Demonstration of two binding sites for ADP on the isolated β‐subunit of the Rhodospirillum rubrum R1F0F1‐ATP synthase

Abstract
Two ADP binding sites have been demonstrated on the reconstitutively active β‐subunit, that was removed from the Rhodospirillum rubrum membrane‐bound ATP synthase. One is a high affinity site (K d = 0.7 μM) that does not require MgCl2 and is unaffected by it. The second is a low affinity binding site (K d = 80 μM) that is absolutely dependent on MgCl2. For stable binding of ADP to this site, MgCl2 must be present not only during the binding step but also during the elution‐centrifugation step used to separate the β‐subunit bound [3H]ADP from the free ligand. When MgCl2 is removed together with the free ligand [3H]ADP dissociates very rapidly from this second binding site.