Demonstration and quantitation of catalytic and noncatalytic bound ATP in submitochondrial particles during oxidative...

Techniques are described for studying the labeling of ADP and ATP bound to the ATP synthase complex of beef heart submitochondrial particles catalyzing oxidative phosphorylation. These suffice for measurements of bound nucleotides during the time required for a single turnover, during steady state net ATP synthesis, or under quasiequilibrium conditions of ATP formation and hydrolysis. Results show that the "tightly bound" ATP associated with isolated submitochondrial particles does not become labeled by medium [32P]Pi rapidly enough to qualify as an intermediate in ATP synthesis. In contrast to chloroplast preparations, little or no bound [32P]Pi committed to ATP formation is present on particles during steady state synthesis. Also, highly active particles synthesizing ATP from [32P]Pi and filtered after EDTA addition have no detectable bound [32P]ATP even though several ATPs have been made per synthase complex. However, under quasiequilibrium conditions membrane-bound ADP and ATP are present whose labeling characteristics qualify them as intermediates in ATP synthesis. In addition, a hexokinase-accessibility approach shows the presence of a steady level of bound ATP. Lack of detection of bound intermediates under other conditions is regarded as reflecting the ready reversibility of oxidative phosphorylation, with consequent facile cleavage of bound ATP and release of bound Pi.