ATP Dependent Reversible Conformational Change of Na+, K+-ATPase Modified with N-(p(2-Benzimidazolyl)phenyl)Maleimide1

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Abstract
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 KCl. 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≈K+, none, Mg2+≈ATP, Na++ATP, Na+Na+ + Mg2+, Na++Mg2++ADP≈Na++ADP. The data indicate the presence of multiple conformational states of the enzyme.