H+ and cation movements associated with ADP, ATP transport in mitochondria.

Abstract

H+ and K+ movement associated with the mitochondrial ADP‐ATP exchange have been measured by glass electrode recordings in order to investigate the energy control of the ADP‐ATP exchange. All measurements are taken in the presence of oligomycin and inhibitors of electron transport in order to exclude other types of H+ transfer. ATP exchange against endogenous ADP as induced by addition of ATP gives only a small H+ uptake which is partially suppressed in a KCl medium. H+ uptake is increased strongly on addition of uncoupler. On addition of valinomycin, K+ is taken up instead of H+. With carbonylcyanide p‐trifluoromethoxyphenylhydrazone (FCCP) and valinomycin respectively, equivalent amounts of H+ and K+ are taken up over a wide range of protein and ATP concentration. The ATP dependence gives a ratio H+ per ATP added = 0.38 and K+ per ATP added = 0.27. Saturation with ATP is reached at about 14 μmol ATP/g protein for H+ uptake and 20 μmol ATP/g protein for K+ uptake.The FCCP‐facilitated, ATP‐induced H+ uptake increases with endogenous content of ADP, giving a ratio H+/ADP = 0.67. Kinetics of the H+ uptake and ATP‐induced nucleotide exchange are closely correlated to about 0.62 H+/nucleotide released. The temperature dependence of the H+ uptake rate measured between 4 and 11 °C gives an activation energy = 33 kcal (138 kJ), similar to that found for the exchange rate. In the exchange of ADP against endogenous ATP as started by addition of ADP, H+ are released in the absence of FCCP. In the presence of FCCP, H+ release is only marked when endogenous nucleotides are preloaded with external ATP. This H+ release is decreased by Pi and enhanced by N‐ethylmaleimide, blocking Pi transfer. It varies according to endogenous ATP content reaching the ratio H+ released/endogenous ATP = 0.3 to 0.5. These data have been interpreted to indicate that the ATPe‐ADPi exchange is largely electrical and the opposite ADPe‐ATPi exchange is only between 50 to 70% electrical. The implications for energy transfer are discussed.