2.7. ATP synthase activity measurement

A commercially available ATP synthase enzyme ELISA assay (Abcam, Cambridge, MA) was used with modifications to determined ATP synthase activity from cell lysates or from purified recombinant proteins. The kit is designed to immunocapture ATP synthase proteins using the same anti-ATP5A1 antibody that we previously applied for the biotin-switch S-sulfhydration assay. The immunocaptured ATP synthase enzyme operates in the “reverse” direction as the physiological role of ATP synthase: it hydrolyzes ATP to ADP and phosphate. This production of ADP is, in turn, coupled to the oxidation of NADH to NAD⁺, which is monitored as a decrease in absorbance at 340 nm. ATP hydrolysis is inhibited by oligomycin (a specific inhibitor of ATP synthase that inhibits through binding to the F_o domain of the protein). During the cell lysate preparation, the entire ATP synthase enzyme is immunocaptured; we considered the oligomycin-inhibitable component of the ATP consumption as specific ATP synthase activity.