The Mechanism of Ion Translocation in Mitochondria. 3. Coupling of K+ Efflux with ATP Synthesis

A system is described for the study of the synthesis of ATP by the K+ concentration gradient. The system consists of mitochondria which accumulate K+ phosphate aerobically in the presence of valinomycin and then slowly release K+ upon addition of rotenone. The release of K+ is accelerated several folds by the addition of ADP and during the phase of ADP stimulated K+ efflux a net synthesis of ATP is observed. The amount of ATP synthesised may amount to 15–20 μmoles/g protein. The reaction proceeds for about 2 min and during this period a constant ratio of 4 K+/ATP is observed. The apparent Km for ADP and Pi are 25 and 150 μM, respectively. The synthesis of ATP is strongly dependent on the Ki+/Ko+ ratio; above an external concentration of 3 mM K+ the synthesis of ATP is abolished. The rate of ATP synthesis is also markedly effected by the pH of the medium in that it is stimulated at pH 6.5 and inhibited at pH above 7.5. A high rate of K+ efflux is observed in the presence of ADP, after an osmotic swelling and in alkaline media. With the use of mersalyl it is shown that the K+ efflux can be largely accounted for by H+ uptake. The high rate of proton translocation and the synthesis of ATP during K+ efflux are assumed to involve a reversal of the proton pump which operates the energy linked accumulation of cations.