Binding of Two Sr2+ Ions Changes the Chemical Specificities for Phosphorylation of the Sarcoplasmic Reticulum Calcium ATPase through a Stepwise Mechanism*

The sequential binding of Sr2+ and Ca2+ to the cyto- plasmic transport sites of the sarcoplasmic reticulum calcium ATPase allows the formation of two different mixed complexes: "E. Sr-Ca, with Sr2+ bound to the '*inner" site and Ca" bound to the "outer" site, and 'E. Ca. Sr, with Ca" bound to the inner site and Sr2+ bound to the outer site (pH 7.0,26 "C, 10 mM MgC12, 100 mM KCl). Both "E. Sr . 46Ca and 'E e 46Ca. Sr react with ATP to internalize one 46Ca/phosphoenzyme. The value of = 83 PM Sr2+ for activation of the enzyme for phosphorylation by ATP is much larger than = 28 p~ Sr2+ for inhibition of phosphoenzyme formation from inorganic phosphate ( n ~ = 1.0-1.3). These results are consistent with the sequential binding of two strontium ions with negative cooperativity and dissociation constants of Ksrl = 35 I.IM and Ksr2 = 65 PM. The species 'E.Sr2 and 'E-Caz react rapidly with ATP but not inorganic phosphate. However, enzyme with one strontium bound, %-Sr, does not react with either inorganic phosphate or ATP. Therefore, the conformational changes in the enzyme that alter the chemical specificity for phosphorylation by ATP and by inorganic phosphate are different. This re- quires the existence of at least three forms of the unphosphorylated enzyme

