ATP SYNTHESIS CATALYZED BY THE ATP SYNTHASE OF ESCHERICHIA-COLI RECONSTITUTED INTO LIPOSOMES

The H+-translocating F0F1 ATPase from Escherichia coli (EF(0)F(1)) was purified and reconstituted into preformed reverse-phase liposomes prepared from egg yolk phosphatidylcholine/phosphatidic acid. The EF(0)F(1) liposomes were energized by an acid/base transition (pH(out) = 8.3; pH(in=)5.0) and a superimposed K+/valinomycin diffusion potential ([K+](out) = 100 mM; [K+](in) = 0.6 mM) yielding a maximum rate (turnover number) of ATP synthesis of 27 +/- 8 mol ATP.mol EF(0)F(1)(-1).s(-l)), i.e. 27 +/- 8 s(-l). This reaction was inhibited by NH4Cl or by addition of the F0F1 inhibitor N,N'-dicyclohexylcarbodiimide. The rate of ATP synthesis measured as a function of the phosphate and ADP concentrations, can be described by Michaelis-Menten kinetics with a K-m of 0.7 +/- 0.2 mM for phosphate([ADP] = 200 mu M) and a K, of 27 +/- 7 mu M for ADP ([phosphate] = 5 mM), respectively.