Conformational changes of renal sodium plus potassium ion-transport adenosine triphosphatase labeled with fluorescein.

We studied conformational changes of purified renal sodium plus potassium ion-transport adenosine triphosphatase (ATP phosphohydrolase, EC 3.6.1.3) labeled with fluorescein isothiocyanate. Fluorescein covalently binds to the alpha-subunit of the enzyme and inhibits the ATPase but not the p-nitrophenylphosphatase activity. Four unphosphorylated and three phosphorylated conformations were distinguished by the level of fluorescence and by the rate of its change (relative fluorescence is shown in percentages). Fluorescence of the ligand-free form (E1, 100%) was increased by Na+ (E1.Na form, 103%) and quenched by K+ (E2.K, 78%) at a site of high affinity (K0.5 for K+ = 0.07 mM). Mg2+ did not alter fluorescence of E1 or E1.Na but raised that of E2.K (E2.K.Mg form, 85-90%). Addition of excess Na+ to the E2.K.Mg form restored high fluorescence but the rate of transition from E2.K.Mg to E1.Na became progressively slower with increasing Mg2+ concentration. Two phosphorylated conformations, (E2-P).Mg (82%) and (E2-P).Mg.K (82%) were differentiated by a faster turnover of the latter form. A third conformation, (E2-P).Mg.ouabain, had the lowest fluorescence (56%) and its formation allowed the binding of ouabain to the phosphoenzyme. Reversible blocking of sulfhydryl groups with thimerosal inhibited the formation of E2.K and (E2-P).Mg.ouabain but not that of the other conformations of the fluorescein-enzyme. The thimerosal-treated fluorescein-enzyme retained K+-p-nitrophenylphosphatase activity, inhibition of this activity by ouabain and ouabain binding. The unphosphorylated enzyme had low (K0.5 = 1.2 mM) and the phosphoenzyme had high affinity (K0.5 = 0.03 - 0.09 mM) for Mg2+ in the absence of nucleotides. Since low and high affinity for Mg2+ alternates as the enzyme turns over, Mg2+ may be bound and released sequentially during the catalytic cycle.

We studied conformational changes of purified renal sodium plus potassium ion-transport adenosine triphosphatase (ATP phosphohydrolase, EC 3.6.1.3) labeled with fluorescein isothiocyanate. Fluorescein covalently binds to the a-subunit of the enzyme and inhibits the ATPase but not the p-nitrophenylphosphatase activity. Four unphosphorylated and three phosphorylated conformations were distinguished by the level of fluorescence and by the rate of its change (relative fluorescence is shown in percentages). Fluorescence of the ligand-free form (El, 100%) was increased by Na+ (EI-Na form, 103%) and quenched by K+ (E2-& 78%) at a site of high affinity for K+ = 0.07 m). M&+ did not alter fluorescence of El or El Na but raised that of E2*K (E2*K*Mg form, 85-90%). Addition of excess Na+ to the Et* K O Mg form restored high fluorescence but the rate of transition from E2*K-Mg to El -Na became progressively slower with increasing M&+ concentration. Two phosphorylated conformations, (E2-P) Mg (82%) and (E2-P)*Mg*K (82%) were differentiated by a faster turnover of the latter form. A third conformation, (E2-P)-Mg.ouabain, had the lowest fluorescence (56%) and its formation followed the binding of ouabain to the phosphoenzyme.
Reversible blocking of sulfhydryl groups with thimerosal inhibited the formation of E2-K and (E2-P) Mgeouabain but not that of the other conformations of the fluorescein-enzyme. The thimerosal-treated fluorescein-enzyme retained K+-p-nitrophenylphosphatase activity, inhibition of this activity by ouabain and ouabain binding.
Since low and high affinity for M&+ alternates as the enzyme turns over, Mg2+ may be bound and released sequentially during the catalytic cycle.
