Activity of the wild-type and mutant forms of SecA. (A) Equilibrium titration of wild-type and mutant SecA into 29 nM SecY_268FlEG in the presence of 1 mM AMPPNP. Data were fitted to a binding equation (Eq. 1) giving the K_d values shown. The results are the average of three independent experiments. (B) (Top) steady-state ATPase activity of 0.3 µM wild-type and mutant SecA in the presence of 0.3 µM wild-type or mutant SecYEG proteoliposomes, measured with and without 1 µM proOmpA. (Bottom) relative levels of translocation activity achieved in each case analyzed by anti-proOmpA immunoblot and quantification of translocation activity using ImageJ software; n = 3. See Fig. S1 for a representative blot and time dependency of this experiment.