Binding of monovalent cations to Na+,K+-dependent ATPase purified from porcine kidney. III. Marked changes in affinities for monovalent cations induced by formation of an ADP-insensitive but no an ADP-Sensitive phosphoenzyme.
We measured the amounts of Rb+ ions (a K+ congener) as well as Na+ and K+ ions bound to the ATPase during the ATPase reaction at pH 7.5 and 0 degrees C. The affinity of the Na+-binding sites for three Na+ ions decreased markedly but that of the K+-binding sites for two K+ or Rb+ ions increased markedly upon formation of an ADP-insensitive phosphorylated intermediate. Furthermore, the present experiment did not give any indication of a change in the Hill coefficient of 2, and showed an increase in the affinity of the K+-binding sites for Rb+ ions of about 28 times upon the formation of an ADP-insensitive EP. The enzyme state with a high affinity for Rb+ was maintained after the disappearance of EP. When the ATPase was treated with N-ethylmaleimide (NEM), almost all the EP formed was ADP-sensitive. The formation of an ADP-sensitive EP with the NEM-treated enzyme induced no change in the affinities of the ATPase for Na+ and Rb+ ions.
D000244:Adenosine Diphosphate / Q000494:pharmacology*; D000818:Animals; D001665:Binding Sites; D005033:Ethylmaleimide / Q000494:pharmacology; D007668:Kidney / Q000201:enzymology; D007700:Kinetics; D010766:Phosphorylation; D011188:Potassium / Q000378:metabolism*; D011485:Protein Binding; D012413:Rubidium / Q000378:metabolism*; D012964:Sodium / Q000378:metabolism*; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism*; D013552:Swine
D000244:Adenosine Diphosphate; D012964:Sodium; D000254:Sodium-Potassium-Exchanging ATPase; D012413:Rubidium; D005033:Ethylmaleimide; D011188:Potassium