Figure 5

Comparison of glycerol uptake and incorporation into PC between of WT and PbAQP-null merozoites. (A) Uptake of [³H]glycerol by WT and PbAQP(-) hepatic merozoites isolated from merosomes. The uptake of radiolabeled glycerol by hepatic merozoites (close circles for WT and open circles for knockout) was measured after 2, 4, 6, 10, 30 and 60 min incubation at 37 °C, followed by rapid spin through oil and radioactivity determination of the samples. In parallel assays performed on WT merozoites, a 10-times excess of unlabeled glycerol (corresponding to 10 mM) was added to the incubation medium (triangles). Data in cpm normalized to mg protein, are means ± S.D., n = 4 independent assays. (B) Incorporation of [³H]glycerol and [¹⁴C]choline into PC of WT and PbAQP(-)merozoites. After 4 h incubation at 37°C, parasites were washed, their lipid extracted and run on TLC plates for PC separation. Spots corresponding to PC revealed by iodine vapor were cut and counted for radioactivity. Data in cpm for [³H]PC and [¹⁴C]PC normalized to mg protein, are means ± S.D., n = 3 independent assays. *p < 0.001. (C) Quantification of PC in WT and PbAQP(-)merozoites using a commercial enzymatic kit containing phospholipase, choline oxidase, peroxidase, and 4-aminoantipyrin. The quantification of PC was performed after 15 min by measuring the absorption at 492 nm. Values are means ± S.D., n = 3 independent assays. *p < 0.05.