By a centrifugation method with purified Na + , K + -ATPase and 22 Na + or 42 K + , the specific binding of Na + or K + to the enzyme was determined as ouabain-sensitive binding, the difference between that in the absence and presence of 0.1 m m ouabain. The ouabain-sensitive K + binding at 50 μ m KCl was 5.7 nmol/mg protein ([ 3 H]-ouabain binding=2.8 nmol/mg protein). It was completely inhibited by 10 m m NaCl but not affected by 10 m m choline chloride. The binding was partially inhibited by 0.2 m m ATP and by 5 m m MgCl 2 , but not by 0.2 m m P 1 . At KCl concentrations of more than 20 μm the ouabain-sensitive K + binding reached a saturation level twice as high as that of ouabain binding. The apparent K d for K + was 6 μM. A Scatchard plot of K + binding showed a curved line, suggesting positive cooperativity of two binding sites for K + (Hill coefficient n H = 1.72); the two K d 's were estimated to be 36 μM and 1 μm. The ouabain-sensitive Na + binding at 0.5 m m NaCl was 5.3 nmol/mg protein (ouabain binding=2.5 nmol/mg protein). It was specifically inhibited by 0.1 m m KCl but not affected by 10 m m choline chloride. The Na + binding was inhibited by 5 m m MgCl, 2 , but not by 0.2 m m ATP or by 0.2 m m P 1 , contrasting with the K + binding. Oligomycin increased the Na + binding to 6.4 nmol/mg protein. This stimulation was much more pronounced at 0.1 m m NaCl; from 1.6 nmol to 6.1 nmol Na + /mg protein. Without oligomycin the Na + binding at NaCl concentrations of less than 1 m m did not reach saturation because of the enzyme's low affinity for Na + . With oligomycin the binding at NaCl concentrations of more than 0.2 m m reached a saturation level which was 2.7-fold that of ouabain binding. The apparent K d 's for Na + in the absence and presence of oligomycin were about 340 μ m and 60 μ m respectively. The affinity enhancement by oligomycin appears to be due to an increase in cooperativity between the three binding sites for Na + (n H = 1.23→1.54). Simultaneous measurement of Na + and K + binding at a constant Na + concentration of 0.5 m m or 0.1 m m with oligomycin and increasing concentrations of K + showed a decreasing pattern of ouabain-sensitive Na + binding which was mirrored exactly by an increasing pattern of ouabain-sensitive K + binding. It is concluded that 3Na + and 2K + do not bind simultaneously to the same enzyme molecule but alternatively to the different conformers of the enzyme, namely E 1 or E 2 .
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Matsui et al. (1982) studied this question.