A Mutant ATP Synthetase of Escherichia coli with an Altered Sensitivity to N,N′‐Dicyclohexylcarbodiimide: Characterization in Native Membranes and Reconstituted Proteoliposomes

Dicyclohexylcarbodiimide‐resistant mutants of Escherichia coli were isolated and characterized. In one mutant the mutation is closely linked to the unc genes and affects the membrane‐integrated part of the ATP synthetase. The sensitivity of ATP synthetase functions to N,N′‐dicyclohexylcarbodiimide was compared in wild‐type and mutant membranes. The membrane‐integrated part of the wild‐type ATP synthetase is highly sensitive to 50 μM dicyclohexylcarbodiimide, as measured by inhibition of ATPase activity, ATP‐dependent membrane energization and restoration of lactate‐dependent energization of ATPase‐depleted membranes. In mutant membranes this concentration has only a slight effect on these activities whereas a severe inhibition is obtained at 200 μM. Using the highly water‐soluble 1‐ethyl‐3(3‐dimethylaminopropyl)‐carbodiimide the activities of wild‐type and mutant membranes are inhibited to the same extent. The ATP synthetase of wild‐type and mutant was partially purified and incorporated into liposomes. These showed an uncoupler‐sensitive ATP‐³²P_i exchange and ATP‐dependent quenching of acridine‐dye fluorescence. The activities of mutant and wild‐type proteoliposomes exhibit the same pattern of sensitivity to dicyclohexylcarbodiimide as the corresponding membranes.