FIGURE 5: T. gondii protein extract dose-dependently diminishes caspase 9 activity triggered by cytochrome c and ATP in a reconstituted in vitro system.

(A, C) Recombinant human Apaf-1 and caspase 9 were incubated with increasing amounts of T. gondii protein lysate or were left untreated. After 1 hour, apoptosome formation was triggered by adding cytochrome c and ATP, and 15 minutes later, caspase 9 activity was determined by fluorimetric measurement of LEHD-AFC cleavage.

(B, D) Recombinant Apaf-1 and caspase 9 were induced to form apoptosomes using cytochrome c and ATP. After 1 hour, preassembled apoptosomes were incubated with increasing amounts of T. gondii protein extract for 45 minutes. Fifteen minutes later, caspase 9 activity was fluorimetrically measured as above.

(C, D) Data represent the increase in substrate cleavage over time from samples as described in (A, B); results are expressed as means ± S.E.M. (n = 3). Background activity in samples without cytochrome c/ATP is indicated by an open circle. Significant differences between activated samples without T. gondii proteins and those incubated with T. gondii proteins have been identified by Student’s t-test (* P < 0.05; ** P < 0.01; *** P < 0.001).

(E) Recombinant and/or purified components were incubated with increasing amounts of T. gondii lysate according to the protocol depicted in (A) but without adding ATP. After SDS-PAGE and Western blotting, proteins were analyzed by immunostaining using specific antibodies as indicated and peroxidase-conjugated secondary antibodies. Immune complexes were visualized by enhanced chemiluminescence. Results are representative for two experiments.