FIGURE 3.

SDS-PAGE analysis of purified ATP synthase preparations from the wild-type and single Lys-180 mutant strains. A, the identity of the a-subunit band was determined on a wild-type ATP synthase sample by an acidified chloroform/methanol extraction, which has been shown to extract both a- and c-subunits (48). The extract is shown in the right lane; for comparison, the purified B. pseudofirmus OF4 F₁ is shown on the left, and the purified F₁F₀ is in the middle lane. B, patterns for each of the mutant enzymes side-by-side with the wild-type preparation. The ATP synthases were purified as detailed under “Experimental Procedures.” The samples (2 μg) were resolved on 11% gels and silver stained. C, to facilitate comparison of the a-subunit bands, the region of the small subunits from the gel in B was cropped out, subjected to auto levels in Adobe Photoshop to intensify the bands, and enlarged. The bromphenol blue front was run off the gels in A and B to optimize resolution of the δ- and a-subunits; as a consequence, trace amounts of c-monomer in samples in B and the substantial amount of c-monomer in the chloroform/methanol extract in A were not visualized. The F₁ preparation in A is not His-tagged, and therefore its β-subunit migrated more rapidly than its counterpart in the F₁F₀ preparation.