chemical specificity of "E. Ca has not been fully characterized; however, cE. Ca, which was formed transiently, does not react with ATP (9). The positive cooperativity for the binding of two calcium ions makes it difficult to determine if the changes in the chemical specificities for phosphorylation by ATP and inorganic phosphate occur concurrently or independently.
The binding of strontium to the cytoplasmic transport sites also acts as a switch that changes the catalytic specificity of the enzyme: the binding of strontium permits the formation of phosphoenzyme from ATP and inhibits the formation of phosphoenzyme from inorganic phosphate (10). Strontium is transported by the calcium ATPase at a rate that is comparable to the rate of calcium transport (11)(12)(13)(14). Incubation of the enzyme with calcium for several seconds is known to cause a conformational change from the E species to the ' E species, which is defined as the stable form of the enzyme in the presence of calcium. This conformational change is accompanied by an increase in the rate of phosphorylation by A T P %.ATP-Ca2 forms phosphoenzyme with a rate constant of k = 220 s" compared with k = 70 s-l for E . ATP. Ca2. Strontium is also apparently able to catalyze the E to "E conformational change since the stable species of enzyme with bound strontium is also phosphorylated by ATP with k = 220 s-l (15). The binding of strontium and calcium to the cytoplasmic transport sites also causes similar changes in the intrinsic fluorescence of the enzyme and the fluorescence of 1-anilino-8-naphthalenesulfonate covalently bound to the enzyme (13,16). At pH 7.4, the Hill slope of n H = 1.64 observed with strontium for the change in the intrinsic fluorescence of the enzyme is slightly lower than the value of n H = 1.83 observed with calcium (13). However, at pH 7.0, the Hill slope of n H = 0.93 observed with strontium for the change in the fluorescence of ANS-bound to enzyme is significantly lower than the value of n H = 1.8 observed with calcium (16).
The value of 1:l for the stoichiometry of Sr2+ uptake per ATP hydrolyzed, which was calculated from a comparison of the rate of "Sr uptake and ATP hydrolysis, is lower than the value of 2:l for Ca2+ uptake (12,13). However, a value of 2:1 for the stoichiometry of Srz+ uptake per ATP hydrolyzed was determined recently by using the pulsed pH-stat method, which allows this stoichiometry to be measured in a single assay (14).
Results reported here indicate that strontium binds to both cytoplasmic transport sites but with little or no positive cooperativity; this conclusion is consistent with Hill slopes of n H = 1.0-1.3 and a stoichiometry of 2:l for STz+ uptake per ATP hydrolyzed. The changes in chemical reactivity toward phosphorylation by either ATP or Pi caused by the binding of strontium do not occur with the high degree of positive cooperativity that is observed for the binding of calcium; Hill slopes of n H = 1.0-1.3 were observed with strontium, whereas Binding of Strontium to the S R Calcium-ATPase Hill slopes of n H = 1.6-2.0 have been reported for calcium (9,17,18). Therefore, enzyme with one strontium ion bound to the cytoplasmic transport sites can be formed at equilibrium; however, enzyme with one calcium ion bound cannot be formed at equilibrium in large quantities.
Three different chemical specificities were observed in the presence of strontium: free enzyme reacts only with inorganic phosphate, enzyme with one strontium ion bound does not react with either inorganic phosphate or ATP, and enzyme with two strontium ions bound reacts only with ATP. Therefore, the changes in chemical specificities caused by strontium occur in a stepwise manner. The binding of one strontium ion prevents phosphorylation by inorganic phosphate but does not activate the enzyme for phosphorylation by ATP. This agrees with the finding that enzyme with one calcium ion bound, which was formed transiently, does not react with ATP (9); however, it is not known if this species can react with inorganic phosphate. The binding of the second strontium ion or the second calcium ion activates the enzyme for phosphorylation by ATP. Although strontium binds to the cytoplasmic transport sites with much weaker affinity than calcium, the changes in catalytic specificity caused by strontium appear to be identical to those caused by calcium.
It is known that the dissociation of two calcium ions from the cytoplasmic transport sites is sequential: the calcium ion bound to the "inner" transport site' cannot dissociate unless the "outer" transport site is unoccupied (9,(19)(20)(21)(22)(23). Two different species of enzyme with one strontium ion and one calcium ion bound to the cytoplasmic transport sites, "E. Sr .
Ca and ' E . Ca. Sr, were formed; these mixed species are able to react with ATP and internalize one calcium ion and, presumably, one strontium ion. The formation of two different mixed species of enzyme with calcium and strontium demonstrates that strontium can bind to both the inner and the outer transport sites.
Tightly sealed sarcoplasmic reticulum vesicles were prepared from rabbit white back and hind leg skeletal muscle by a slight modification of the MacLennan procedure (24), as described previously (25). The total amount of phosphoenzyme observed with saturating concentrations of calcium and ATP was 1.7-4 nmol (mg of total protein)".
The preparations hydrolyzed 3-5 pmol of ATP (mg of total protein)" min" when the vesicles were made permeable to calcium either by addition of the ionophore A23187 or by treatment with alkali (26). The ionophore A23187 in 100% ethanol (2 mM) was added to reaction mixtures to give a final concentration of 2 pM.
Methods-Reactions were carried out at 25 "C with 5 mM MgC12, 100 mM KC1, and 40 mM MOPS at pH 7.0, unless otherwise noted.
All reported concentrations of Ca2+ include 10 p~ Ca2+, which is the concentration of contaminating calcium measured under similar conditions (27).
[:3zP]H3P04 was treated with 1 N HCI for 20 min at 100 "C to hydrolyze any pyrophosphate and polyphosphate impurities. To 0.5 ml of ["PIPi (1 mCi/ml) was added 50 pl of 12 N HCI and 14 p1 of 1 M KH,PO,; this solution was heated to 100 "C for 20 min, with a marble covering the vial to prevent evaporation. The mixture was Petithory and Jencks (9) refer to the first calcium ion to dissociate from the cytoplasmic transport sites as the outer calcium ion and to the second calcium ion as the inner calcium ion. allowed to cool to room temperature and was neutralized with 0.6 ml of 0.98 N KOH. Acid-washed Norit A charcoal (50 mg) and a small piece of Whatman No. 1 filter paper (-50 mm2) were added, and this suspension was forced through a 25-mm Millex-GV filter unit using a I-ml syringe. The Whatman No. 1 filter paper was added to bind a contaminant that increases the background levels in the phosphoenzyme measurement^.^ The charcoal and filter paper were washed with 0.5 ml of distilled water; this suspension was forced through the same Millex-GV filter unit. The first and second filtrates were combined and added to 29 ml of 5 mM Pi.
Phosphoenzyme levels were measured essentially as described by Verjovski-Almeida et at. with slight modifications (29, 30). Enzyme that had been phosphorylated by [y-32P]ATP was quenched with acid to give a final concentration of 0.5 M HCIO, and 13 mM KH2P04. Enzyme that had been phosphorylated by [""PIPi was quenched with acid to give a final concentration of 0.5 M HCIO,. All subsequent manipulations and solutions were at 0-4 "C. Bovine serum albumin and ATP were added to the acid-quenched reaction mixtures to give final concentrations of -0.3 mg/ml total protein and -1 mM ATP. After 5 2 h at 0 "C, the samples were centrifuged at 1,500 X g for 15 min, and the pellets were resuspended in 0.5 M HC104 and 15 mM KH2P0,. The protein was collected on Whatman GF/C glass fiber filters by vacuum filtration and rinsed with 15 ml of the resuspension solution. The radioactivity was measured by liquid scintillation counting in glass vials containing 7 ml of Aquasol-2.
Experiments to measure the uptake of were carried out essentially as described previously (9). The reaction mixtures were quenched by the addition of EGTA and CaCI2 to give final concentrations of 5 mM EGTA and 3.3 mM CaCl,. The vesicles were collected within 5 s on nitrocellulose filters (Millipore HAW/P 0.45 pm) by vacuum filtration; the filters had been soaked with a solution of 10 mM CaCI2, 100 mM KC1, 5 mM MgSO,, and 40 mM MOPS, pH 7.0. The collection tube and filters were rinsed with 15 ml of 15 mM EGTA, 10 mM CaC12, and 40 mM MOPS, pH 7.0. The filters were counted in glass vials containing 7 ml of Aquasol-2.
Uptake of 45Ca and formation or decay of phosphoenzyme were followed with a rapid mix-quench apparatus that can be used with either three or four syringes of 1-ml volume, as described previously (9,31).
Apparent dissociation constants of 3.9 X M for the calcium-EGTA complex and 8.9 X M for the strontium-EGTA complex were used to calculate the concentration of free Ca2+ and Sr2' (32, 33). Concentrations of free metal ions, MgATP, MgPi, and SrPi were calculated using the dissociation constants and the computer program of Fabiato (33-36).

