated P falciparum trophozoites at 20°C. (A) Effect of isoleucine, leucine, or methionine on the initial (glucose-independent) uptake of [¹⁴C]isoleucine. Transport was measured over 15 seconds and is expressed in terms of the [¹⁴C]isoleucine distribution ratio. Prior to the experiment, isolated parasites were washed twice in amino acid–free saline (solution B), washed once more, then incubated (15 mL; ≥ 15 minutes; 37°C) in either solution B (for the nominally zero-trans samples) or solution B supplemented with 1 mM unlabeled isoleucine, leucine, or methionine (for the exchange samples). At the end of the incubation period the parasites were washed and resuspended in the same solution supplemented with protein synthesis inhibitors [cycloheximide (40 μM) and anisomycin (150 μM)]. The 15-second uptake measurement commenced with the suspension of the parasites at time zero in a solution containing radiolabeled isoleucine, protein synthesis inhibitors, and a sufficient concentration of the appropriate unlabeled amino acid to give a final extracellular amino acid concentration of 1 mM. The black bars (labeled A, C, and E) indicate the results of the nominally zero-trans experiments in which the unlabeled amino acids were (at time zero) present in the extracellular medium but nominally absent from the parasite cyosol. The gray bars (labeled B, D, and F) indicate the results of the exchange experiments in which the parasites were preloaded with the unlabeled amino acids, and the amino acid was therefore present at similarly high concentrations at both the extracellular (cis) and intracellular (trans) faces of the parasite plasma membrane throughout the 15-second uptake period. The data are averaged from 4 separate experiments performed on different days and are shown ± SEM. (B) Exchange of intracellular [¹⁴C]isoleucine for extracellular leucine. Isolated parasites depleted of amino acids (as above) were washed twice and incubated (50 mL; 15-20 minutes; 37°C) in glucose-free saline (solution C) to deplete the cells of ATP, then resuspended (for > 10 minutes at 20°C) in solution C supplemented with protein synthesis inhibitors together with [¹⁴C]isoleucine to preload the cells with radiolabel. A sample was taken to assess the equilibrium concentration of radiolabel then, at time zero, cell suspension (1.08 mL) was mixed with 10 μL of either solution C (•) or solution C containing 108 mM unlabeled leucine to give a final extracellular leucine concentration of 1 mM (○). The concentration of radiolabel within the parasite is expressed in terms of the [¹⁴C]isoleucine distribution ratio. The data are averaged from 4 separate experiments performed on different days and are shown ± SEM. (C) Exchange of intracellular [¹⁴C]leucine for extracellular isoleucine. The efflux of [¹⁴C]leucine from isolated parasites depleted of amino acids and ATP was measured using a protocol similar to that outlined above. The time course was commenced by the addition of either solution C (•) or solution C containing unlabeled isoleucine to give a final extracellular isoleucine concentration of 1 mM (○). The concentration of radiolabel within the parasite is expressed in terms of the [¹⁴C]leucine distribution ratio. The data are averaged from 3 separate experiments performed on different days and are shown ± SEM. Asterisks indicate significant difference between the distribution ratios measured in the presence and absence of extracellular amino acid at the different time points, with ** and * denoting P values less than .005 and less than .01, respectively (paired student t test).