Simultaneous Binding of Three Na+and Two K+ Ions to Na+, K+-Dependent ATPase and Changes in Its Affinities for the Ions Induced by the Formation of a Phosphorylated Intermediate1

Abstract

Na+, K+-dependent ATPase [EC 3.6.1.3] was purified from porcine kidney by the method of Lane et al. [(1973) J. Biol. Chem. 248, 7197–7200] with slight modifications. A phosphorylated intermediate was formed by the reaction of the enzyme with 0.5 mM [γ-32P]ATP in 100 mM NaCl and 4 mM MgCl2 at pH 7.5 and 20°C, and its amount was taken as that of the active site. It was 0.90–2.37 μmol/g protein. The amounts of Na+and K+bound to the ATPase were measured by a modified membrane filtration method. The reaction mixture (50 μl), containing 2–18 mg/ml enzyme, 0.05–2.0 mM 22NaCl or 0.24–5.0 mM 42KC1, 50 mM 3H-D-glucose, 0.6 mM EDTA, and 50–75 mM choline-Cl, was incubated at pH 7.5 and 0°C, then filtered through a Nucleopore filter (=0.4 μm) by sucking for 5 s. The filtrate (about 3 μl) was trapped on a Millipore filter. The concentration of free Na+ions in the reaction mixture was calculated from the volume of the filtrate determined from the radioactivity of 3H-D-glucose and the amount of Na+or K+ions in the filtrate determined from that of 32Na or 42K. We found two kinds of cation-binding sites on the ATPase molecule. One consisted of Na+-binding sites (about three mol per mol of active site). Na+ions bound to the sites cooperatively (Hill coefficient; about 2.5), and the apparent dissociation constant was about 0.2 mM. The other consisted of K+-binding sites (about two mol per mol of active site). The apparent dissociation constant of the K+binding to the sites was less than 0.2 mM, while the affinity of Na+was much less than that of K+(the ratio of dissociation constants of Na+and K+was 62: 1). The affinity of the Na+-binding sites for Na+ions decreased markedly but that of the K+-binding sites for K+ions increased markedly upon the formation of a phosphorylated intermediate. The enzyme state with changed affinities for monovalent cations was maintained after the disappearance of the phosphorylated intermediate.