ABSTRACT
Bovine heart submitochondrial particles (smp) were incubated with p-fluorosulfonylbenzoyl-5'-adenosine (FSBA) in order to study the binding of this ligand and its effect on ATP synthesis and ATP hydrolysis in smp and to compare the results with those obtained with isolated F1. The binding was measured with the 14C-labeled compound. ATP hydrolysis was in all cases as much inhibited as succinate-driven ATP synthesis and ITP hydrolysis was more inhibited than ATP hydrolysis. The binding experiments show that modification of three nucleotide binding sites results in nearly complete inhibition of ATPase activity. In the presence of pyrophosphate up to 6 mol [14C]SBA/mol F1 can be bound. FSBA preferentially modifies amino acids of the alpha-subunits but also beta-subunits are modified. It is concluded that modification of both subunits results in inhibition of activity. The results are very well comparable with the results obtained with isolated F1, which indicates that our preparation of F1 is a good model for F1 in the intact system. Furthermore it is concluded that each alpha-subunit of F1 in smp, just like in the isolated form, contains two pockets where adenosine moieties can bind, one located above the P-loop, modifying alpha-Tyr-244 and alpha-Tyr-300 and the other one located below the P-loop where also the adenosine moiety of AD(T)P binds, modifying beta-Tyr-368.
Subject(s)
Adenosine/analogs & derivatives , Affinity Labels/metabolism , Affinity Labels/pharmacology , Proton-Translocating ATPases/antagonists & inhibitors , Proton-Translocating ATPases/metabolism , Adenosine/metabolism , Adenosine/pharmacology , Adenosine Triphosphate/biosynthesis , Adenosine Triphosphate/metabolism , Animals , Binding Sites , Cattle , Diphosphates/pharmacology , Enzyme Inhibitors/metabolism , Enzyme Inhibitors/pharmacology , Hydrolysis , In Vitro Techniques , Inosine Triphosphate/metabolism , Mitochondria, Heart/enzymology , Proton-Translocating ATPases/chemistry , Submitochondrial Particles/enzymologyABSTRACT
The action of sulfite on ATP hydrolysis and synthesis activities is investigated in membrane vesicles prepared from the cyanobacterium Synechococcus 6716, chromatophores from the photosynthetic purple bacterium Rhodospirillum rubrum, membrane vesicles from the related non-photosynthetic bacterium Paracoccus denitrificans, and bovine heart submitochondrial particles. Without any further pretreatment ATP hydrolysis is stimulated by sulfite in all four membrane preparations. Typically ATP synthesis in the cyanobacterial membrane vesicles is inhibited by sulfite, whereas ATP synthesis in chromatophores and the submitochondrial particles is not. These differences in sensitivity of ATP synthesis to sulfite, however, correspond well with the distribution of (photosynthetic) sulfur oxidizing pathways in the remaining three organisms/organelles compared in this study.
