Rapid filtration analysis of nucleotide binding to Na,K-ATPase

Transient kinetic analysis of nucleotide binding to pig kidney Na,K-ATPase using a rapid filtration technique shows that the interaction between nucleotide and enzyme apparently follows simple first-order kinetics both for ATP in the absence of Mg2+ and for ADP in the presence or absence of Mg2+. Rapid filtration experiments with Na,K-ATPase membrane sheets may nevertheless suffer from a problem of accessibility for a fraction of the ATPase binding sites. Accordingly, we estimate from these data that for ATP binding in the absence of Mg2+ and the presence of 35 mM Na+ at pH 7.0 at 20 degreesC, the bimolecular binding rate constant k(on) is about 30 muM(-1).s(-1) and the dissociation rate constant k(off) is about 8 s(-1). In the presence of 10 mM Mg2+, the binding rate constant is the same as that in the absence of Mg2+. For ADP or MgADP the binding rate constant is about 20 muM(-1) . s(-1) and the dissociation rate constant is about 12 s(-1). Results of rapid-mixing stopped-flow experiments with the fluorescent dye eosin are also consistent with a one-step mechanism of binding of eosin to the ATPase nucleotide site. The implication of these results is that nucleotide binding to Na,K-ATPase both in the absence and presence of Mg2+ appears to be a single-step event, at least on the time scale accessible in these experiments.