THE BOUND ADENINE-NUCLEOTIDES OF PURIFIED BOVINE MITOCHONDRIAL ATP SYNTHASE

The experiments in this study were directed towards defining the nucle otide content of purified beef-heart mitochondrial F1F0 ATP synthase d uring binding and hydrolysis of ATP. The purified, soluble synthase as prepared contained 2 mol ATP and 2 mol ADP/mol enzyme. Three of these four nucleotides were exchangeable on incubation with radiolabelled M gATP. Passage of the ATP synthase through a column of Sephadex G-50 re adily removed 1 mol ADP/mol. The remaining bound nucleotides were not displaced by incubation with 1 mM GTP or 5 mM sodium sulfite, the latt er an activator of the ATPase activity of the synthase. Incubation of the synthase with 250 mu M MgATP in the presence of 3 mM sodium azide, an inhibitor of the ATPase, resulted in the transitory formation of a form of the enzyme in which 5-6 nucleotide-binding sites were loaded with ATP and/or ADP, thus showing that the ATP synthase, like the solu ble F-1 ATPase, contained a minimum of six nucleotide-binding sites. T he presence of an ATP-regenerating system during incubation with MgATP resulted in the loading of 5-6 sites to yield a form of the enzyme co ntaining 3-4 mol ATP and 2 mol ADP/mol synthase even after passage thr ough a centrifuged column. Following hydrolysis of the medium MgATP, t he enzyme reached a stable form containing 2 mol ATP and 2 mol ADP/mol synthase. Like the form of the enzyme originally prepared, 1 mol ADP/ mol synthase was readily released. However, this ADP remained bound to the synthase in the presence of GTP if azide was present. These resul ts are discussed in the context of current ideas about nucleotide-bind ing sites on the F-1 ATPase portion of the F1F0 ATP synthase. It is co ncluded that the properties of the sites on the F1F0 synthase show som e differences from those on the F-1 ATPase.