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

Abstract

1. 8-Azido-ATP (8-N3ATP) is a substrate of (Na+ + K+)-ATPase from pork kidney and photoinactivates it by binding to the Mr = 100000 .alpha.-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 .mu.M). K+ ions protect the enzyme against photoinactivation as does excess ATP. 2. To see whether the Mg2+ -requirement of the photoinactivation is due to the action of free Mg2+ or to the existence of an Mg .times. 8-azido-ATP complex, the action of the stable Mg .times. ATP complex analogue, chromium .cntdot. 8-N3 ATP (Cr .cntdot. 8-N3 ATP), was studied. Cr .cntdot. 8-N3 ATP photoinactivates (Na+ + K+)-ATPase in the absence of Mg2+, but the photoinactivation is enhanced by Mg2+, indicating that the formation of a Mg .cntdot. 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 wiht MgATP and free Mg induce conformational changes in the purine subsite of the high-affinity ATP binding site. 3. Controlled trypsinolysis of the [.alpha.-32P]8-N3ATP-photolabelled enzyme in the presence of K+ results in the formation of an Mr = 56000 radioactive peptide, whereas trypsinolysis of a [.gamma.-32P] Cr .cntdot. ATP-labelled enzyme under identical conditions forms an Mr = 41000 radioactive peptide. Extensive trypsinolysis of the [.alpha.32P]- 8-N3ATP-photolabelled .alpha.-subunit leads to the formation of a radioactive peptide of Mr = 1800.