RESULTS
The Binding of 45Ca to 'E .Sr2-The binding of calcium to the cytoplasmic transport sites was assayed by measuring the amount of 45Ca internalized after the addition of ATP and EGTA ( Fig. 1). It is known that the addition of ATP to enzyme with two calcium ions bound results in the formation of phosphoenzyme and the internalization of both calcium ions into the vesicles (37, 38). The simultaneous addition of ATP and EGTA to CE.45Caz results in the rapid binding of ATP to form "E. ATP .45Caz, which partitions between the formation of phosphoenzyme, with k = 220 s-l, and dissociation of 45Ca, with k = 80 s-'; therefore, the addition of ATP and EGTA to CE.45Caz results in a single turnover of the enzyme with the internalization of 220/(220 + 80) = 0.7 of the 45Ca bound to the cytoplasmic transport sites and provides a measure of the amount of 45Ca that was bound prior to the addition of ATP and EGTA (9,39). The open circles and open squares in Fig. 1 show that the addition of 0.4 mM ATP and 15 mM EGTA to enzyme that had been incubated in the presence of 190-250 p M 45Ca for 5 s results in the internalization of -3.6 nmol of ''Ca/mg of protein. The amount of 45Ca that is internalized after the addition of ATP and EGTA is dependent on both the amount of 45Ca bound and the kinetic partitioning ratio between phosphorylation and dissociation of 45Ca. A value of K0. 5   2.0 p~ Ca" was measured by Petithory and Jencks' (9) for the formation of %-Ca2 under the conditions used for this work; therefore, >99% of the enzyme will be present as 'E-"Ca2 in the presence of -200 pM 45Ca. The addition of ATP and EGTA to "E.'Ta2 results in the internalization of -70% of the "Ca bound to the cytoplasmic transport sites (9). A value of 5.2 nmol of "Ca bound per mg of protein was calculated from the observed value of 3.6 nmol of "Ca/mg of protein internalized after the addition of ATP and EGTA and the value of 70% for the kinetic partitioning toward phosphorylation under these conditions. A value of EtoM = 2.6 nmol/mg was calculated from the total concentration of '%a bound and the ratio of two calcium ions bound per E -1 (9); this value is within the range of E, , = 1.7-4.0 nmol/mg that was observed for different preparations of enzyme. Therefore, 3.6 nmol of "Ca/mg of protein is internalized after the addition of ATP and EGTA when 2 mol of '5Ca are bound per mol of enzyme and 70% of the enzyme undergoes phosphorylation, as indicated on the right axis of Fig. 1.
The filled circles (0) in Fig. 1 show that the addition of 190 PM "Ca to enzyme that had been incubated with 730 PM Sr2' results in the binding of "Ca with biphasic kinetics: one Etot,l binds rapidly within 50 ms, followed by much slower binding of a second "Ca/Ebb1 over several seconds. The solid line is calculated for rapid binding of one "Ca/Ebbl, with k = 150 s-', and slow binding of 0.83 46Ca/Ebb~, with k = 0.7 s-'. The value of 3.3 nmol of "Ca/mg observed at t = 5 s is slightly lower than the value of 3.6 nmol of 'Ta/mg observed after the addition of 190-250 PM "%a to enzyme that had been incubated in the presence of either EGTA or unlabeled calcium. This decrease of 8% in the end point value may represent either competition at equilibrium between the binding of SP+ and 45Ca for the cytoplasmic transport sites or competitive inhibition by Sr2+ at the M e binding site.
The open circles (0) show that the addition of 250 pM 'Ta to enzyme that had been incubated with 39 p~ 40Ca also results in the binding of 'Ta with biphasic kinetics: one "Ca binds rapidly within 50 ms, followed by much slower binding of a second 45Ca/Etotal over several seconds. The dashed line is calculated for the binding of one 45Ca/Ebbl, with k = 50 s"', followed by binding of a second 45Ca/Ebbl, with k = 0.1 s-l, in agreement with previous results (9,21). Therefore, the reactions of 'E Sr2 and 'E. Caz with 45Ca appear to be qualitatively similar, which suggests that the mechanisms for the binding and dissociation of these ions at the cytoplasmic transport sites are similar.
It is known that calcium binds to and dissociates from the cytoplasmic transport sites in a sequential mechanism (9,19,21). The rate-limiting step in the exchange of 45Ca into cE.40Caz is the dissociation of 40Ca from the cytoplasmic transport sites. The dissociation of calcium from the outer binding site is rapid, with k-c. = 50 s-' , and is not affected by the presence of calcium in the external medium, but the dissociation of calcium from the inner binding site is slower and is inhibited by the presence of calcium in the external medium (9). This inhibition is caused by occupancy of the outer calcium binding site: the outer calcium binding site must be empty before the inner calcium ion may dissociate. This sequential mechanism for the dissociation of calcium has been proposed to explain the biphasic kinetics observed for the binding of 45Ca to %.Can (9,21).
The biphasic kinetics that are observed for the binding of '%a to 'E-SrZ (0, Fig. 1) are consistent with an ordered and sequential mechanism for the dissociation of strontium from the cytoplasmic transport sites, as shown in Scheme 1. A rate constant of k-s, = 115 & 15 s-' was observed for the dissociation of strontium from "E. Sr2 in the presence of 15 mM EGTA (15); this value agrees within experimental error with the value of k-s, = 150 & 30 s-' observed for the dissociation of one strontium ion in the presence of 190 PM calcium. Therefore, the dissociation of the outer strontium ion is not inhibited by the presence of 190 PM calcium in the external medium and results in the rapid formation of %.Sr, with strontium bound to the inner binding site. The binding of calcium to the outer binding site of 'E. Sr results in the formation of 'E. Sr + Ca. The dissociation of the inner strontium ion can occur only when the outer binding site is unoccupied. Therefore, the dissociation of the inner strontium ion is very slow in the presence of 190 pM calcium and occurs over several seconds. The solid line in Fig. 1 is drawn for dissociation of the outer and inner strontium ions with rate constants of 150 s-' and 0.7 s-', respectively. The open squares (0) in Fig. 1 show that the addition of 190 pM 45Ca to enzyme that had been incubated with EGTA results in the formation of "E .45Caz with biphasic kinetics. The dotted line is calculated for the formation of 0.6 E,,I with k = 75 s-', followed by the formation of 0.4 "E. 45Caz/Etotal with k = 3 s-'. Petithory and Jencks (27) measured a faster overall rate for the reaction of 120 p~ 45Ca with enzyme that had been incubated in the presence of EGTA and 5 mM M e and showed that two calcium ions must be bound to the transport sites before phosphorylation by ATP can occur. It is possible that the slower rate for the reaction of 190 p~ 45Ca with enzyme that had been incubated in the presence of EGTA and 10 mM Mg2+ (0, Fig. 1) results from binding of magnesium to the cytoplasmic transport sites and slow dissociation of M$+ in the presence of 190 p M Ca2+.