Several reactive states of (Na,K)-ATPase' (ATP phosphohydrolase, EC 3.6.1.3) could be distinguished by kinetic meas-* This work was supported by the Danish Medical Research Council and by United States Public Health Service Grant 1 R 0 1 16611, National Heart, Lung and Blood Institute, National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. The abbreviations used are (Na,K)-ATPase, sodium plus potassium ion-transport adenosine triphosphatase; (K)-NPPase, K+-stimulated p-nitrophenylphosphatase; thimerosal, sodium ethylmercurithiosalicylate; E l , conformations of high fluorescence; E2, conformations of low fluorescence. The symbol for each conformation includes the inducing ligands also. urements (1) but these measurements did not provide information about the underlying changes in protein structure. Ligand-induced structural differences in the a-subunit (molecular weight, about 100,000) of the enzyme were directly detected by digesting the purified enzyme with trypsin (2-4) or chymotrypsin ( 5 ) since different sensitive bonds were exposed as the conformation changed. By analyzing the rate of inactivation and the split products three major conformations of the a-subunit of (Na,K)-ATPase were found: a Na form ( E , ) (2, 3) a K form (E2) (2, 3), and an ouabain-induced form ( 5 ) . Phosphorylation from ATP (2, 3) or ouabain-binding ( 5 ) induced conformations similar to the K form. Another convenient and sensitive indicator of conformational changes in the protein is fluorescence of intrinsic tryptophan (6,8) or of extrinsic fluorescent probes (9,10). An extrinsic probe, fluorescein isothiocyanate binds convalently to the a-subunit at or near the ATP-binding site and, although it prevents reaction of the enzyme with ATP, it does not inhibit conformational changes induced by cations or by phosphorylation (9,10). Experiments with these intrinsic and extrinsic probes have allowed the estimation of the rate of conformational changes (6,9,10).
While the role of Na' and K' in the catalytic cycle and in the conformational transitions has been examined in detail, the role of Mg2+ is not clear, mostly because in kinetic studies it is difficult to separate the effects of free Mg2+ from those of the Mg-ATP and Mg-ADP complexes (11)(12)(13)(14). The fluorescence technique mentioned earlier allows a study of the interactions of both the dephospho forms and phospho forms of (Na,K)-ATPase with Mg2+ in the absence of nucleotides. It has also become feasible to examine whether the protein conformations of the K-bound form, the phosphoenzyme, and the ouabain-bound form of (Na,K)-ATPase are identical or whether they represent subconformations of the enzyme protein. Another interesting possibility is the use of selective chemical modification to study the underlying chemical mechanism of the transitions in the protein structure.
In this study we used purified, membrane-bound renal (Na,K)-ATPase (15) labeled with fluorescein isothiocyanate. The preparation contained exclusively the protein of (Na,K)-ATPase and fluorescein was bound only to the a-subunit (9,10). The fluorescein-labeled (Na,K)-ATPase was therefore an ideal substrate for a detailed examination of the structural changes in the a-subunit. We have measured the level and the rate of change in fluorescence in response to reaction with specific ligands to monitor differences in protein structure between the K-or Na-bound forms, the phosphorylated forms, and the ouabain-bound forms. Reversible modification with thimerosal (ethylmercurithiosalycilate) (16)(17)(18) has been used to assess the importance of sulfhydryl groups for transitions between the cation-induced conformations and the conformations of the phospho forms of the protein. The results show that subconformations of the Ez form can be characterized by their steady state fluorescence intensity. Our observations also allow a tentative classification of conformational changes by the underlying chemical mechanism since some conformational transitions were blocked by thimerosal, whereas other conformations remained unaffected.
The fluoresceinenzyme was also suitable for examining the interaction of Mg2' with different conformations in the absence of ATP. The phosphoenzyme had high and the unphosphorylated enzyme had low affinity for Mg". Our results provide evidence for tight binding of MgZf to the protein during transition between the phosphorylated conformations. Mg2+ affected the rate of dissociation of K-bound forms or Rb-bound forms of the protein in a way suggesting that the "occluded" K form of the enzyme also contained magnesium.

