Demonstration of an Mg2+‐induced conformational change by photoaffinity labelling of the high‐affinity ATP‐binding site of (Na++ K+)‐ATPase with 8‐azido‐ATP

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Abstract
8‐Azido‐ATP (8‐N3ATP) is a substrate of (Na++ K+)‐ATPase from pork kidney and photoinactivates it by binding to the Mr= 100 000 α‐subunit. The photoinactivation requires the presence of Mg2+ even though 8‐azido‐ATP is recognized by the high‐affinity ATP binding site (Kd= 3.1 μM). K+ ions protect the enzyme against photoinactivation as does excess ATP. To see whether the Mg2+‐requirement of the photoinactivation is due to the action of free Mg2+ or to the existence of an Mg · 8‐azido‐ATP complex, the action of the stable Mg · ATP complex analogue, chromium · 8‐N3ATP (Cr · 8‐N3ATP), was studied. Cr · 8‐N3ATP photoinactivates (Na++ K+)‐ATPase in the absence of Mg2+, but the photoinactivation is enhanced by Mg2+, indicating that the formation of a Mg · ATP complex is an absolute requirement for photoinactivation. However, the interaction of Mg2+ 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. Controlled trypsinolysis of the [α‐32P]8‐N3ATP‐phostolabelled enzyme in the presence of K− results in the formation of an Mr= 56000 radioactive peptide, whereas trypsinolysis of a [γ‐32P]Cr · ATP‐labelled enzyme under identical conditions forms an Mr= 41000 radioactive peptide. Extensive trypsinolysis of the [α‐32P] 8‐N3ATP‐photolabelled α‐subunit leads to the formation of a radioactive peptide of Mr= 1800.
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