Preparation of Na+,K+-ATPase with Near Maximal Specific Activity and Phosphorylation Capacity: Evidence That the Reaction Mechanism Involves All of the Sites

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Abstract
The phosphorylation capacity of Na+,K+-ATPase preparations in common use is much less than expected on the basis of the molecular weight of the enzyme deduced from cDNA sequences. This has led to the popularity of half-of-the-sites or flip-flop models for the enzyme reaction mechanism. We have prepared Na+,K+-ATPase from nasal salt glands of salt-adapted ducks which has a phosphorylation capacity and specific activity near the theoretical maxima. Preparations with specific activities of >60 μmol (mg of protein)-1 min-1 at 37 °C had phosphorylation capacities of >60 nmol/mg of protein, and the rate of turnover of the enzyme was 9690 min-1, within the range reported for the enzyme from other sources. The fraction of the maximal specific activity of the enzyme compared well with the fraction of the protein on SDS−PAGE which was α and β chains, especially at the highest specific activity which indicates that all of the αβ protomers are active. The gels of the most reactive preparations contained only α and β chains, but less active preparations contained a number of extraneous proteins. The major contaminant was actin. The preparation did not contain any protein which migrated in the molecular weight range of the γ subunit. The subunit composition of the enzyme was α1 and β1 only. This is the first report of a pure, homogeneous, fully active preparation of the protein. Reaction models which incorporate a half-of-the-sites or flip-flop mechanism do not apply to this enzyme.