The relatively rapid binding of the second 45Ca ion to enzyme that had been incubated with EGTA (0) demonstrates that the slow binding of the second calcium ion to enzyme that had been incubated with strontium is not caused by slow association of 190 p M calcium with "E .Ca. We conclude that it is caused by the slow dissociation of strontium from "E. Sr-45Ca, which is required before 45Ca can bind to the inner transport site. The plateau in the binding of 45Ca that is observed between 0.03 and 0.25 s demonstrates that ' E .Sr .45Ca is a kinetically stable intermediate that can be phosphorylated by ATP, which results in the occlusion of one 45Ca and, presumably, one strontium ion.
The addition of ATP and EGTA to "E. Sr 45Ca results in the internalization of 1.8 nmol of 45Ca/mg of protein. This value is the same as the value observed after the addition of ATP and EGTA to "Ea4'Ca .45Ca and is equivalent to onehalf of the value of 3.6 nmol of 45Ca/mg that was observed after the addition of ATP and EGTA to "E. 45Caz. These data indicate that the reactivity of "E + Sr . Ca after the simultaneous addition of ATP and EGTA is identical to that of "E .Caz, which is known to form 70% phosphoenzyme because of the kinetic partitioning between phosphorylation and dissociation of calcium under these conditions, as described above (39). This result suggests that the replacement of Ca2+ for Sr2+ at the inner transport site has little or no effect on the rate constant for the dissociation of Ca2+ from the outer transport site, with k = 80 s-', in spite of the much lower affinity of SP+ for the cytoplasmic binding sites.
The Dissociation of 45Ca from cE-45Caz in the Presence of Strontium-The rate of dissociation of 45Ca from "E .45Ca~ was measured in the presence of 50 pM 40Ca, 300 p M Srz+, or 5 mM EGTA as shown in Fig. 2. The amount of calcium bound to the cytoplasmic transport sites was assayed by measuring the internalization of 45Ca after the addition of ATP and EGTA as described above (Fig. 1). The addition of 0.3 mM ATP and 15 mM EGTA to enzyme that had been incubated for 15 s in the presence of a saturating concentration of 45ca, 40 pM, results in the internalization of 3.0 nmol of 45Ca/mg. A value of 4.3 nmol of 45Ca bound per mg of protein was calculated from the observed value of 3.0 nmol of 46Ca/mg internalized and the fraction of the bound 46Ca that is internalized after the addition of ATP and EGTA, 70%. A value of = 2.1 nmol/mg was calculated from the total concentration of 45Ca bound, 4.3 nmol/mg, and the ratio of two calcium ions bound per Etotel (9); this value of E,,, is within the range of E, , I = 1.7-4.0 nmol/mg that is observed for these preparations of enzyme. Therefore, 3.0 nmol of 45Ca/ mg is internalized when 2 mol of 45Ca are bound per mol of enzyme, as indicated on the left and right axes of Fig. 2, and 70% of the enzyme undergoes phosphorylation. The filled circles (0) in Fig. 2 show that the dissociation of 45Ca from ' E -*Ca2 in the presence of 300 p~ Sr2+ is biphasic. The solid line is calculated for the rapid dissociation of one 45Ca/E,ml, with k = 50 s-', followed by slow dissociation of the second Ca/E,,l, with k = 4 9-l. The open circles (0) in Fig. 2 show that the dissociation of 45Ca from 'E. 45Caz in the presence of 50 pM Ca2+ is biphasic, which is consistent with previous results (9,21). The dashed line is drawn for dissociation of 1.5 nmol of 45Ca/mg, with k = 50 s-' , followed by dissociation of 1.5 nmol of 45Ca/mg with k = 0.4 s-'. Therefore, the dissociation of 45Ca from eE.45Caz is qualitatively similar in the presence of 300 p M strontium or 50 p M 40ca.
The biphasic dissociation of 45Ca in the presence of 50 phi 40Ca or 300 p~ Sr2+ is consistent with an ordered sequential mechanism for the dissociation of 45Ca (9,19,21). The rapid dissociation of the first 45Ca/Etotal represents the dissociation of 45Ca from the outer binding site, which is not inhibited by the presence of calcium in the medium (9,19,21). The slow dissociation of the second 45Ca/Etotal represents the dissociation of '%a from the inner binding site. The dissociation of calcium from "E. Ca is known to occur with k = 30 s-' in the absence of calcium and strontium (27). The slow dissociation of the inner calcium ion in the presence of 50 p M 40Ca or 300 p~ Sr2+ indicates that Ca2+ and Sr2+ can bind to 'E."Ca to form ' E . 45Ca. 40Ca and 'E. 45Ca. Sr, respectively. The 46Ca ion bound to the inner transport site cannot dissociate when either Ca2+ or Sr2+ is bound to the outer transport site.
The dotted line in Fig. 2 is drawn for the dissociation of 45Ca from CE-45Caz in the presence of 5 mM EGTA, with k = 50 s-'. This first-order reaction corresponds to the dissociation of 45Ca from the outer binding site, which results in the formation of "E .45Ca. The addition of ATP and EGTA to enzyme with one 45Ca ion bound, cE.45Ca, does not result in the formation of phosphoenzyme and, therefore, does not result in the internalization of 45Ca under these conditions (9). Therefore, both 45Ca ions of the species 'E. 45Caz lose their reactivity to ATP after the dissociation of one 45Ca ion, with k = 50 s-', under these conditions. The filled circles (0) in Fig. 2 show that only one 45Ca ion of the species "E. 45Caz loses its reactivity to ATP with a rate constant of k = 50 s-' in the presence of 300 pM Sr". This result indicates that one strontium ion binds to the outer transport site of 'E. 4'Ca to form 'EE. 45Ca. Sr and activates the enzyme for phosphorylation by ATP, as shown in Scheme 2. The dissociation of the inner calcium ion is 87% slower in the presence of 300 p~ Sr2+, with hoba = 4 & 2 s-l, compared with the dissociation of calcium from "E.Ca in the presence of 5 m M EGTA, with k c a = 30 s-l (9). This suggests that in the presence of 300 p~ Sr2+ the ratio of "E. Ca to "E. Ca which indicates that two Ca2+ ions bind to the cytoplasmic transport sites with a high degree of positive cooperativity and that this binding prevents reaction of the enzyme with inorganic phosphate' (17). This inhibition by calcium of phosphoenzyme formation from inorganic phosphate is a measure of the affinity of the cytoplasmic transport sites for calcium. The affinity of the cytoplasmic transport sites for strontium was determined by measuring the inhibition of the formation of phosphoenzyme from inorganic phosphate at equilibrium, as shown in Fig. 3, A and B. The value of = 28 p~ for inhibition by Sr2+ is much larger than the value of Koa = 1.8 pM for inhibition by calcium under similar conditions and confirms that strontium binds to the cytoplasmic transport sites with a much weaker affinity than calcium.' The Hill plot in Fig. 3B shows that the inhibition by strontium is consistent with a Hill slope of n H = 1.0 (dashed line); the data are not consistent with a Hill slope of nH = 2.0 (dotted line). The simplest model consistent with the data shown in Fig. 3, A and B , consists of one strontium ion binding to the cytoplasmic transport sites. The dashed lines in Fig. 3, A