MATERIALS AND METHODS
(Na,K)-ATPase was purified in membrane-bound form from the red outer medulla of pig kidney as described (15). Specific activity of (Na,K)-ATPase was 28-44 pmol of P,/mg of protein/min at 37 "C. The enzyme was stored at -75 "C in 10% (w/v) sucrose, 1 mM EDTA, and 25 mM imidazole-HC1, pH 7.5. Labeling with fluorescein isothiocyanate was done by a slight modification of the method of Karlish (8). Aliquots of (Na,K)-ATPase were sedimented by centrifugation for 10 min at 148,000 X g in a Beckman Airfuge and resuspended in 100 mM NaC1, 1 mM Tris-EDTA, and 50 mM Tris-C1, pH 9.2, to a final concentration of 1.0-1.4 mg of protein/ml or about 5-6 p~ (Na,K)-ATPase. (Molar concentration of the enzyme was estimated by assuming a molecular weight of 280,000 and one binding site of high affinity for ATP and ouabain, respectively.) Fluorescein isothiocyanate was dissolved in dimethyl formamide, to a concentration of 1.5 mM, and was added to the enzyme suspension with continuous stirring to a final concentration of about 20 p~. The final concentration of dimethyl formamide was less than 2% (v/v). After incubation at 20 "C for 45 min, the enzyme was sedimented in a Beckman Airfuge at 148,000 X g for 10 min and was washed four times with 50 mM Tris-C1 and I mM Tris-EDTA, pH 8.0 (Buffer E) by centrifugation and resuspension. The final sediment was resuspended in 10% (w/v) sucrose in Buffer E to a final concentration of 1 mg of protein/ml, divided in 100-pl portions, and stored at -75 "C, where it was stable for at least 1 month. The fluorescein-(Na,K)-ATPase could also be stored in Buffer E at 0 "C for 2 weeks without much loss of activity.
To block sulfhydryl groups with thimerosal, fluorescein-(Na,K)-ATPase (0.08 mg of protein/ml) was incubated with 2 mM thimerosal, 1 mM Tris-EDTA, and 25 mM imidazole-HCI, pH 7.5, in an ice bath. After 60 min the enzyme was sedimented at 140,000 X g and 4 "C for 3 h in a Beckman type 65 rotor; the sediment was resuspended in Buffer E and stored at 4 "C. To reverse inhibition by thimerosal this enzyme was incubated with 3 mM dithiothreitol in Buffer E at room temperature for 60 min and assayed without removing dithiothreitol.
To estimate total phosphorylation 1 ml of the reaction mixture contained: 0.048 mg of fluorescein-(Na,K)-ATPase or 0.056 mg of thimerosal/fluorescein-(Na,K)-ATPase, 1 pmol of MgCI2, 1 pmol of ["PIPt (70 cpm/pmol), 0.5 pmol of ouabain, and 40 pmol of Tris-C1 (pH 8.0). To estimate nonspecific phosphorylation, MgCI2 was replaced by 1 pmol of Tris-EDTA in an otherwise identical mixture. After incubation at room temperature for 10 min, the reaction was stopped with 5 ml of ice-cold 5% (w/v) trichloroacetic acid containing 5% (v/v) phosphoric acid. The precipitated protein was collected by centrifugation at 4,500 X g for 30 min and washed three times with 5 ml of the same acid mixture. The final sediment was dissolved in 1 N NaOH, protein was measured by the Lowry procedure (20), and radioactivity was measured by scintillation counting. Specific phosphorylation of (Na,K)-ATPase is the difference between "total" and "nonspecific" phosphorylation. Nonspecific phosphorylation was less than 20% of the total.
Binding of ['Hlouabain in the presence of 5 mM MgC12 and 5 mM Tris-P04 was measured and corrected for nonspecific binding as described (21). Dissociation constant and the number of binding sites were calculated from Scatchard plots (22).
