Figure 1
Extracellular ATP triggers intracellular protease activity in P. yoelii and P. berghei . (A) Graphical representation of resonance energy transfer (FRET) peptide hydrolysis (peptide Abz-AIKFFARQ-EDDnp) in the presence of ATP (200 μM) and the control in isolated (saponin treated) free mixed blood stages of P. berghei parasites. (B) Fluorescence trace and confocal imaging of Abz-AIKFFARQ-EDDnp hydrolysis. ATP (200 μM) addition is indicated by the arrow. Fluorescence (fl) and phase contrast (ph) before and after ATP addition are below the trace. (C and D) Bar graph analyses of peptide hydrolysis at different concentrations of ATP (25, 50, 200 and 250 μM) relative to the control (1.2 ± 0.03, n = 12, P = 0.0332; 1.5 ± 0.07, n = 9, P = 0.0007; 1.7 ± 0.075, n = 17, P < 0.0001 and 1.33 ± 0.04, n = 10; P = 0.0048) and (1.17 ± 0.07, n = 5, P = 0.425; 1.4 ± 0.06, n = 12, P = 0.0039; 1.7 ± 0.15, n = 16, P = 0.0267 and 1.7 ± 0.2, n = 8; P = 0.0228) for P. berghei and P.yoelii parasites, respectively. P values were calculated by comparison with the control (ctr) (1.05 ± 0.08, n = 6) and (1.22 ± 0.03, n = 6), respectively. Isolated parasites (108 cells ml-1) were incubated in MOPS buffer with 1 mM calcium in a 1 ml cuvette. The fluorescence was measured continuously (acquisition rate: every 0.5 seconds)1 min after addition of the peptide Abz-AIKFFARQ-EDDnp (10 μM) for 400 seconds.