The activation of lymphocyte plasma membrane (Na,K)-ATPase by EGTA is explained better by zinc than calcium chelation.

We have observed that maximal lymphocyte membrane (Na,K)-ATPase activity occurred only when a chelating agent for divalent cations such as ethylene glycol bis(P-aminoethyl ether)-N,N,N*,N*-tetraacetic acid (EGTA) was present in the reaction mixture. In the absence of EGTA, the ionized calcium was 2 PM and the (Na,K)-ATPase was 25 to 40% of maximum. Twentyfive p~ EGTA reduced the ionized Ca to 0.6 PM and caused maximal (Na,K)-ATPase activity. Three observations indicated that Ca chelation was not causal in the enzyme activation. First, an excess of additional Ca, 9 as compared to 2 p ~ , was required to inhibit the (Na,K)-ATPase to its base-line activity. Second, when 250 PM histidine, which does not chelate Ca, was substituted for EGTA, the (Na,K)-ATPase was activated comparably. Third, the addition of Ca to the ATPase assay fully activated by histidine caused no inhibition at ionized Ca concentrations as high as 12 p ~ . These data indicate that EGTA binds a cation other than Ca to activate the (Na,K)-ATPase. Trace amounts of Zn (2 PM) were found by atomic absorption analysis of the assay system. Three observations supported the causal role of Zn as the inhibitor. First, when Zn was added to the (Na,K)-ATPase, partially activated by 50 PM histidine, inhibition of the ATPase was observed. Second, at 35 p~ added Zn, the calculated ionized Zn concentration was 2 PM, similar to the initial Zn value, and the inhibition of the ATPase was similar to that in the absence of chelator. Third, mixtures of histidine and Zn calculated to produce partial activation of the (Na,K)-ATPase produced results predicted by the free Zn concentration achieved. These data established that enhancement of the activity of the lymphocyte (Na,K)-ATPase by EGTA is not explained by chelation of Ca; rather, the chelation of Zn activates the (Na,K)-ATPase under the conditions of these studies.