Fluorescence (excitation, 495 nm; emission, 519 nm) of fluoresceinlabeled enzymes was measured on a Perkin-Elmer MPF 44A spectrofluorimeter at constant temperature (25 * 1 "C) in a continuously stirred cuvette. Ligands or reagents were added to the samples in 6297 small volumes from Hamilton syringes mounted on pushbutton repetitive dispensers to minimize changes in fluorescence because of dilution. The total additions never increased the original volume by more than 5%. Intensity of fluorescence was expressed in two ways: ( a ) as a percentage of the fluorescence observed in the absence of cations ("relative fluorescence") or ( b ) in arbitrary units ("fluorescence change"). To follow rapid changes in fluorescence the fluorimeter was modified and its time constant was lowered to 0.003 s. The fastest rate of change that could be measured depended on the speed of mixing. It was about 5 s-l as estimated by mixing 10 pl of 10% rhodamine B solution into 2.5 ml of water. The rate constants were estimated by graphic analysis of the records as follows. A smooth curve that best approximated the tracings was drawn with a curve ruler. The limiting value of fluorescence that this curve approached asymptotically was estimated and the time necessary to reach half of this value ( t 1~4 was read from the chart. Since the changes in fluorescence could be described by a single exponential term the rate constant ( k ) was calculated from the first order rate equation ( k = 0.693/t1/2). For each value of k the mean * S.E. and the number of the determinations are given. The free Mg'+ concentration was calculated on the basis of published equilibrium constants (23).

RESULTS
Fluorescein binds at or near the ATP-binding site and prevents reaction with nucleotides but not with phosphatase substrates (9, 10). In our experiments a 4-fold molar excess of fluorescein inhibited 98% of the ATPase but none of the (K)-NPPase activity of the enzyme. We routinely monitored fluorescence at the optimal wavelengths of excitation (495 nm) and emission (519 n m ) (see emission spectrum on indicating that the conformation in Tris buffer was close to that of the Na-bound form (El.Na) (cf: Ref. 2). Mg" (1-3 mM), Pi (1-4 mM), or ouabain (0.1 mM) alone did not alter fluorescence of E, or E l .Na (not shown). When added together to E,, 3 mM pi plus 2 mM Mg'+ reduced fluorescence to 82% (Fig. 1, Lower tracing), The addition of P,, Mg2+ and ouabain quenched fluorescence to 56%, a level which was lower than that obtained with any other combination of ligands (Fig. 1, lower tracing).
Conformational Changes of the Unphosphorylated Fluorescein-(Na,K)-ATPase-To determine the relative affinities of the unphosphorylated conformations for cations the fluorescein-enzyme was titrated with K' , alone or in the presence of Mg" (Fig. 2) or Na+ (Fig. 3). In the absence of other cations quenching of fluorescence by K' followed a hyperbola (Fig. 2) and the data fitted a single straight line in a double-reciprocal plot (not shown), indicating that K' was bound to a single site. Koa for K' at this site was 0.07 & 0.01 mM (n = 5 ) . With increasing Mg2+ concentration the titration curves with K' became sigmoid. (In Hill plots of the data the Hill-coefficients were 1.2-1.5 (not shown).) The titration curves with K' became sigmoid also in the presence of Na+ (Hill coefficients varying between 1.3-1.5) and Ko.5 for K' was also a sigmoid function of Na+ concentration (Fig. 3, inset). K' therefore appeared to act at a single site in the absence of other cations but at multiple interacting sites in the presence of Mg2+ or Na+.
