Demonstration of an Mg 2+ ‐induced conformational change by photoaffinity labelling of the high‐affinity ATP‐binding site of (Na + + K + )‐ATPase with 8‐azido‐ATP

1 8‐Azido‐ATP (8‐N 3 ATP) is a substrate of (Na + + K + )‐ATPase from pork kidney and photoinactivates it by binding to the M r = 100 000 α‐subunit. The photoinactivation requires the presence of Mg 2+ even though 8‐azido‐ATP is recognized by the high‐affinity ATP binding site ( K d = 3.1 μM). K + ions protect the enzyme against photoinactivation as does excess ATP. 2 To see whether the Mg 2+ ‐requirement of the photoinactivation is due to the action of free Mg 2+ or to the existence of an Mg · 8‐azido‐ATP complex, the action of the stable Mg · ATP complex analogue, chromium · 8‐N 3 ATP (Cr · 8‐N 3 ATP), was studied. Cr · 8‐N 3 ATP photoinactivates (Na + + K + )‐ATPase in the absence of Mg 2+ , but the photoinactivation is enhanced by Mg 2+ , indicating that the formation of a Mg · ATP complex is an absolute requirement for photoinactivation. However, the interaction of Mg 2+ with a low‐affinity site also enhances the photoinactivation. It is therefore concluded that interactions with MgATP and free Mg induce conformational changes in the purine subsite of the high‐affinity ATP binding site. 3 Controlled trypsinolysis of the [α‐ 32 P]8‐N 3 ATP‐phostolabelled enzyme in the presence of K − results in the formation of an M r = 56000 radioactive peptide, whereas trypsinolysis of a [γ‐ 32 P]Cr · ATP‐labelled enzyme under identical conditions forms an M r = 41000 radioactive peptide. Extensive trypsinolysis of the [α‐ 32 P] 8‐N 3 ATP‐photolabelled α‐subunit leads to the formation of a radioactive peptide of M r = 1800.