Dg. Ward et Jd. Cavieres, SOLUBILIZED ALPHA-BETA NA,K-ATPASE REMAINS PROTOMERIC DURING TURNOVERYET SHOWS APPARENT NEGATIVE COOPERATIVITY TOWARD ATP, Proceedings of the National Academy of Sciences of the United Statesof America, 90(11), 1993, pp. 5332-5336
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.