Unique relationships between the rates of oxidation and phosphorylation and the protonmotive force in rat-liver mitochondria

Abstract

The rate of ATP synthesis ( J P) in isolated rat-liver mitochondria was strongly dependent on the magnitude of the protonmotive force ( Δ~ μ H + ) across the mitochondrial inner membrane. Addition of different concentrations of various uncouplers or malonate to mitochondrial incubations in State 3 led to a depression of Δ~ μ H + and a concomitant decrease in J P. A unique relationship between J( P and Δ~ μ H + was obtained, which was independent of the way in which Δ~ μ H + was varied. This unique relationship was observed when K + (in the presence of valinomycin) was used as a probe for Δψ. Different relationships between J P and Δ~ μ H + were observed when K + was used as a probe for Δψ and when K + was measured after separation of the mitochondria by centrifugation without silicone oil. This led to a serious underestimation of Δψ, specifically when uncouplers were present, and non-unique flow-force relationships were thus obtained. Anomalous relationships between J P and Δ~ μ H + were also found when TPMP + was used as a probe for Δψ. However, in uncoupler incubations the presence of TBP − strongly affected the TPMP + accumulation ratio without any effect on the K + accumulation or on J P and in the presence of TBP − unique relationships between J P and Δ~ μ H + were again obtained. This indicates that the accumulation of TPMP + inside the mitochondria is not a straightforward function of Δψ but also depends on conditions like the presence of TBP − or uncouplers. We conclude that there is a unique relationship between the rate of phosphorylation and the protonmotive force in mitochondria and that under some conditions the behaviour of TPMP + is anomalous.