2.2. ATPase activity measurements

The ATPase enzymatic activities of full-length Hsp72 were assessed by a colorimetric malachite green assay in which orthophosphate ions can directly be detected, as described previously (Yokoyama et al., 2021 ▸). The ATPase reaction was directly performed in microplate wells using a 40 µl sample composed of 0.3 µM full-length Hsp72 (WT, Y149A or T204A), 40 mM Tris–HCl pH 8, 50 mM KCl, 10 mM MgCl₂ and 15–1000 µM ATP in the presence or absence of the co-chaperones DnaJB1 and BAG1. The reaction samples were incubated at 310 K for 180 min (WT and T204A) or 40 min (Y149A) and were then stained by the addition of 160 µl malachite green reagent composed of one volume of 4.2%(w/v) ammonium molybdate in 4 N HCl and three volumes of 0.045%(w/v) malachite green. The absorbance at 620 nm was measured using a FilterMax F5 (Molecular Devices). The values of the Michaelis constant K _m and k _cat were determined by a Lineweaver–Burk double-reciprocal plot for ATP as a substrate.