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.

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.
(Na,K)-ATPase activity of human lymphocyte membrane vesicles does not reach maximal velocity unless ethylene glycol his@-aminoethyl ether)-N,N,N',N"tetraacetic acid is present in the reaction mixture (1). Many assays of erythrocyte * This work was supported by United States Public Health Service Research Grant CA-12790, by the University of Rochester Blood Research "Jimmy" Fund, by National Institute of Environmental Health Services Grant ES01247, and by the Ruth Estrin Goldberg Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore he hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
$ To whom reprint requests should he addressed at, the University of Rochester, Department of Pediatrics, Box 777,601 Elmwood Ave., Rochest,er, NY 14642.
(Na,K)-ATPase activity have contained chelators of divalent cations (2-5) but others have omitted these agents without apparent adverse effect on enzyme activation (6-8). Ca is an inhibitor of the erythrocyte (Na,K)-ATPase only when added in concentrations of 50 to 100 /AM (9). In the present studies, we initially tested the hypothesis that Ca in the reaction mixture inhibited lymphocyte (Na,K)-ATPase activity in vztro and that Ca chelation explained the activating effect of EGTA.' The data, however, suggested that Zn rather than Ca inhibited the (Na,K)-ATPase under these conditions.

RESULTS
Chelator Activation of (iVu,K)-ATPase EGTA-(Na,K)-ATPase activity was measured in membranes exposed to EGTA from 0 to 100 p~ (Fig. 1). "Ca before ATP" indicates the ionized calcium during the 10-min preincubation before the addition of ATP to the reaction mixture.
The preincubation is necessary for maximal activation of the ATPase. "Ca after ATP" indicates the ionized calcium after ATP was added to start the assay.
In the absence of EGTA at an ionized Ca before and after the addition of ATP of 9 and 2 p~, respectively, the mean (Na,K)-ATPase activity of three membrane preparations was 0.3 nmol of P,/pg of protein/3O min. Maximal ATPase activity of 1.3 nmol of Pi/pg of protein/30 min occurred at an EGTA concentration of 25 /AM and an ionized Ca before and after ATP of 3 and 0.6 /AM, respectively.
Histidine-Histidine, a chelating agent with a high affinity for many divalent cations bound by EGTA, has a negligible affinity for Ca. (Na,K)-ATPase activity was measured at histidine concentrations from 0 to lo00 PM (Fig. 2). The ionized Ca of 9 before and 2 p~ after the addition of ATP remained unchanged regardless of the histidine concentration. The mean ATPase activity in four membrane preparations in the absence of histidine was 0.8 nmol of Pi/pg of protein/30 min and maximal activation, 2.2 nmol of P,/pg of protein/30 min, was achieved a t a concentration of 250 FM histidine.
The (  Conc. (pM) FIG. 1. EGTA activation of (Na,K)-ATPase. The phosphohydrolysis of ATP by the (Na,K)-ATPase was measured in the presence of increasing concentrations of EGTA. The ionized Ca before (E-ATP) and after the addition of ATP @-ATP) are shown below the corresponding concentrations of EGTA. The ionized Ca in this study was measured using a Radiometer Ca electrode. The data represent the mean -C S.E. of 3 lymphocyte plasma membrane populations, each measured in triplicate.
in velocity.
Histidine-In the absence of histidine and added Ca, the ATPase activity was 0.8 nmol of Pi/pg of protein/30 min. The addition of 10 mM histidine maximally increased the (Na,K)-ATPase to 1.8 (Fig. 4). No significant change in the histidineactivated ATPase was observed a t a concentration as high as 100 pM added Ca, corresponding to an ionized Ca greater than 50 before and 12 p~ after the addition of ATP, approximately 6 times the initial ionized Ca concentration.
The Effect of Zn on (Na,K)-ATPase Atomic absorption analysis of the reaction mixture revealed no detectable Cd or Pb and trace amounts of Zn. We examined, therefore, the effect of added Zn in the assay system (Fig. 5 ) . The (Na,K)-ATPase activity without added Zn or histidine was 0.5 nmol of Pi/yg of protein/30 min at an ionized Zn of 2 FM calculated from the atomic absorption measure-

