LOCALIZATION OF ADENINE NUCLEOTIDE-BINDING SITES ON BEEF-HEART MITOCHONDRIAL ATPASE BY PHOTOLABELING WITH 8-AZIDO-ADP AND 8-AZIDO-ATP

1. 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. 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. 3. In the absence of Mg2+, 8-azido-ADP binds almost equally to the α and β subunits of F1, whereas in the presence of Mg2+ the α subunits are predominantly labelled. 4. 4. The ATPase activity is completely inhibited when two molecules of 8-azido-ADP are bound per molecule F1. 5. 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. 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. 7. 8-Azido-ATP is also a suitable photoaffinity label for F1 in the isolated ATPase complex and submitochondrial particles, photolabelling leading to inactivation of ATPase activity. 8. 8. As is the case for isolated F1 the α and/or β subunits of F1 in the ATPase complex or particles are specifically labelled with 8-azido-ATP. 9. 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. 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. © 1979.