Solubilized alpha beta Na,K-ATPase remains protomeric during turnover yet shows apparent negative cooperativity toward ATP.

A prominent feature of the Na,K-ATPase reaction is an ATP dependence that suggests high- and low-affinity ATP requirements during the enzymic cycle. As only one ATP-binding domain has been identified in the alpha subunit and none has been identified in the beta subunit, it has seemed likely that the apparent negative cooperativity results from subunit interactions in an (alpha beta)2 diprotomer. To test this possibility, we have examined the behavior of solubilized alpha beta protomers of Na,K-ATPase down to 50 nM [gamma-32P]ATP. Active-enzyme analytical ultracentrifugation shows that the protomer is the active species and that no oligomerization occurs during turnover. However, we find that dual ATP effects can be clearly demonstrated and that nonhydrolyzable ATP analogs can stimulate the Na,K-ATPase activity of the soluble protomer. We conclude that the apparent negative cooperativity is inherent to the alpha beta protomer and that this should explain some of the complexities found with membrane-bound Na,K-ATPase and, perhaps, other P-type cation pumps.