Molecular Pharmacology, Vol 18, 53-56, Copyright © 1980 by the American Society for Pharmacology and Experimental Therapeutics

Ouabain-Induced Changes in the Tertiary And the Quaternary Conformations of (Na⁺ + K⁺)-Activated Adenosine Triphosphatase

¹ Department of Pharmacology, Medical College of Ohio, Toledo, Ohio 43699

Purified preparations of (Na⁺ + Ki⁺)-activated adenosine triphosphatase (EC 3.6.1.3) contain two major polypeptides: the catalytic subunit (-subunit), and a glycoprotein (-subunit) of unknown function. Effects of ouabain on the chemical crosslinking of the subunits in the presence of o-phenanthroline and Cu²⁺ were studied, and the following results were obtained: (1) When the native enzyme was exposed to 0.25 mM Cu²⁺ and 1.25 mM o-phenanthroline, a crosslinked ,-dimer was obtained in the presence of Na⁺ + ATP, but not in the presence of either Na⁺, or K⁺, or K⁺ + ATP. Exposure of the preformed ouabain-enzyme complex to the same reagent resulted in the formation of the , -dimer. The level of this dimer was lower than that obtained from the native enzyme in the presence of Na⁺ + ATP, and was not affected by Na⁺, ATP, and K⁺. (2) When the native enzyme was preincubated with Na⁺ + Mg²⁺ + ATP and then exposed to 0.25 mM Cu²⁺ and 1.25 mM o-phenanthroline, the ,-dimer was obtained. Addition of K⁺ to the preincubated enzyme prior to the addition of the crosslinking reagent prevented dimer formation. Addition of ouabain to the preincubated enzyme, did not affect dimer formation but did block the K⁺ effect on dimer formation. (3) When the native enzyme was exposed to 0.25 mM Cu²⁺ and 0.5 mM o-phenanthroline, an ,-dimer was obtained under all ligand conditions except when K⁺ + ATP was present. With the preformed ouabain-enzyme complex, the dimer was obtained, and its formation was not affected by K⁺ + ATP. (4) The formation of an ,-dimer in the presence of Cu²⁺ and o-phenanthroline was not affected by ouabain. These findings indicate that the minimum quaternary structure of ₂₂ existS in both the native enzyme and the ouabain-enzyme complex, but that ouabain binding induces changes in -subunit conformation and in -subunit interactions within the oligomeric structure of the enzyme.