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

Solubilized Rhodospirillum 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 Mg(2+)-ATP or Mg(2+)-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 faster koff constants for two of the Mg-dependent nucleotide binding sites. This could be the reason for the tenfold lower Mg2+ than Ca(2+)-ATPase activity observed with native RrF1, as with epsilon-depleted, activated CF1. An almost complete conversion of both RrF1 and CF1 from Ca(2+)- to Mg(2+)-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.