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 Mg(2+) and for ADP in the presence or absence of Mg(2+). 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 Mg(2+) and the presence of 35 mM Na(+) at pH 7.0 at 20 degrees C, the bimolecular binding rate constant k(on) is about 30 microM(-1) x s(-1) and the dissociation rate constant k(off) is about 8 s(-1). In the presence of 10 mM Mg(2+), the binding rate constant is the same as that in the absence of Mg(2+). For ADP or MgADP the binding rate constant is about 20 microM(-1) x 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 Mg(2+) appears to be a single-step event, at least on the time scale accessible in these experiments.
Fedosova
Natalya U
NU
Department of Biophysics, University of Aarhus, Ole Worms Allé 185, DK-8000 Aarhus C, Denmark.
Champeil
Philippe
P
Esmann
Mikael
M
D000244:Adenosine Diphosphate / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D004801:Eosine Yellowish-(YS) / Q000378:metabolism; D005374:Filtration; D066298:In Vitro Techniques; D007668:Kidney / Q000201:enzymology; D007700:Kinetics; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry / Q000378:metabolism*; D013552:Swine
D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D000254:Sodium-Potassium-Exchanging ATPase; D004801:Eosine Yellowish-(YS)