ATP dependent reversible conformational change of Na+,K+-ATPase modified with N-(p-(2-benzimidazoly)phenyl)maleimide.
Na+,K+-ATPase [EC 3.6.1.3] from pig kidneys was treated with the fluorescent reagent N-(p-(2-benzimidazolyl)phenyl)maleimide (BIPM) in the presence of CKl. The resultant preparation showed 70% of the activity with only a small change in the apparent affinity for ligands of the enzyme. The addition of Na+ to the treated preparation induced a -2.1 +/- 0.1% change of the total fluorescence intensity observed in the absence of Na+. Further addition of both Mg2+ and ATP transiently increased the fluorescence to +0.5 +/- 0.1%. After the exhaustion of ATP, the fluorescence decreased to -3.1 +/- 0.1%. This cycle can be repeated by the readdition of ATP but not by ADP. Ouabain inhibits the fluorescence change. The ligands used reduced the fluorescence intensity as follows: Mg2+ + Na+ + ATP approximately K+, none, Mg2+ approximately ATP, Na+ + ATP, Na+ approximately Na+ + Mg2+, Na+ + Mg2+ + ADP approximately Na+ + ADP. The data indicate the presence of multiple conformational states of the enzyme.
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate; D000818:Animals; D007668:Kidney / Q000201:enzymology*; D008301:Maleimides / Q000494:pharmacology*; D010042:Ouabain / Q000494:pharmacology; D011485:Protein Binding; D011487:Protein Conformation; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013552:Swine
D008301:Maleimides; C000245:N-(4-(2-benzimidazolyl)phenyl)maleimide; D010042:Ouabain; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D000254:Sodium-Potassium-Exchanging ATPase