Arginase activity determination

PC (1×10⁵) were obtained from TE and PBS injected animals or from control and infected animals and the arginase activity was measured in cell lysates as described elsewhere [38, 39]. Briefly, cells were lysed with 50 μl of 0.1% Triton X-100 containing protease inhibitors and the mixture was stirred for 30 min and then 50 μl of 10 mM MnCl₂ with 50 mM Tris-HCl were added to activate the enzyme for 10 min at 56 °C. Arginine hydrolysis was initiated by the addition of 25 μl of 0.5 M L-arginine, pH 9.7 at 37 °C for 45 min. The reaction was stopped using a mixture of acids, and the urea concentration was measured at 540 nm after the addition of 25 μl of α-isonitrosopropiophenone (dissolved in 100% ethanol), which was followed by heating at 95 °C for 45 min. These results are expressed as μg of urea per μg of protein.