Colorimetric ATPase Activity Assay

A malachite green/molybdate‐based colorimetric assay (Biomol Green reagent, Enzo Life Sciences) was used to assess ATPase activity as described previously by us and others (D'Arcy et al., 2019 (link); Rule et al., 2016 (link)). Briefly, reactions containing 20 μM enzyme were assembled in 20 mM HEPES pH 8.5, 13 mM NaCl, 1% glycerol, 5 mM ATP, and 5 mM MgCl₂ in a total volume of 30 μl. Reactions were initiated by the addition of ATP and MgCl₂, and at the indicated time points (0, 15, 30, 45, and 60 min), 5 μl aliquots were removed and quenched by diluting into 245 μl 1 × HNG buffer followed by flash freezing in liquid nitrogen. To determine the amount of orthophosphate released, Biomol Green reagent was added to each well and absorbance was measured at 620 nm using a Tecan Infinite M1000 Pro multimode plate reader. The mean ± SEM was derived from the cumulative data generated from three separate experiments.