Involvement of intramitochondrial adenine nucleotides and inorganic phosphate in oxidative phosphorylation of extramitochondrially added adenosine-5'-diphosphate.

Possible direct, product/substrate mediated interactions between the phosphate carrier and/or the adenine nucleotide translocator and the mitochondrial H+-ATPase were investigated by tracer flux experiments. For this purpose the specific radioactivity of ATP synthesized was measured and compared with those of intra- and extramitochondrial precursors. Two experimental strategies were applied: 1) preloading of mitochondria with [32P]Pi before starting oxidative phosphorylation of external ADP; 2) simultaneous addition of tracer amounts of [3H]ADP and [32P]Pi to phosphorylating mitochondria under steady-state conditions. The observed participation of the intramitochondrial Pi pool in oxidative phosphorylation excludes a directly functional interaction between Pi carrier and H+-ATPase. At 5 degrees C an at least partial compartmentation of intramitochondrial ADP during oxidative phosphorylation was found, whereas at 25 degrees C a complete equilibrium of intra- and extramitochondrial [32P]Pi and [3H]ADP could be observed, excluding a functionally important compartmentation of these species in the intramitochondrial space under physiological conditions.