The role of ionic interactions in the adenosinetriphosphate (ATP) dependent Na binding by rat liver microsomes was investigated. In the concentration range of 0 to 20 mM, Mg and Ca are demonstrated to compete strongly against Na for microsome binding sites. In the presence of Ca, the nonbiological complexing agent ethylenediaminetetraacetate (EDTA) produced a marked increase in Na binding accompanied by a concomitant decrease in Ca binding. Under similar conditions ATP, which is a weaker complexing agent than EDTA, produced quantitatively smaller but qualitatively similar changes in binding. The data show that the effect of ATP on Na binding is not dependent upon the formation of a hypothetical Na binding intermediate in the hydrolysis of ATP as other investigators have postulated. Rather, the effect of ATP is demonstrated to depend upon the presence of unhydrolyzed ATP and its ability to complex divalent cations, and thereby to reduce divalent cation competition against monovalent cations for membrane binding sites.
In the past decade since the demonstration of a crab nerve membrane (Na + K) dependent Mg-activated adenosine triphosphatase system, and the suggestion that this system may be involved in the active extrusion of Na from the nerve fiber (Skou, '57), considerable evidence has accumulated indicating that it is closely linked to the cellular Na and K transporting mechanism (Skou, '65).
Efforts to formulate a reasonable ion transport model intimately involving this ATPase system have led to much speculation that a labile phosphorylated intermediate molecule capable of selectively binding Na or K is formed in the hydrolysis of adenosine triphosphate and may act as the "carrier" in the transporting mechanism. Several investigators have, in fact, presented evidence of ATPdependent binding of Na ( J b e f e l t , '61; J b e f e l t and von Stedingk, '63; Charnock and Post, '63; Charnock, Rosenthal and Post, '63; Walz and Chan, '66) and Rb (Hashimoto and Yoshikawa, '63; Yoshikawa and Hashimoto, '65) by cellular membrane materials in the presence of added Mg. These data have been interpreted as being consistent with the hypothesis that a Na or K binding phosphorylated intermediate is formed in the ATPase catalyzed hydrolysis of ATP.
However, investigations in this laboratory (Sanui and Pace, '65a) recently