Biochemical detection of enzymes NTPDase in tachyzoites of Toxoplasma gondii and possible functional correlations

Abstract

This study aimed to investigate the presence and activity of NTPDase enzymes in tachyzoites of Toxoplasma gondii (RH strain), as well as to test an inhibitor of this enzyme. Initially, the strain was properly reactivated through successive passages in mice, followed by a final passage on 10 mice, which were monitored in 5 days. Then, all the animals were anesthetized and decapitated, and the peritoneal fluid containing T. gondii tachyzoites was collected. The parasites were separated from the other cells, and a pellet of tachyzoites was obtained. The protein concentration in the pellet each was determined as detailed in the text. The NTPDase activity in the parasite was tested, using 0 (control), 0.025, 0.05, 0.1, 0.2, 0.4, 0.7, and 1.0 mg mL−1 concentrations. Activity of this enzyme in all concentrations tested was observed. The NTPDase activity (substrates ATP and ADP) increased progressively according to protein increase, up to a certain point, then immediately had reduced activity when higher concentrations of protein were tested. The NTPDase activity (hydrolysis of ATP and ADP) had variations depending on temperature, pH, and concentration of the substrate in the reaction. In tests with an NTPDase inhibitor (azide), it was observed an inhibition in the enzyme activity increased when the concentration of the inhibitor also increased. Based on these results, we conclude that the enzyme NTPDase is present in tachyzoites of T. gondii, and they could be detected biochemically by their activities. Azide is able to inhibit the enzyme detected in this study, which might be an option for chemotherapy.