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

Abstract

Dicyclohexylcarbodiimide-resistant mutants of E. coli were isolated and characterized. In 1 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 .mu.M dicyclohexylcarbodiimide, as measured by inhibition of ATPase [EC 3.6.1.3] 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 activites but a severe inhibition is obtained at 200 .mu.M. The highly water-soluble 1-ethyl-3(3-dimethylaminopropyl)-carbodiimide inhibited the activities of wild-type and mutant membranes 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-32Pi 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.