Localisation of adenine nucleotide-binding sites on beef-heart mitochondrial ATPase by photolabelling with 8-azido-ADP and 8-azido-ATP
Wagenvoord, R.J.; Van Der Kraan, I.; Kemp, A.
Biochimica et Biophysica Acta 548(1): 85-95
In addition to the previosuly studied 8-azido-ATP, 8-azido-ADP is a suitable photoaffinity label for beef-heart mitochondrial ATPase (F1). 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. 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 labeled. The ATPase activity is completely inhibited when 2 molecules of 8-azido-ADP are bound/molecule F1. 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. 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. 8-Azido-ATP is also a suitable photoaffinity label for F1 in the isolated ATPase complex and submitochondrial particles, photolabeling leading to inactivation of ATPase activity. As is the case for isolated F1 and .alpha. and/or .beta. subunits of F1 in the ATPase complex or particles are specifically labeled with 8-azido-ATP. Oxidative phosphorylation and the ATP-driven reduction of NAD+ by succinate are also inhibited by photolabeling Mg-ATP particles with 8-azido-ATP. In contrast to the uncoupled ATPase activity, where the 2 ATP-binding sites do not interact, cooperation between the 2 sites is required for ATP hydrolysis coupled to reduction of NAD+ by succinate.