Preparation of Na+,K+-ATPase with near maximal specific activity and phosphorylation capacity: Evidence that the reaction mechanism involves all of the sites

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 prepa red 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 mu mol (mg of protein)(-1) mi n(-1) at 37 degrees C had phosphorylation capacities of >60 nmol/mg of prot ein, and the rate of turnover of the enzyme was 9690 min(-1), within the ra nge reported for the enzyme from other sources. The fraction of the maximal specific activity of the enzyme compared well with the fraction of the pro tein on SDS-PAGE which was alpha and beta chains, especially at the highest specific activity which indicates that all of the alpha beta protomers are active. The gels of the most reactive preparations contained only alpha an d beta chains, but less active preparations contained a number of extraneou s proteins. The major contaminant was actin. The preparation did not contai n any protein which migrated in the molecular weight range of the gamma sub unit. The subunit composition of the enzyme was alpha(1) and beta(1) only. This is the first report of a pure, homogeneous, fully active preparation o f the protein. Reaction models which incorporate a half-of-the-sites or fli p-flop mechanism do not apply to this enzyme.