If fluorescence was maximally quenched (to 78%) by 10 mM KCI, the addition of 2 mM MgC12 increased fluorescence to 90% and slowed the rate of increase in fluorescence induced by 100 mM NaCl (Fig. 4, left panel). In the absence of Mg"' the rate of the Ez.K to E l .Na transition was 1.73 & 0.03 s-' ( n = 7) and this rate was progressively slowed by increasing concentrations of Mg"' (Fig. 4, rightpanel). Mgz+ concentration at half-maximal rate was about 1.2 mM. This value is an estimate of the apparent affinity of the unphosphorylated enzyme for Mg". Rb', a congener of K' , quenched fluorescence to the same extent as K' but the rate of the E s . Rb to E , . Na transition was slower than the rate of the E2. K to E,. Na transition, both in the presence and in the absence of Mg2+ (Fig. 4, right panel). High concentrations (more than 3 mM) of ATP slightly enhanced the rate of the EB. K to EZ .Na   24) that, in the presence of Mg", Pi quenched fluorescence by phosphorylating the enzyme (Fig. 1, lower tracing). We also confumed the observation of Karlish that the fluoresceinenzyme can be phosphorylated with ["PIPi. Additions of small amounts of Mg2' in the presence of a saturating concentration of Pi (3 mM) reduced fluorescence stepwise (Fig. 5 ) each plateau probably corresponding to a new steady state level of the phosphoenzyme. The minimum level of fluorescence after phosphorylation was 82%, slightly higher than that of the Ez. K form (78%). Quenching by phosphorylation was half-maximal a t 0.15 mM Mg"+ in the presence of 3 mM Pi (Fig. 5) and at 0.4 mM Pi in the presence of 1 mM Mg'+. The (Ez-P) -Mg conformation with low fluorescence was formed at a rate of 0.14 & 0.005 s" (n = 8) as estimated from the time course of quenching following the addition of P, plus Mg'+. Na+, added together with Mg2+ but before P,, prevented quenching just as it prevents phosphorylation from Pi (1,12). If quenching by P, We could detect another conformation of the phosphorylated fluorescein-(Na,K)-ATPase, (E2-P). Mg. K, by measuring the rate of quenching by P, and Mg2' and the rate of Na+induced reversal in the presence of K' . Since both phosphorylation and K' reduced fluorescence, we chose conditions in which quenching by K+ was excluded. First we added 2 mM MgC12 to fluorescein-(Na,K)-ATPase then 1-2 mM K' . This much Mg2' prevented quenching by K' and quenching started only after the addition of 3 mM Pi. K' had two effects; first, it increased the rate of quenching by Pi plus Mg2+ from 0.14 f 0.005 s-' (n = 8) to 1.60 f 0.23 s-' (n = 5). Second, K' increased the rate of reversal of this quenching after addition of 100 mM Na' from 0.20 f 0.05 s" (n = 8) to 0.82 2 0.20 s-' (n = 5). Similar effects of K+ on the rate of phosphorylation were observed previously (1,12). The turnover rate for phosphate of the (Ea-P) e Mg . K conformation seems therefore to be faster than that of the (E,-P). Mg form.
If the rate of phosphorylation is slower than the rate of ouabain-binding (e.g. a t low Mg2+ concentration), ouabain can "trap" the phosphoenzyme, so that dephosphorylation is negligible relatlvp to phosphorylation. Under these conditions it is possible to determine the initial rate of formation of the (E,-P). Mg . ouabain conformation and the affinity of this form for Mg". In the presence of 4 mM P, and 0.1 mM ouabain the rate of formation of (E,-P). Mg . ouabain could be measured after the addition of as little as 0.01 mM Mg2+ (Fig. 6). Mg2+ concentration a t half-maximal rate, measured on two fluorescein-(Na,K)-ATPase preparations, was between 0.03 and 0.09 mM. These changes in fluorescence were not due to the presence of ouabain, because ouabain does not absorb light at 495 nm nor emits light at 519 nm.
Conformational Changes of Fluorescein-(Na,K)-ATPase after Treatment with Thimerosal-Thimerosal reversibly blocks some sulfhydryl groups of (Na,K)-ATPase and inhibits the ATPase but not the (K)-NPPase activity of the enzyme (16)(17)(18). If the reaction with thimerosal took place at 0 "C, thimerosal actually increased the hydrolysis of p-nitrophenylphosphate by both the native (18) and the fluorescein-enzyme (Fig. 7), not only in the presence but also in the absence of K' . The maximal (K)-NPPase activity (at 40 mM K') of both the fluorescein-enzyme and the thimerosal/fluoresceinenzyme was completely inhibited by 0.1 mM ouabain (not shown). The basic reaction medium did not contain any K' or its congeners and in this medium the native or fluoresceinenzyme had no ouabain-sensitive nitrophenylphosphatase activity.