18480
Binding of Strontium to the SR Calcium-ATPase transport sites as shown in Figs. 1 and 2.

4)
Increasing the concentration of S ? ' above the value of = 28 pM causes a second conformational change with K0.5 = 83 p~, which activates the enzyme for phosphorylation by ATP, as shown in Fig. 4; this second conformational change must involve the binding of a second strontium ion, as discussed below. E P The data shown in Fig. 3, A and B are consistent with the binding of two strontium ions to the enzyme with negative cooperativity; the solid lines are drawn for the sequential binding of two strontium ions as shown in Scheme 4, with values of Ksrl = 35 FM and Ksrz = 55 p~. In this scheme, the strontium ions bind to the cytoplasmic transport sites in a sequential mechanism analogous to the mechanism for the binding of two calcium ions (9, 19, 21, 41) or the binding of one calcium ion and one strontium ion to form a mixed complex as shown in Figs. 1 and 2 A Hill slope of n~ -2 is predicted when the quadratic term in Equation 1 is much larger than the linear term, [Sr2+] >> KsrZ. At these high concentrations of strontium, the binding of the second strontium ion is highly favorable, and the ratio of ["E Sr] to ["E. Sr2] will be low. These two forms of enzyme, "E. Sr and "E. Sr2, do not react with inorganic phosphate. If the concentration of "E. Srz is much greater than the concentration of 'E. Sr, then most of the enzyme that is unreactive toward inorganic phosphate must dissociate two strontium ions to become reactive to inorganic phosphate; this is indicated by the Hill slope of nH -2.0. A Hill slope of n H -1 is predicted when the linear term is much larger than the quadratic term, [SrZ+] << Ksrz. At these low concentrations of strontium, the binding of a second strontium ion is highly unfavorable, and the ratio of ["E -Sr] to ["E. Sr2] will be high.
If the concentration of "E. Sr is much greater than the concentration of "E -Sr2, then most of the enzyme that is unreactive toward inorganic phosphate must dissociate only one strontium ion to become reactive to inorganic phosphate; this is indicated by the Hill slope of nH -1.0. The Hill plot shown in Fig. 3B is most accurate near the half-maximal concentration of Koa = 28 pM for strontium. Data outside the range -1  Table I, and the values of Ksr1 = 35 phi and Ksrz = 55 pM. Therefore, the presence of 2.5 mM Pi is predicted to decrease the apparent affinity of the enzyme for strontium by 17%, according to the model shown in Scheme 4.

( E P m a x -E P ) ] < 1-
The inhibition of E P formation from inorganic phosphate by calcium and strontium shows that phosphate cannot react with the enzyme when calcium or strontium is bound to the transport site. However, it is not known whether the binding of these divalent ions to the transport sites inhibits the binding of inorganic phosphate, as shown in Scheme 4, or whether this binding inhibits the formation of covalent phosphoenzyme but does not inhibit the noncovalent binding of phosphate to the enzyme, as shown in Scheme 5.  The Binding of Strontium to the Cytoplasmic Transport Sites at Equilibrium: Formation of ' E . Srz-The affinity of the cytoplasmic transport sites for strontium was also determined by incubating enzyme with different concentrations of strontium and then measuring the concentration of phosphoenzyme formed after the addition of [y3'P]ATP and EGTA, as shown in Fig. 4, A and B. The addition of ATP and EGTA to "E. Srz results in the rapid formation of "E a ATP. Srz, which partitions between the formation of phosphoenzyme with k = 220 s-l and the irreversible dissociation of Sr2+ with k-sr = 115 s" (15). The fraction of 'E. Srz that undergoes phosphorylation in a single turnover after the addition of ATP and EGTA depends on the rate constants for the formation of phosphoenzyme, kp, and for the dissociation of strontium, k-s,, as shown in Equation 5.
Values of k, = 220 s" and k-s, = 115 s" are calculated to result in 65% phosphorylation of %.Sr2. The addition of EGTA prevents the binding of additional strontium to the transport sites; therefore, the amount of phosphoenzyme formed is proportional to the concentration of "E. Srz that is present prior to the addition of ATP and EGTA.
The activation of phosphoenzyme formation from ATP by strontium is consistent with the solid lines in Fig. 4, A and B , which were drawn for a value of Ko.5 = 83 p~. The value of Ko.5 = 83 p~ Sr2+ is much larger than the value of = 2.0 p M Ca2+ for activation of phosphoenzyme formation from ATP, which confirms that strontium binds to the cytoplasmic transport sites with a much weaker affinity than calcium. The concentration of free Sr2+ that is present after the addition of  (0). Therefore, the formation of phosphoenzyme is a result of the binding of strontium to the cytoplasmic transport sites before, and not after, the addition of ATP and EGTA.
The Hill plot in Fig. 4B shows that activation of the enzyme by Sr2+ for reaction with ATP is consistent with a Hill slope of n H = 1.0 (dashed line) but is not consistent with a Hill slope of n H = 2.0, shown by the dotted line. Therefore, the binding of two strontium ions does not occur with the high degree of positive cooperativity that is observed for the binding of two calcium ions, with n H -2 (6, 9, 17-19).
The data shown in Fig. 4, A and B, are consistent with the binding of two strontium ions with negative cooperativity. The solid lines are in good agreement with the data and are drawn for the sequential binding of two strontium ions, as shown in Scheme 6, with values of Ksrl = 35 p~, Ksrz = 55 p~ and other equilibrium constants from Table I; these lines were calculated by using Equation 6, which is derived in the Appendix. Binding of Strontium to the SR Calcium-ATPase

