4.4. GTPase Activity Assay

The amount of Pi released during the assembly of FtsZ was measured using BIOMOL Green phosphate reagent (Biomol, Milan, Italy) as described earlier [7]. Briefly, FtsZ (6 µM) was incubated with different concentrations of GTP, ranging from 0 µM to 250 µM, either in the absence or in the presence of 35 µM TL, TRIL and TRILF in 25 mM PIPES/NaOH, pH 6.8 for 30 min at 30 °C. The reaction was performed for 10 min and stopped by addition of 100 µL BIOMOL Green reagent. The Pi release was determined after incubation at 25 °C for 25 min by measuring the absorbance at 620 nm. The background was subtracted from all the readings. The experiment was performed in duplicate. Kinetic parameters were fitted by nonlinear regression with GraphPad Prism 4Project.