| Abstract: | Mutations in the uncA gene of Escherichia coli cause loss of both oxidative phosphorylation and ATP-driven generation of the transmembrane proton gradient. The uncA gene encodes the alpha-subunit of the F1-sector of the E. coli membrane proton-ATPase. F1-alpha-subunit from normal (unc+) E. coli binds ATP tightly (KD = 0.1 microM) and undergoes a large ATP-induced conformational change, but the functional role of the ATP-binding site is currently unknown. There is disagreement in the literature as to whether the ATP-binding site is present or lacking in F1-alpha-subunit from uncA mutant strains. One obstacle in studying this question is the difficulty of purifying mutant alpha-subunits in native form. In order to circumvent this difficulty we have studied ATP binding and ATP-induced conformational changes in mixtures of F1 subunits obtained by dissociating uncA mutant F1. Anti-alpha antibody was used in conjunction with immunoblotting to identify the alpha-subunits in the mixtures. Retention of native conformation by the alpha-subunits was demonstrated by the fact that the dissociated alpha-subunits were fully competent to repolymerize with other F1 subunits to yield intact F1 aggregate. The results show that, contrary to previous reports, alpha-subunits from three catalytically defective uncA mutants do indeed bind ATP and do undergo an ATP-induced conformational change. The binding affinity of alpha-subunit for ATP was lower than normal in each of the three mutants, but this is not likely to be a significant factor under physiological conditions. |
