Tight nucleotide binding sites and ATPase activities of theRhodospirillum rubrum RrF1-ATPase as compared to spinach chloroplast CF1-ATPase

SolubilizedRhodospirillum rubrum RrF₁-ATPase, depleted of loosely bound nucleotides, retains 2.6 mol of tightly bound ATP and ADP/mol of enzyme. Incubation of the depleted RrF₁ with Mg²⁺-ATP or Mg²⁺-AMP-PNP, followed by passage through two successive Sephadex centrifuge columns, results in retention of a maximal number of 4 mol of tightly bound nucleotides/mol of RrF₁. They include 1.5 mol of nonexchangeable ATP, whereas all tightly bound ADP is fully exchangeable. A similar retention of only four out of the six nucleotide binding sites present on CF₁ has been observed after its passage through one or two centrifuge columns. These results indicate that the photosynthetic, unlike the respiratory, F₁-ATPases have fasterk _off constants for two of the Mg-dependent nucleotide binding sites. This could be the reason for the tenfold lower Mg²⁺ than Ca²⁺-ATPase activity observed with native RrF₁, as with -depleted, activated CF₁. An almost complete conversion of both RrF₁ and CF₁ from Ca²⁺- to Mg²⁺-dependent ATPases is obtained upon addition of octylglucoside, at concentrations below its CMC, to the ATPase assay medium. Thus, octylglucoside seems to affect directly the RrF₁ and CF₁ divalent cation binding site(s), in addition to its proposed role in relieving their inhibition by free Mg²⁺ ions. The RrF₁-ATPase activity is 30-fold more sensitive than CF₁ to efrapeptin, and completely resistant to either inhibition or stimulation by the CF₁ effector, tentoxin. Octylglucoside decreases the inhibition by efrapeptin and tentoxin, but exposes on CF₁ a low-affinity, stimulatory site for tentoxin.Abbreviations: CF₁, EcF₁, MF₁, and TF₁, the soluble F₁-ATPase from chloroplasts, PE. coli, mitochondria,R. rubrum, and the thermophilic bacterium PS3, respectively: AMP-PNP, adenylyl-, -imidodiphosphate; CMC, critical micellar concentration; DTT, dithiothreitol, LDAO, lauryl dimethylamine oxide.Dedicated to Professor Achim Trebst in honor of this 65th birthday.