Figure 2.

Formation of the γ−ɛ−cc′ cross-link through disulfide bonds in the E. coli F₁F_o mutant. (A) Purified E. coli F₁F_o mutant was incubated with 100 μM CuCl₂ to induce the cross-linking. As a control, 1 mM DTT was added instead of CuCl₂. The samples were loaded on SDS/polyacrylamide (12–20%) gel, subjected to electrophoresis, and stained with Coomassie brilliant blue. The dissociation buffer for SDS/PAGE contained 40 mM N-ethylmaleimide but no reducing agent. (B) Anti-γ subunit immunoblotting. Inner membranes from the wild-type and mutant F₁F_o were exposed to 100 μM CuCl₂. The γ−ɛ–cc′ cross-link was confirmed with anti-γ, ɛ, and c subunit immunoblotting. (The data from the anti-ɛ and c subunit antibodies are not shown.)