1 Introduction p Plasmodium falciparum [1–4] Toxoplasma gondii [5,6] [7] P. falciparum [8] [9–13] P. falciparum [14] P. falciparum [15] 2 Materials and methods 2.1 Chemicals 3 −1 −1 3 −1 −1 3 −1 −1 d l d d d d 2.2 Parasite culture P. falciparum 2 2 2 d [16] [17,18] [19] 2.3 Uptake assay of radiolabelled folates 7 8 7 8 All cells, washed extensively to remove folate present in the culture medium, were mixed with or without glucose (20 mM final concentration) and inhibitors as appropriate, together with radiolabel (normally 1 μCi/ml, equivalent to 38 nM, unless otherwise stated) to make up a total volume of 100 μl with PBS or alternative buffer. All the components were prewarmed to 37 °C before the start of the assay, the reaction mix incubated at 37 °C normally for 30 min, and stopped by addition of 1 ml ice-cold PBS. When assaying for folate uptake by whole parasitised red cells, these were spun down after the uptake period and the pellet washed with 1 ml ice-cold PBS at least three times to bring extracellular label down to the background level. The washed parasite pellet obtained by subsequent saponin treatment was then lysed with 0.02% SDS before counting. Uptake by naked parasites was determined in the same way. Unless otherwise indicated, the uptake values represent labelled folate that has been imported into synchronised trophozoite stage parasites. All assays were routinely performed in triplicate on different batches of parasites unless otherwise indicated, and the data expressed as the mean ± S.D. 2.4 Extraction and affinity purification of folate derivatives g [20] 3 2.5 pH measurements 3 [21] 8 2 [21] 3 Results 3.1 Uptake of folates over the asexual erythrocytic cycle P. falciparum [22] Fig. 1 Fig. 1 P. falciparum 3 [23] Fig. 1 3.2 Kinetics of folate uptake R 2 Fig. 2 Fig. 2 Fig. 2 [24] Fig. 2 [25,26] Toxoplasma [6] K m Fig. 2 K m v max 7 7 3.3 Export of folinic acid Fig. 3 Lactobacillus salivarius [27,28] P. falciparum [2,23,20] [23,20] [23,20] 3.4 The dependence of folate uptake on carbon source Fig. 4 P. falciparum Fig. 4 d d P. falciparum [29,30] l d d d Fig. 4 P. falciparum [31] 3.5 Effect of pH on folate uptake [21,32] [21] Fig. 5 Fig. 5 Fig. 5 [21] 2 3.6 Effect of perturbing the plasma membrane proton gradient on folate uptake 50 Fig. 6 Table 1 + + Fig. 6 [33] P. falciparum [21,32] 50 Fig. 6 3.7 Metal ion dependency of folate uptake + + 2+ 3.8 Effect of antifolate drugs and other inhibitors on folate uptake Table 1 [4] 50 50 Table 1 [21] [34,35] [15] [36–38] [39–42] [11] 50 50 Fig. 7 Table 1 50 50 Fig. 7 4 Discussion P. falciparum [7] P. falciparum Leishmania [43–45] P. falciparum Lactobacillus casei L. salivarius [27] [2,23,20] K m P. falciparum Lactobacillus [27] Xenopus [46] Leishmania [47] P. falciparum v max [11] [48,49,10] v max [50] [39,41,42] [51–53] [51] [25,53] [54] [34] [4] P. falciparum d [21,8] d [30] l d d d P. falciparum [21] + + 2+ Lactobacillus [55,27] [32] [21,32] P. falciparum + [21,32] [56] P. falciparum [57,58] [32] [32] [8] [12] + + + P. falciparum [59] http://www.plasmodb.org Leishmania [47,45] Synechocystis Arabidopsis [60] Toxoplasma gondii [6] [59] P. falciparum