SCHEME 6
The value of = 83 p~ for the formation of "E. Sr2 was measured in the presence of 2.5 mM unlabeled inorganic phosphate, which was added to allow comparison with the experiment shown in Fig. 3, as described below. This value of K O . 5 was calculated by using equilibrium constants from Table  I and Equation 7, which is derived in the Appendix. Prior to the addition of [Y-~'P]ATP and EGTA, the incubation medium was identical to that used for the experiment shown in Fig. 3, except that the inorganic phosphate was unlabeled. The addition of [y-32P]ATP and EGTA measures the concentration of "E. Srz that is present before the addition of ATP and EGTA; therefore, a direct comparison can be made between the results shown in Figs. 3 and 4, as described below (Fig. 5 ) . Equation 8, which is derived in the Appendix, describes the relationship between the Hill slope for the formation of "E Srg and [Sr"] for Scheme 6.

IEn,l
A Hill slope of nH -2 is predicted when the concentration of strontium is much less than the value of KSrl. At these low concentrations of strontium, the binding of the first strontium ion is unfavorable, and the ratio of [E] to ["E -Sr] is high. These two forms of enzyme, E and 'E, Sr, do not react with ATP; only enzyme with two strontium ions bound, 'E. Srz, can react with ATP. If the concentration of E is much larger than the concentration of "E. Sr, then most of the enzyme that is unreactive to ATP must bind two strontium ions to become reactive to ATP, and this is reflected in the Hill slope of nH -2.0. A Hill slope of nH -1 is predicted when the concentration of strontium is much greater than Ksrl at low concentrations of inorganic phosphate, [Pi] 5 Kp. At these high concentrations of strontium, the binding of the first strontium ion is highly favorable, and the ratio of [E] to ["E. Sr] will be very low. If the concentration of "E. Sr is much greater than the concentration of E, then most of the enzyme that is unreactive to ATP must bind only one strontium ion to become reactive to ATP, and this is reflected in the Hill slope of nH -1.0. The Hill plot shown in Fig. 4B Table I  K0.5 = 28 p~ for the inhibition of phosphoenzyme formation from inorganic phosphate. These results confirm that two strontium ions can bind to the cytoplasmic transport sites and that binding of the second strontium ion is required for phosphorylation of the enzyme by ATP under these conditions.
The solid lines are drawn for the sequential binding of two strontium ions as indicated in Scheme 6, with negative cooperativity, n H -1.3, and values of KSrl = 35 pM and Ksr2 = 55 p M (Equations 1 and 6). The value of K0. 5 = 28 p~ Sr2+ for inhibition of the reaction with inorganic phosphate and the value of = 83 p~ Sr2+ for activation of the reaction with ATP indicate that the binding of the first strontium ion occurs with higher affinity than the binding of the second strontium ion. Fig. 5 demonstrates that the unphosphorylated enzyme has three distinct chemical reactivities. The area to the left of both curves represents enzyme with no bound strontium ions, which reacts with inorganic phosphate to form phosphoenzyme. The area between the two curves represents enzyme with one bound strontium ion, which does not react with either inorganic phosphate or ATP. The area to the right of both curves represents enzyme with two bound strontium ions, which reacts with ATP to form phosphoenzyme but does not react with inorganic phosphate. These results demonstrate that the binding of the first strontium ion causes a conformational change, which inhibits phosphorylation of the enzyme by inorganic phosphate. The binding of the second strontium ion causes a different conformational change, which activates the enzyme for phosphorylation by ATP.

Two Different Chemical Specificities for Phosphorylation Have Been Observed with Calcium, but Three Different Chemical Specificities for Phosphorylation Are Observed with Strontium-The
sarcoplasmic reticulum calcium ATPase can undergo phosphorylation by either inorganic phosphate or ATP (42)(43)(44)(45)(46). The chemical specificity of the enzyme is altered by the binding of two calcium ions to the cytoplasmic transport sites with positive cooperativity. Enzyme with two calcium ions bound reacts with ATP, whereas the free enzyme reacts with inorganic phosphate. However, a third form of the enzyme, in addition to these two chemical reactivities, was observed in the presence of strontium. The novel form of the unphosphorylated enzyme has one strontium ion bound to the cytoplasmic transport sites, 'E-Sr, and is unreactive to both ATP and inorganic phosphate, as shown in Scheme 7. The species with one bound calcium ion, 'E .Ca, which was generated transiently, is also known to be unreactive to ATP (9). However, it is not known if "E .Ca is able to react with inorganic phosphate because two calcium ions bind with positive cooperativity to the transport sites, as shown in Scheme 8, so that only a small fraction of the enzyme is present as 'E. Ca  The two forms of chemical specificity that were previously identified in the presence of calcium also exist in the presence of strontium: enzyme with no ions bound to the cytoplasmic transport sites, E, reacts with inorganic phosphate but does not react with ATP. Phosphorylation by 10 mM inorganic phosphate is inhibited by the binding of calcium to the cytoplasmic transport sites with = 1.8 p~ (18); whereas phosphorylation by 2.5 mM inorganic phosphate is inhibited by a much higher concentration of strontium, with = 28 pM (Fig. 3). Phosphorylation of the enzyme by ATP under these conditions requires that two ions are bound to the cytoplasmic transport sites. The species 'EE.Caz and 'E. Srz are both phosphorylated by ATP but do not react with inorganic phosphate. The corrected value of Ko.5 = 2.0 p~ Ca2+ for the formation of %.Cap (9)4 is much smaller than the value of K0.5 = 83 p~ Sr2+ for the formation of 'E, Srz (Fig.   4). Although two strontium ions bind with much weaker affinity than calcium and with little or no cooperativity (nH SCHEME 7

