Modification of membrane-bound F1 by p-fluorosulfonylbenzoyl-5'-adenosine: sites of binding and effect on activity.
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.
van der Zwet-de Graaff
I
I
E.C. Slater Institute, University of Amsterdam, The Netherlands.
Hartog
A F
AF
Berden
J A
JA
D000241:Adenosine / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000096:biosynthesis / Q000378:metabolism; D000345:Affinity Labels / Q000378:metabolism* / Q000494:pharmacology*; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D011756:Diphosphates / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000378:metabolism / Q000494:pharmacology; D006868:Hydrolysis; D066298:In Vitro Techniques; D007293:Inosine Triphosphate / Q000378:metabolism; D008929:Mitochondria, Heart / Q000201:enzymology; D006180:Proton-Translocating ATPases / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism*; D013367:Submitochondrial Particles / Q000201:enzymology
D000345:Affinity Labels; D011756:Diphosphates; D004791:Enzyme Inhibitors; D007293:Inosine Triphosphate; C010959:5'-(4-fluorosulfonylbenzoyl)adenosine; D000255:Adenosine Triphosphate; D006180:Proton-Translocating ATPases; D000241:Adenosine