Binding of Monovalent Cations to Na+, K+-Dependent ATPase Purified from Porcine Kidney

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Abstract
We previously measured the amounts of Na+ and K+ ions bound to the Na+ , K+-ATPase [EC 3.6.1.3] purified from porcine kidney by a modified membrane filtration method [(1979)J. Biochem. 86, 509–523]. In this study, we improved the method for measuring the amount of the active site and measured the amount of Rb+ ions (a K+ congener) bound to the ATPase as well as those of Na+ and K+ ions to get more accurate information on the K+-and Na+-binding sites. The following results were obtained. Two kinds of cation-binding sites were found to exist on the ATPase molecule. One was the Na+-binding sites (3 mol per mol of active site). Na+-ions were bound to the sites cooperatively (Hill coefficient, 2.5–-3), and the apparent dissociation constant was 0.20–0.32 mM Three moles of Na+ ions bound to the sites was displaced by 1 mol of K+ ions bound to the ATPase (φK, 24 μM). The other was the K+-binding sites (2 mol per mol of active site). Two moles of K+, Rb+ or Na+ ions was bound to the sites cooperatively (Hill coefficient, 1.5–2), and their apparent dissociation constants were 0.044, 0.024, and 2.2 m respectively. We measured the amounts of Na+ and Rb+ions bound to the ATPase in the presence of 0.8 mi NaCI and 0.13 msi RbCI, and obtained unequivocal evidence for the simultaneous binding of 3 mol of Na+ ions and 2 mol of Rb+ ions per mol of active site of the ATPase.