18484
Binding of Strontium to the SR Calcium- ATPase   = 1.0-1.3), the catalytic properties of the enzyme with two bound strontium ions, 'E. Srz, are identical to those of "E. Can: both species are phosphorylated by ATP with k = 220 8" (15, 39).
The three different chemical specificities for reaction of the unphosphorylated enzyme in the presence of strontium (Scheme 7) are not consistent with the El-E2 model. According to the El-E2 model, the El conformation binds calcium on the cytoplasmic side of the membrane and reacts with ATP but not with inorganic phosphate, whereas the E2 conformation binds calcium on the lumenal side of the membrane and reacts with inorganic phosphate but not with ATP. However, there are three conformations of the enzyme in the presence of strontium, as shown in Fig. 5: the area to the left of both curves represents free enzyme, which reacts with inorganic phosphate but not with ATP; the area to the right of both curves represents enzyme with two strontium ions bound, which reacts with ATP but not with inorganic phosphate; the area between the two curves represents enzyme with one strontium ion bound, which cannot react with either ATP or inorganic phosphate. The value of 28 PM Sr2+ for 50% inhibition of phosphorylation by inorganic phosphate is much lower than the value of 83 PM Sr2+ for 50% activation of phosphorylation by ATP (Fig. 5 ) . The E1-E2 model predicts that the half-maximal concentrations of strontium for these two processes should be identical because there are only two conformations, El and E2. The changes in chemical reactivities that are caused by strontium are not consistent with the E1-E2 model and require the presence of a third species of unphosphorylated enzyme, which is not able to be phosphorylated by either ATP or inorganic phosphate. This species is formed when one strontium ion is bound to the cytoplasmic transport sites. Therefore, the binding of strontium to the cytoplasmic transport sites changes the chemical specificities of the enzyme in a stepwise mechanism, which agrees with the proposal that a conformational transition may be associated with nearly every step in the catalytic cycle (48). Several other predictions of the E1-E2 model are not consistent with experimental results (18, 27,48, 49).

Calcium and Strontium Bind to the Cytoplasmic Transport
Sites with Different Degrees of Cooperativity-Two calcium ions bind to the cytoplasmic transport sites with positive cooperativity: Hill slopes of n H = 1.6-2.0 have been measured for the binding of calcium to the cytoplasmic transport sites at equilibrium using a variety of methods including direct binding assays, spectroscopy, and changes in chemical specificities (6, 7, 9, 19). The Hill slopes measured for the binding of strontium to the cytoplasmic transport sites at equilibrium are lower than those for the binding of calcium. Values of n H = 1.0-1.3 for the change in chemical specificities caused by strontium (Figs. 3B and 4 B  Although negative cooperativity is observed with strontium, the properties of the outer cytoplasmic transport sites appear to be identical for both 'E. Sr and "E .Ca: the association constants calculated from Table I for the binding of Sr2+ to "E. Sr and 'E-Ca are not significantly different, 1.8 X lo4 M-' and 2.2 X lo4 M-', respectively. The conformational change caused by the binding of one calcium ion is known to be critical for the positive cooperativity that is observed for the binding of two calcium ions to the cytoplasmic transport sites (6, 7, 19, 27, 50, 51). Therefore, the E to "E conformational change, which can be induced by the binding of either one calcium ion or one strontium ion, specifically increases the affinity of the enzyme for calcium but has little effect on the affinity of the enzyme for strontium.
The negative cooperativity that is observed with strontium cannot easily be explained by electrostatic repulsion. The unfavorable electrostatic interaction between two ions bound to the cytoplasmic transport sites is expected to be similar with strontium and with calcium: they are both divalent cations, and the distance between the two binding sites has been estimated to be -10 A, which is much larger than the radius of either ion (52-56). Therefore, any electrostatic repulsion that is produced upon the binding of two strontium ions to the cytoplasmic transport sites should also be observed in the binding of two calcium ions. The negative cooperativity that is observed with strontium is more likely to arise from either the steric requirements of the outer transport sites or the ability of calcium, which has a higher charge density, to interact more strongly with ligands at this site.