Fluorescence measurements showed that thimerosal selectively inhibited some conformational changes of both the unphosphorylated and the phosphorylated enzyme. Thimer- osal inhibited quenching by K' (i.e. the El to E2 -K transition), but not the Na'-induced increase in fluorescence (i.e. the E, to E,. Na transition). (Fig. 8, upper tracing). Incubation of the thimerosal/fluorescein-enzyme with 3 mM dithiothreitol, as described under "Materials and Methods," restored quenching by K' (Fig. 8, lower tracing). Fig. 9 shows the effects of thimerosal on the phosphorylated forms. Thimerosal did not alter, or in some experiments slightly reduced, quenching by Pi plus Mg'+ and did not affect reversal of this quenching by Na+. When present, the slight reduction of quenching could be reversed with dithiothreitol. (Fig. 9, tracings T-F-E and T-F-E + DTT). The thimerosal/ fluorescein-enzyme could be phosphorylated with ['j2P]Pi (not shown). In contrast to its small effect on the (E2-P) .Mg form, thimerosal completely prevented formation of the (E2-P) Mg . ouabain form. After thimerosal treatment the rate and the extent of quenching by Pi, Mg' and ouabain were the same as those by P, and Mg2' (Fig. 9, tracings T-F-E and T-F-E   + 0.1 mM ouabain). Though (Fig. 9).
An equimolar mixture ofp-nitrophenylphosphate and Mg'+ (final concentration, 1.56 mM) reduced fluorescence of the thimerosal/fluorescein-(Na,K)-ATPase to 83% but that of the fluorescein-(Na,K)-ATPase only to 96% (Fig. 10). This decrease in fluorescence was probably related to the hydrolysis of p-nitrophenylphosphate because fluorescence of the fluorescein-enzyme, which does not hydrolyzep-nitrophenylphosphate in the absence of K' , was only slightly reduced (upper tracing on  quenched fluorescence of the thimerosal/fluorescein-enzyme more rapidly ( k = 0.21 + 0.01 s -I , n = 8) than Mg2+ plus Pi (0.14 s-'; see above). This difference in rates was statistically significant ( p < 0.01). Because of the instability of p-nitrophenylphosphate, the reaction mixture contained a small amount of Pi that, however, was not enough to quench fluorescence measurably (final concentration of Pi in the experiment shown in Fig. 10,0.005 mM). Quenchings of fluorescence by Pi and p-nitrophenylphosphate were similar in two ways: both required Mg2+ and both were accelerated by K'. This analogy suggests that p-nitrophenylphosphate might have phosphorylated the enzyme as P, did (25,26), but we did not attempt to measure this phosphorylation. Fig. 11 summarizes schematically all forms of fluorescein-(Na,K)-ATPase and the paths of their interconversion that we could distinquish by the intensity and by the rate of change of their fluorescence with two exceptions. E1 -Mg and Mg. El. Na are probably not separate conformations relative to E, and E Na, respectively, and they are included in Fig. 11 only to show that addition of Mg'+ to El or El -Na did not change the level of their fluorescence.

Conformations of Fluorescein-(Na,K)-ATPase
To study conformational changes of (Na,K)-ATPase we used fluorescein as reporter group (9, 10) and purified plasma membranes as source of the enzyme. These membranes contained only the a and ,&subunits of (Na,K)-ATPase (15) and thus artifacts, due to labeling of other membrane proteins, were absent. Since fluorescein binds only to the a-subunit, we assumed that any change in fluorescence reflected conformational changes of primarily this subunit. Such conformational changes were studied earlier by digestion with proteolytic enzymes (see Refs. 2-5 and the introduction to the text) or by the fluorescence of intrinsic tryptophan (6)(7)(8). Fluorescein as a reporter group offered several advantages: the fluorescent signal was much larger and more stable than that of tryptophan (6)(7)(8) and, because of the fast response, the rate of conformational change could be measured, which measurement is not possible with proteolysis. The disadvantage of fluorescein is that it blocks interaction with ATP (9, 10) and prevents the study of conformations induced by ATP or other nucleotides. Conformations of the fluorescein-enzyme are summarized on Fig. 11 and their properties are discussed as follows.
The El Conformation-El, the form of the enzyme in Tris buffer, was highly fluorescent. A small, but significant increase in this fluorescence by Na' (Fig. 1) indicated that the conformation of the Na-bound form was similar to E,. Earlier El and El. Na were described as a single conformation, the Na form, because of the identical pattern of inactivation by trypsin (2, 3, 5) and the similarity of tryptophan fluorescence (6,7). ATP-binding experiments, however, showed a Na+-dependent increase in the amount of ATP-binding sites ( Table  I in Ref. 27). Our fluorescence measurements provided evidence that this change in ATP binding is accompanied by a change in the structure of the a-subunit.
