V-ATPase activity assay

ATPase activity was measured as the liberation of ³²Pi from [γ−³²P]ATP²¹. The assay was carried out in a total volume of 200 μl under the following conditions: 4 μl phosphatidylserine (26 μM concentration), 5 μl V-ATPase in buffer C (at 10 nM concentration), and 189 μl ATPase assay solution A (30 mM KCl, 50 mM Tris-MES, pH 7.0, 3 mM MgCl₂, and 3 mM [γ−³²P]ATP (400 cpm/nmol)) and 2 μl of ethanol or ethanol dissolved bafilomycin A1 (Sigma-Aldrich, 1 μM concentration), The V-ATPase was first incubated with phosphatidylserine for 2 min, and then the reaction was started by addition ATPase assay solution A and ethanol/bafilomycin A1, and continued for 10 min at 37 °C. The ATP hydrolysis reaction was terminated by adding 1.0 ml of 1.25 N perchloric acid, and the released ³²Pi was extracted and counted in a Beckman scintillation counter⁴⁴. The results were expressed as specific activity (μmol of Pi/min per mg of protein), The experiment has been repeated twice, and the results were performed using GraphPad Prism8.