Figure 2
Calcium is required for FRET activation triggered by ATP in P. berghei and P. yoelii isolated (saponin treated) free mixed blood stages parasites. (A and B) The rate of peptide hydrolysis induced by ATP (50 μM) as measured by fluorescence was inhibited after incubation with the extracellular and intracellular calcium chelators: EGTA (5 mM) for 5 min (1.0 ± 0.07, n = 6, P = 0.0004) and BAPTA/AM (100, 200 or 500 μM) for 40 min (1.2 ± 0.03, n = 11, P = 0.0002; 1.18 ± 0.01, n = 13, P < 0.0001; 1.2 ± 0.03, n = 5, P = 0.005, respectively) P values were calculated by comparison with the ATP (50 μM) data (1.5 ± 0.07, n = 9) in P. berghei parasites. P. yoelii parasites were also incubated with EGTA (5 mM) (1.02 ± 0.08, n = 7, P = 0.0009) and BAPTA/AM (25, 100, 200 or 500 μM) for 40 min (1.4 ± 0.07, n = 7, P = 0.823; 1.2 ± 0.03, n = 10, P = 0.002; 1.2 ± 0.03, n = 10, P = 0.017; 1.2 ± 0.03, n = 10, P = 0.022, respectively). P values were calculated by comparison with the ATP (50 μM) data (1.4 ± 0.06, n = 12). Isolated parasites (108 cells ml-1) were incubated in MOPS buffer without CaCl2. The fluorescence was measured continuously (acquisition rate - every 0.5 seconds) for 400 seconds.