The EQ. K Conformation-This is the second major conformation detected by tryptic digestion (2, 3, 5) and by tryptophan fluorescence (6)(7)(8). K', as a single ligand, induced this conformation of fluorescein-(Na,K)-ATPase at a fast rate (Fig.  1) and at a single site of high affinity = 0.07 mM). The E2-K.Mg Conformation-This was a new conformation that could be differentiated from El and E2. K by three properties: first, the level of fluorescence of E,. K. Mg was between that of El and E , . K; second, Ea. K -Mg was converted to E, .Na more slowly than E2.K (Fig. 4), and third, titration of fluorescein-(Na,K)-ATPase showed that, in the absence of Mg2+, K+ bound to a single site but, in the presence of Mg2+, K+ bound to multiple sites (see Fig. 2 and "Results"). The composition of E2 K -Mg is therefore E2. K,-Mg, where the most likely value for n is 2, suggested by the stoichiometry of Na+-K+ transport (1). These fluorescence measurements were in agreement with the results of structural studies made by tryptic digestion which showed that Mg" does not induce the K form (7,8).
The (E2-P). Mg Conformation-This conformation had about the same low level of fluorescence as EB. K (Fig. 1) and structural similarity between the two conformations was shown earlier by tryptic digestion (3, 5, 7) and tryptophan fluorescence (7,8). Direct measurement of the incorporation of [32P]Pi confinned an earlier observation of Karlish (10, 24) that the fluorescein enzyme, though it does not have ATPase activity, can be phosphorylated by P,. The steady state level of the phosphorylated fluorescein-enzyme was however much lower than that of the native enzyme. It is likely therefore that fluorescein at least partially inhibited phosphorylation. The conformational transition was probably proportional to phosphorylation because both processes were half-maximal at about the same concentration of Mg" and P, (see Fig. 5, "Results," and Ref. 12).
The (E2-P). Mg. K Conformation-This could be differentiated from (E2-P) -Mg by the faster rate of conformational transitions from E, to (E,-P) .Mg e K and from (Ez-P) -Mg-K to E,. Na (see "Results"). K' increased the rate of this transition the same way as it increases phosphorylation from P, (12).
The (E2-€') .Mg.ouabain Conformation-This was characterized by its low level of fluorescence and by the sensitivity of its formation to the blocking of some sulfhydryl groups. These results, together with the previous observation that ouabain binding exposes a peptide bond within the a-subunit to chymotrypsin digestion (5), show that ouabain-binding induces an unique conformation of the a-subunit of (Na,K)-ATPase. The rate of the conformational transition, E, to (E2-P) .Mg.ouabain, was similar to the rate of ouabain-binding which was apparently the rate-limiting step. Fluorescein-(Na,K)-ATPase, treated with thimerosal, could assume the (E2-P) . Mg but not the (E2-P) . Mg ouabain conformation even though this enzyme could bind ouabain and its (K)-NPPase activity was inhibited by ouabain ( Fig. 9 and "Results''). This apparent paradox can be resolved by assuming that binding of ouabain to (E*-P) -Mg was sufficient for inhibition without further conformational change at the ATP binding center though ouabain normally induced such a change.
The Chemical Basis of Conformational Changes AS sensed with proteolytic enzymes (2-5) and fluorescent probes (6)(7)(8)(9)(10) the structural transitions between dephosphoforms (El .Na and E2.K) and phosphoforms (EI-P and E2-P) appear to be identical. Specific inhibition of functional groups of the enzyme, however, showed that notable differences in the structure of these forms can be detected. Thimerosal, the inhibitor we used, reacted with sulfhydryl groups only, because its effects could be reversed with dithiothreitol (Figs. 9 and 10). In the fluorescein-enzyme, thimerosal selectively blocked the El to E2.K and the (E,-P) .Mg to (E,-P) .Mg. ouabain transitions.
These two conformational changes are therefore distinguished either by a need for reactive sulfhydryl groups or by a need for free space around those groups that thimerosal