1. It is not clear how the Pryrophosphate Reagent can be used for kinase activity measurement. This reagent is supposed for measuring pyrophosphate content.
2. The acceptor substrate for PhoQ histidine kinase assay was not specified in the paper. It is mentioned in the Introduction that the kinase can phosphorylate itself and PhoP. Is PhoQ kinase activity is measured on itself? If so, the signal must be very week, as 25 umol/L of the kinase was used in the reaction. Assuming the reaction volume is 100 uL in the 96-well plate, the maximal reduction of NADH will be 2.5 nmol, which corrsponds to ~15 mili OD (using absorbance coefficient of 6180 M-1), which is unlikely to give out a good signal during the assay in the presence of an inhibitor and only portion of NADH is consumed. Please explain how the kinase activity was measured.
