SOLUBILIZED ALPHA-BETA NA,K-ATPASE REMAINS PROTOMERIC DURING TURNOVERYET SHOWS APPARENT NEGATIVE COOPERATIVITY TOWARD ATP

A prominent feature of the Na,K-ATPase reaction is an ATP dependence t hat suggests high- and low-affinity ATP requirements during the enzymi c cycle. As only one ATP-binding domain has been identified in the alp ha subunit and none has been identified in the beta subunit, it has se emed likely that the apparent negative cooperativity results from subu nit interactions in an (alphabeta)2 diprotomer. To test this possibili ty, we have examined the behavior of solubilized alphabeta protomers o f Na,K-ATPase down to 50 nM [gamma-P-32]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 prot omer. We conclude that the apparent negative cooperativity is inherent to the alphabeta protomer and that this should explain some of the co mplexities found with membrane-bound Na,K-ATPase and, perhaps, other P -type cation pumps.