1. 1. Tightly bound ATP and ADP, found on the isolated mitochondrial ATPase, exchange only slowly at pH 8, but the exchange is increased as the pH is reduced. At pH 5.5, more than 60% of the bound nucleotide exchanges within 2.5 min.
2. 2. Preincubation of the isolated ATPase with ADP leads to about 50% inhibition of ATP hydrolysis when the enzyme is subsequently assayed in the absence of free ADP. This effect, which is reversed by preincubation with ATP, is absent on the membrane-bound ATPase. This inhibition seems to involve the replacement of tightly bound ATP by ADP.
3. 3. Using these two findings, the binding specificity of the tight nucleotide binding sites was determined. iso-Guanosine, 2′-deoxyadenosine and formycin nucleotides displaced ATP from the tight binding sites, while all other nucleotides tested did not. The specificities of the tight sites of the isolated and membrane-bound ATPase were similar, and higher than that of the hydrolytic site.
4. 4. The nucleotide specificities of ‘coupled processes’ nucleoside triphosphate-driven reversal of electron transfer, nucleoside triphosphate-32Pi exchange and phosphorylation were higher than that of the hydrolytic site of the ATPase and similar to that of the tight nucleotide binding sites.
5. 5. The different nucleotide specificities of uncoupled ATP hydrolysis and coupled processes can be explained even if both processes involve a single common site on the ATPase molecule. This model requires that energy can be ‘coupled’ only when it is released/utilised in the nucleotide binding steps of the mechanism.
6. 6. Adenosine β,γ-imidotriphosphate (AMP-PNP) is not a simple reversible inhibitor of the ATPase, since incubation requires preincubation and is not reversed when the compound is diluted out, or by addition of ATP. This compound inhibits the isolated and membrane-bound ATPase equally well. Its guanosine analogue does not act in this way.
7. 7. In submitochondrial particles, ADP inhibited uncoupled hydrolysis of ATP much more effectively than coupled hydrolysis, the latter being measured both directly (from ATP hydrolysis in the absence of uncoupler) or indirectly, by monitoring ATP-driven reduction of NAD+ by succinate.
8. 8. The effects of ADP and AMP-PNP were interpreted as providing evidence for two of the intermediates in the proposed scheme for coupled triphosphate hydrolysis.
Abbreviations: ε-ATP, N1,N6-ethenoadenosine triphosphate; 8-BrATP, 8-bromoadenosine triphosphate; AMP-PNP, adenosine β,γ-imidotriphosphate; GMP-PNP, guanosine β,γ-imidotriphosphate; N1,O-ATP, adenosine-N1-oxide triphosphate; rro-ATP 2,2′[1-(9-adenyl)-1′-(triphosphoryl-oxymethyl)-dihydroxydiethyl ether; and similarly for the respective diphosphates; NTP, NDP, nucleoside tri-, diphosphate; ANS, 1-anilino-8-naphthalene sulphonate; FCCP, carbonylcyanide p-trifluoromethoxyphenylhydrazone; HEPES, N-2-hydroxyethylpiperazine-N′-2-ethane sulphonic acid; MES, 2-(N-morpholino)-ethane sulphonic acid; TES, tris(hydroxymethyl)methylamino ethane sulphonic acid
