FIGURE 3.

The 47-kDa adduct involving subunit 6 is associated to the oligomeric forms of the ATP synthase upon oxidation of either mitochondria or digitonin extracts. A and C, mitochondria (A) and digitonin extracts (C) were incubated with or without 2 mm CuCl₂ for 15 min at 30 °C. The reactions were stopped upon the addition of 10 mm EDTA and 10 mm NEM. Mitochondria were then extracted with digitonin at the indicated digitonin-to-protein ratios in the presence of 10 mm NEM. Digitonin extracts were submitted to CN-PAGE. The slab gels were revealed by the in-gel ATPase activity procedure. Fragments of gels corresponding to monomeric and supramolecular forms of ATP synthase were cut, and proteins were separated by SDS-PAGE on a 16% polyacrylamide gel. The slab gel was analyzed by Western blot. B, analysis of fragments from A. su.6., subunit 6. D, analysis of fragments from C. The blots were revealed with a polyclonal antibody raised against the yeast subunit 6.