Localisation of adenine nucleotide-binding sites on beef-heart mitochondrial ATPase by photolabelling with 8-azido-ADP and 8-azido-ATP.
1. In addition to the previously studied 8-azido-ATP, 8-azido-ADP is a suitable photoaffinity label for beef-heart mitochondrial ATPase (F1). 2. Photolysis at 350 nm of 8-azido-ADP in the presence of isolated F1 leads to inactivation of ATPase activity. Both ATP and ADP (but not AMP) protect against the inactivation. 3. In the absence of Mg2+, 8-azido-ADP binds almost equally to the alpha and beta subunits of F1, whereas in the presence of Mg2+ the alpha subunits are predominantly labelled. 4. The ATPase activity is completely inhibited when two molecules of 8-azido-ADP are bound per molecule F1. 5. 8-Azido-ATP and ATP are competitive substrates for F1, indicating that in the presence of Mg2+ 8-azido-ATP binds to the same site as ATP. 6. The amount of tightly bound nucleotides in F1 is not significantly changed upon incubation with 8-azido-ATP either in the light or the dark. 7. 8-Azido-ATP is also a suitadrial particles, photolabelling leading to inactivation of ATPase activity. 9. Oxidative phosphorylation and the ATP-driven reduction of NAD+ by succinate are also inhibited by photolabelling Mg-ATP particles with 8-azido-ATP. 10. In contrast to the uncoupled ATPase activity, where the two ATP-binding sites do not interact, cooperation between the two sites is required for ATP hydrolysis coupled to reduction of NAD+ by succinate.
D000244:Adenosine Diphosphate / Q000031:analogs & derivatives* / Q000494:pharmacology; D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000494:pharmacology; D000345:Affinity Labels; D000818:Animals; D001386:Azides; D001665:Binding Sites; D001667:Binding, Competitive; D002417:Cattle; D007700:Kinetics; D046911:Macromolecular Substances; D008274:Magnesium / Q000494:pharmacology; D008929:Mitochondria, Heart / Q000201:enzymology*; D010782:Photolysis; D011485:Protein Binding; D013367:Submitochondrial Particles / Q000201:enzymology
D000345:Affinity Labels; D001386:Azides; D046911:Macromolecular Substances; C017040:8-azidoadenosine 5'-triphosphate; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; C021622:8-azidoadenosine diphosphate; D008274:Magnesium