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.
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 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 microM). 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 X 8-azido-ATP complex, the action of the stable Mg X ATP complex analogue, chromium X 8-N3ATP (Cr X 8-N3ATP), was studied. Cr X 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 X 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 [alpha-32P]8-N3ATP-photolabelled enzyme in the presence of K+ results in the formation of an Mr = 56 000 radioactive peptide, whereas trypsinolysis of a [gamma-32P]Cr X ATP-labelled enzyme under identical conditions forms an Mr = 41 000 radioactive peptide. Extensive trypsinolysis of the [alpha-32P] 8-N3ATP-photolabelled alpha-subunit leads to the formation of a radioactive peptide of Mr = 1800.
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000378:metabolism; D000345:Affinity Labels / Q000378:metabolism*; D000818:Animals; D001386:Azides / Q000378:metabolism*; D006868:Hydrolysis; D007668:Kidney / Q000201:enzymology; D008274:Magnesium / Q000494:pharmacology*; D010446:Peptide Fragments / Q000032:analysis; D010777:Photochemistry; D011188:Potassium / Q000494:pharmacology; D011487:Protein Conformation / Q000187:drug effects; D011956:Receptors, Cell Surface / Q000378:metabolism* / Q000528:radiation effects; D011983:Receptors, Purinergic; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism* / Q000528:radiation effects; D013552:Swine; D014357:Trypsin
D000345:Affinity Labels; D001386:Azides; D010446:Peptide Fragments; D011956:Receptors, Cell Surface; D011983:Receptors, Purinergic; C017040:8-azidoadenosine 5'-triphosphate; D000255:Adenosine Triphosphate; D014357:Trypsin; D000254:Sodium-Potassium-Exchanging ATPase; D008274:Magnesium; D011188:Potassium