Evidence for a Mg2+-induced conformational change at the ATP-binding site of (Na+ + K+)-ATPase demonstrated with a photoreactive ATP-analogue.
1. The 3'-ribosyl ester of ATP with 2-nitro-4-azidophenyl propionic acid has been prepared and its ability to act as a photoaffinity label of (Na+ + K+)-ATPase has been tested. 2. In the dark 3'-O-[3-(2-nitro-4-azidophenyl)-propionyl]adenosine triphosphate (N3-ATP) is a substrate of (Na+ + K+)-ATPase and a competitive inhibitor of ATP hydrolysis. 3. Upon irradiation by ultraviolet light, N3-ATP photolabels the high-affinity ATP-binding site and is covalently attached to the alpha-subunit and an approximately 12000-Mr component. 4. Photolabeling of the alpha-subunit by N3-ATP irreversibly inactivates (Na+ + K+)-ATPase. 5. Photoinactivation is strictly Mg2+-dependent. Na+ enhances the inactivation. ATP or ADP and K+ protect the enzyme against inactivation. 6. Mg2+, in concentrations required for photoinactivation, protects (Na+ + K+)-ATPase against inactivation by tryptic digestion under controlled conditions. 7. It is assumed that a conformational change of the ATP-binding site of (Na+ + K+)-ATPase occurs upon binding of Mg2+ to a low-affinity site.
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000378:metabolism / Q000528:radiation effects; D000345:Affinity Labels; D000818:Animals; D001386:Azides; D001665:Binding Sites / Q000187:drug effects; D007668:Kidney / Q000201:enzymology; D008274:Magnesium / Q000494:pharmacology*; D010782:Photolysis; D011487:Protein Conformation / Q000187:drug effects; D000254:Sodium-Potassium-Exchanging ATPase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D013552:Swine; D014357:Trypsin; D014466:Ultraviolet Rays
D000345:Affinity Labels; D001386:Azides; C033528:3'-O-(3-(2-nitro-4-azidophenyl)propionyl)adenosine triphosphate; D000255:Adenosine Triphosphate; D014357:Trypsin; D000254:Sodium-Potassium-Exchanging ATPase; D008274:Magnesium