Conc. (pM)
FIG. 2. Histidine activation of (Na,K)-ATPase. The phosphohydrolysis of ATP by the (Na,K)-ATPase was measured in the presence of increasing concentrations of histidine. The data represent the mean f $.E. of 4 lymphocyte plasma membrane populations, each measured in triplicate.
histidine was compared directly in eight lymphocyte populations. The ATPase activity with no additive was 0.83 nmol of Pi/pg of protein/30 min and increased to 1.9 2 0.2 when optimal concentrations of either EGTA or histidine were present.
The Effect of Added Ca on the (Na,K)-ATPase Activated by EGTA or Histidine EGTA--In the presence of 50 PM EGTA and no added calcium, the ATPase was maximally activated to 1.5 nmol of Pi/yg of protein/30 min at an ionized Ca of 1.5 before and 0.4 p~ after ATP (Fig. 3). The addition of Ca caused a progressive decrease in the ATPase activity; however, maximd suppression did not occur until 200 p~ Ca was added. The ionized calcium in the presence of 200 p~ Ca was 33 before and 9 p~ after the addition of ATP, approximately 4 times the ionized Ca present in the absence of EGTA when ATPase was similar The phosphohydrolysis of ATP by the (Na,K)-ATPase was measured in the presence of increasing concentrations of Ca. The concentration of histidine in these studies was 10 mM. The data represent the mean -C S.E. of 3 lymphocyte plasma membrane populations, each measured in triplicate. ment. A partially activating concentration of histidine, 50 p~, reduced the ionized Zn to 3 n~ and increased the ATPase to 1.2. The addition of Zn caused an increase in the ionized Zn and inhibition of the (Na,K)-ATPase. When 35 p~ Zn was added to the ATPase, activated by 50 p~ histidine, the calculated free Zn concentration was near 2 PM, the starting value, and the inhibition of ATPase was similar to that in the absence of chelator, about 0.5 nmol of P,/pg of protein/30 min (Fig. 5). Once established, the Zn inhibition of the lymphocyte ATPase was not reversed by the addition of 10 mM histidine. The pattern of ATPase activation and inhibition corresponded inversely to the Zn concent.ration in the ATPase assay.
Since added Zn inhibited the (Na,K)-ATPase, we tested whether additional histidine would prevent inhibition of the ATPase activity. From the logarithmic affinity constants K I and K2 of histidine for Zn given under "Materials and Methods,'' we calculated that 230 and 275 p~ additional histidine (total histidine, 280 and 325 PM) would be necessary to reduce the ionized Zn to 3 nM when the total Zn was 35 and 45 pM, respectively. When the additional histidine was added followed by the addition of Zn to produce a total of 35 and 45 PM, the ATPase was restored quantitatively from an inhibited to a partially activated state.

The Effect of Preincubation on (Na,K)-ATPase Actiuity
If Zn in the assay system accounted for the suboptimal activity of the (Na,K)-ATPase, it should produce irreversible inactivation of the (Na,K)-ATPase in the absence of added chelator. To study this, the assay system was preincubated from 5 to 45 min at 37 "C without chelator prior to the addition of both ATP and EGTA (Fig. 6). Under these conditions, preincubation for 5 min led to a decrease in ATPase, and preincubation for 15 min led to maximal reduction of the (Na,K)-ATPase activity. When 50 p~ EGTA was present throughout the preincubation period, the ATPase remained maximally activated at approximately 1.9 nmol of P,/pg of protein/30 min; when no EGTA was present during the prein- cubation or assay, the ATPase activity remained low (Fig. 6). These data indicate that chelator activation of the ATPase requires its presence as soon as the membranes are brought to 37 "C and that the enzyme inhibition is irreversible.

DISCUSSION
Because of the biologic importance of Ca and the larger body of knowledge about the role of Ca as an activator or inhibitor of physiologic systems, the effects of EGTA are most likely to represent the result of Ca chelation, although the chelator has a high affinity for at least 12 divalent cations. In this regard, the addition of Ca to red cell (Na,K)-ATPase has been shown to inhibit its activity. This occurs as the Cadependent Mg-ATPase is activated, and inhibition of the (Na,K)-ATPase is maximal at 180 p~ Ca (9). Some investigators have added chelators such as EGTA in relatively high concentrations, 0.5 m~, to eliminate the Ca-ATPase when studying the (Na,K)-ATPase (2). Also, Ca inhibition was suggested to explain the effect of chelators on (Na,K)-ATPase of rabbit brain (14). For these reasons, we suspected initially that Ca chelation was necessary to permit measurement of the (Na,K)-ATPase in lymphocyte plasma membranes.
Several lines of evidence indicated, however, that Ca chelation was not the explanation for the EGTA activation of the lymphocyte membrane (Na,K)-ATPase. First, histidine binds many of the divalent cations chelated by EGTA; however, histidine does not bind Ca even when present in concentrations as high as 10 mM, and histidine activated the (Na-K)-ATPase in a manner similar to EGTA. Furthermore, the addition of enough Ca to achieve 4 times the ionized Ca concentration present in the EGTA-free reaction mixture was required to reduce the EGTA-activated ATPase to the level observed in the absence of EGTA. Moreover, the addition of Ca to the ATPase maximally activated by histidine showed no inhibition until greater than 6 times the initial ionized Ca was present. Taken together, these data indicate that EGTA bound a cation other than Ca to activate the (Na,K)-ATPase.
The addition of chelators such as EDTA or EGTA to prevent trace metals other than Ca from interfering with the (Na,K)-ATPase has been suggested because of erratic measurements noted early in the study of the erythrocyte enzyme (15). In addition, several investigators have reported enhanced (Na,K)-ATPase activity in beef brain, kidney cortex, and chick brain at EGTA concentrations less than 100 VM (16-18). However, the inhibiting ions present in the assay were not defined specifically. The inhibitors may have differed in the various studies since the addition of EGTA did not alter the enzyme activity under some conditions (8).
Zn at micromolar concentrations is a known irreversible inhibitor of the (Na-K)-ATPase in plasma membranes such as electrophorus (19), and 2 PM Zn was present in our reaction mixture. Several lines of evidence suggested that Zn chelation by either histidine or EGTA stimulated the (Na,K)-ATPase. First, when the free Zn was restored to 2 p~ by the addition of Zn, the ATPase was inhibited to an extent similar to that present in the absence of chelator. Second, the (Na,K)-ATPase activity could be restored after the addition of Zn (prior to warming to 37 "C) only if additional chelator was added to reduce the free Zn to nanomolar concentrations. The inhibition of the lymphocyte (Na,K)-ATPase in the absence of chelator was irreversible once it was established which is consistent with Zn inhibition. These data suggest that Zn is the principal inhibitor of the (Na,K)-ATPase assay under these conditions, and its chelation by EGTA or histidine accounts for the increase in enzyme activity observed.