Strontium and Calcium Ions Bind Sequentially to the Cytophmic Transport Sites and Form Two Different Mixed
Complexes, 'E. Sr -Ca and 'E. Ca. Sr-The mixed species, 'E.
Sr-Ca, has strontium bound to the inner site and calcium bound to the outer site and is formed by the addition of Can+ to "E Sr2. The strontium ion bound to the outer site is rapidly replaced by external Can+, whereas the strontium ion bound to the inner site dissociates much more slowly in the presence of 190 p~ Ca2+. Strontium bound to the inner site cannot dissociate if calcium is bound to the outer site. The other mixed species, 'E Ca. Sr, represents enzyme with calcium bound to the inner site and strontium bound to the outer site and was formed by the addition of Sr" to "E. Can. The calcium ion bound to the outer site is rapidly replaced by strontium, whereas the calcium ion bound to the inner site dissociates much more slowly in the presence of 300 PM S3'. Calcium bound to the inner site cannot dissociate if strontium is bound to the outer site. Therefore, strontium can bind to both the inner and outer transport sites. The sequential binding of one strontium ion and one calcium ion to the cytoplasmic transport sites results in the formation of two different mixed species, 'E. Sr a Ca and "E. Ca-Sr; these forms of enzyme react rapidly with ATP.
The trivalent cations of the lanthanide series have also been used to probe the cytoplasmic transport sites of the calcium ATPase. Recently, Inesi and co-workers observed biphasic dissociation of calcium from "E. Can in the presence of Pr3+, with rapid dissociation of 50% of the bound calcium, followed by slow dissociation of the remaining calcium; they suggested that the lanthanide ion was binding to the outer transport site of 'E. Ca to form the mixed complex, "E-Ca-Pr (57). However, Shigekawa and co-workers (58) observed only a small burst, -10-20%, of calcium dissociation in the presence of Gd3+, with the majority of the calcium dissociating with monophasic kinetics; they concluded that lanthanides do not inhibit the dissociation of calcium by binding to the outer transport site of 'E. Ca under these conditions (58).
Instead, they concluded that lanthanide ions inhibit the dissociation of both the inner and the outer calcium ions of ' E -Can by binding to a site that is distinct from the cytoplasmic transport sites. The biphasic dissociation of calcium observed by Inesi and co-workers may arise from the presence of contaminating calcium in the micromolar range, which would inhibit the dissociation of only the inner calcium ion (9,(19)(20)(21)(22)(23). Therefore, the mixed species of enzyme, 'E I Ca. Pr, may not involve the binding of Pr3+ to a transport site.
It has also been reported recently that the binding of lanthanum activates the enzyme for phosphorylation by ATP (57). Inesi and co-workers observed that incubation of the enzyme with 40 pM LaC13, 20 p M CaC12, 10 mM MgC12,80 mM KC1, and 50 p~ [T-~~PIATP at pH 6.8 for several seconds results in phosphorylation of the enzyme. They observed that this concentration of lanthanum inhibits the binding of *'Ca to the enzyme at equilibrium under these conditions. These data suggested that the binding of lanthanum to the cytoplasmic transport sites activates the enzyme for phosphorylation by ATP. However, in the presence of ATP, the binding of calcium to the transport sites is not at equilibrium because ATP increases the concentration of bound calcium by phosphorylating the enzyme. In fact, LaATP is a particularly good cofactor for trapping calcium at the transport sites because the binding of lanthanum to the catalytic site for phosphoryl transfer greatly decreases the rate of calcium dissociation from both the cytoplasmic transport sites of the unphosphorylated enzyme and the lumenal transport sites of the phosphorylated enzyme (29). The effectiveness of lanthanum as a competitor of calcium for binding to the cytoplasmic transport sites is different in the presence and absence of ATP. The results observed by Inesi and co-workers are consistent with the conclusion that the binding of lanthanide ions to the cytoplasmic transport sites inhibits the formation of phosphoenzyme (28, 58). Therefore, although lanthanide ions can be used to probe the specificity of the cytoplasmic transport sites for binding, the inability of lanthanide ions to change the chemical specificity of the enzyme for phosphorylation by ATP (28,58) limits their utility as probes of the cytoplasmic transport sites for activation of catalysis.
In contrast, strontium may be used to determine the specificity of the cytoplasmic transport sites both for binding and for activation of catalysis. Strontium binds to both the inner and outer cytoplasmic transport sites, and the binding of strontium and calcium to these sites is sequential. The binding of two strontium ions or two calcium ions causes the same changes in chemical reactivity. Strontium does not bind with positive cooperativity and was used to determine the chemical specificity of the enzyme with one ion bound to the cytoplasmic transport sites. The enzyme species with one bound strontium ion can be formed at equilibrium and has a novel chemical specificity: ' E a Sr does not react with either ATP or inorganic phosphate.

APPENDIX: DERIVATION OF EQUATIONS
The Dependence of %. Ca, Concentration on [Ca2+]-Two calcium ions bind to the cytoplasmic transport sites in a sequential mechanism as shown in Scheme A (9,19,21). Two calcium ions must be bound to the transport sites to activate the enzyme for phosphorylation by ATP in the presence of 100 mM KC1 and 40 mM MOPS, pH 7.0, at 25 "C (9

64-51
In the case of infinite positive cooperativity (Kl >> Kz) Equation A.5 simplifies to the following.

(-4.6)
In the case of infinite negative cooperativity (K2 >> KJ >> K l , the Hill slope is nH -1.0. This variation in the value of the Hill slope results from the change in the ratio of [E] and [E .Call. These two forms of enzyme, E and E .Gal, do not react with ATP. If the concentration of E is much greater than the concentration of E. Cal, then most of the enzyme that is unreactive toward ATP must bind two calcium ions to become reactive to ATP. The Hill slope of nH -2.0 is an indication that two calcium ions are binding. If the concentration of E. Cal is much greater than the concentration of E, then most of the enzyme that is unreactive toward ATP must bind one calcium ion to become reactive to ATP. The Hill slope of nH -1.0 is then an indication that only one calcium ion must bind to the predominant species of enzyme that is unreactive to ATP. A value of nH = 1.6 for the formation of ' E . Caa at the halfmaximal concentration of Kos = 2.0 p~ was calculated from the corrected value of Kl = 3 p~. This value is close to the value of nH = 1.9 that was reported by Petithory and Jencks (9).
Inhibition of EP Formation from Inorganic Phosphate by Strontium-Scheme 4 under "Results" shows a model in which the binding of strontium is competitive with the binding of inorganic phosphate. The strontium binds in an ordered sequential mechanism.

the SR Calcium-ATPase
The fraction of enzyme that is phosphorylated by inorganic phosphate is defined as 8.
Pma. is defined as EP,,,/EtOal or @ in the absence of strontium and calcium.
Divide numerator and denominator by E P and substitute from A.15.

(A.21)
The value of Y is defined as the amount of phosphoenzyme formed divided by the maximal amount of phosphoenzyme, E P I E P m a x or PILLl.