Purification and Characterization of and [3H]Ouabain Binding to the Transport Adenosine Triphosphatase from Outer Medulla of Canine Kidney

Abstract

This report describes a rapid procedure for purification of (Na+ + K+)-ATPase from canine kidney outer medulla with a yield of 16 to 18 mg of protein per 100 g of tissue. The purified enzyme manifests the highest specific activity reported to date for a mammalian tissue source and binds between 3,000 and 4,000 pmoles of [3H]ouabain per mg of protein. Polyacrylamide gel electrophoresis of the detergent-solubilized enzyme yields two protein peaks of 89,000 and 56,000 daltons. The former is phosphorylated with [γ-32P]ATP in the presence of magnesium and sodium and the latter is stained with periodic acid-Schiff reagent. [3H]-ouabain reacts with the purified enzyme in a manner similar to that of crude enzyme preparations. Ouabain-enzyme complexes were formed in the presence of different ligands [Mg + ATP + Na], [Mg + Pi], [Mn], [Mg + ATP], and all complexes dissociated at similar rates when chase was carried out in presence of binding ligands. Using ouabainbinding data, we calculate a turnover number of 6,480 min-1.