Lanthanum Inhibits Steady-state Turnover of the Sarcoplasmic Reticulum Calcium ATPase by Replacing Magnesium as the Catalytic Ion*

effective of the of reticulum to in the formation

is reversed by calcium but inhibition of steady-state turnover is not. Therefore, binding of La3+ to the cytoplasmic calcium transport site is not responsible for the inhibition of steady-state ATPase activity. The addition of 6.7 KM LaCls (1.1 pM free La3+) has no effect on the rate of dephosphorylation of phosphoenzyme formed from MgATP and enzyme in leaky vesicles, while 6.7 mM CaC12 slows the rate of phosphoenzyme hydrolysis as expected; 6.7 PM LaC13 and 6.7 mM CaC12 cause 95 and 98% inhibition of steady-state ATPase activity, respectively. This shows that inhibition of ATPase activity in the steady state is not caused by binding of La3+ to the intravesicular calcium transport site of the phosphoenzyme. Inhibition of ATPase activity by 2 pM LaC13 (0.16 pM free La3+, 0.31 pM LaATP) requires >5 s, which corresponds to -50 turnovers, to reach a steady-state level of ~80% inhibition. Inhibition by La3+ is fully reversed by the addition of 0.55 mM CaC12 and 0.50 mM EGTA; this reactivation is slow with tH -9 s. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. goes hydrolysis much more slowly than does magnesium phosphoenzyme.
The slow rate of reactivation by Ca2+ and EGTA corresponds to the slow decay of lanthanum phosphoenzyme.
Co'+ inhibits steady-state turnover by binding to the ionophore A23187, which blocks calcium efflux from the vesicles.
Lanthanum is the first element of the lanthanide series; the elements of this series show only small changes in chemical properties throughout the series. Ions in the lanthanide series have been used extensively as probes of calcium-binding sites in proteins because of the similar ionic radii and ligand specificities of the lanthanides and calcium (for reviews see Martin and Richardson, 1979;Evans, 1983;Horrocks and Albin, 1984). However, the binding of lanthanides is not restricted to binding sites for calcium; replacement of magnesium and other cations by lanthanides has been reported for several proteins (Martin and Richardson, 1979;Morrison and Cleland, 1983;Giradet et al., 1989). Lanthanides have been reported to bind to the SRV' calcium ATPase at the cytoplasmic calcium transport site and at a site that has an association constant of 35-130 M-l for magnesium (Chevallier and Butow, 1971;Highsmith and Head, 1983;Highsmith, 1984;Giradet et al., 1989). Lanthanides are known to inhibit turnover of the SRV calcium ATPase; this inhibition is generally believed to occur by binding of lanthanides to the cytoplasmic site for calcium transport (Krasnow, 1977;Stephans and Grisham, 1979;Highsmith and Head, 1983;Highsmith and Murphy, 1984;Scott 1984).
There are at least four different mechanisms by which divalent and trivalent cations inhibit the calcium ATPase: (a) metal ions (Mgz', Mn*+) can compete with calcium for the cytoplasmic transport site (Yamada and Tonomura, 1972;Kalbitzer et al., 1978), (b) metal ions (Ca"') can replace M$+ as the catalytic ion at the phosphoryl transfer site (Yamada and Tonomura, 1972;Shigekawa et al., 1983;Orlowski et al., 1988), (c) metal ions (Ca2+, MC) can inhibit steady-state turnover by binding to the lumenal transport site of the phosphorylated enzyme (Weber, 1966;Ikemoto, 1975;Bishop and Al-Shawi, 1988), and (d) we have found that metal ions (Co") can bind to ionophore and inhibit the efflux of Ca2+ from intact vesicles that is required in order to maintain steady-state turnover.
The interactions between lanthanide ions and binding sites on the calcium ATPase are important to understand because lanthanide ions have frequently been used as spectroscopic probes for the cytoplasmic calcium-binding site (Stephens and Grisham, 1979;Highsmith and Head, 1983;Scott, 1984;Highsmith and Murphy, 1984;Scott, 1985;Klemens et al., 1986;Gangola and Shamoo, 1987 ml of acetone and, after 10 min, 1.5 ml of 5% ammonium molybdate in 2.5 N HzSO, was added. This mixture was vortexed for 30 s, 3.0 ml of a 1:l isobutyl alcohol/benzene solution that had been saturated with water was added, and the new mixture was vortexed for 60 s. The organic phase was separated from the aqueous phase by centrifugation at 1500 x g for 5 min and 3 ml of the organic phase was added to 7 ml of Aquasol-(Du Pont-New England Nuclear) for scintillation counting. Phosphoenzyme levels were measured essentially as described by Verjovski-Almeida et al. (1978) with slight modifications (Hanel and Jencks, 1990 were followed with a rapid mix-quench process using an apparatus that can be used with either three or four syringes of lml volume, as described previously (Stahl and Jencks, 1984;Petithory and Jencks, 1988). Reaction times greater than 0.3 s were followed by a pulsed quench configuration, as described previously (Hanel and Jencks, 1990). The contents of two or three syringes were mixed and filled an aging tube. After a delay time, the solution in the aging tube was displaced and mixed with an equal volume of quench solution. Concentrations of free metal ions, LaATP and MgATP were calculated using the computer program of Fabiato and Fabiato (1979). The dissociation constants were obtained from Morrison and Cleland (1983) for LaATP; Perrin (1979) for MgSO,, CaS04 and LaSOI; and Fabiato and Fabiato (1979) for MgATP, CaATP, CaEGTA and MgEGTA.

RESULTS
Steady-state turnover of the sarcoplasmic reticulum Ca*'ATPase was found to be inhibited by low concentrations of lanthanide ions, in agreement with previous work (Yamada and Tonomura, 1972;DOS Remedios, 1977;Gangola and Shamoo, 1987;Highsmith and Head, 1983;Scott, 1984). Fig. 1 shows that the K, for inhibition of steady-state turnover by La3+ is linearly dependent on the concentration of SRV, which indicates that La3+ binds to the SRV nonspecifically.
The slope of this plot is 10 nmol of La3+/mg of SRV, which represents the stoichiometry for this binding of La3+ to SRV, and the y intercept is 0.16 FM La3+ (15 nM free La3+, 21 nM LaATP), which represents Ki for La3+ under these conditions extrapolated to zero SRV concentration. Similar stoichiometries for the binding of Tb3+ and Nd3' to SRV have been reported previously (Highsmith and Head, 1983;Highsmith and Murphy, 1984). These binding sites are saturated with La3+ at concentrations that cause only partial inhibition of steady-state turnover; therefore, the binding of La3+ to these sites does not cause inhibition. Fig. 2 shows that inhibition of steady-state ATP hydrolysis by 8 pM TbCl, decreases with time; the rate changes from 94 to 20% inhibition with an approximate tljs of 12 min. This may be caused by a decrease in the concentration of Tb3' as a result of complexation of Tb3+ by inorganic phosphate, a product of the reaction. Increases in the rate of ATP hydrolysis with time were also observed with reactions inhibited by La3+, Nd3+, Sm3+, or Y3+ (data not shown). The concentration of Pi produced, 0.2 mM, is sufficient to explain the increase in the rate of ATP hydrolysis in the presence of 8 pM TbC13 from 0.14 to 2.0 pmol/min/mg.
The solubility of lanthanidephosphate complexes is very low (&, = lo-'* M* for La.PO, (Smith and Martell, 1976)), and in the experiment shown in Fig. 2, the reaction mixture is supersaturated in Cl s. The rate and extent of the decrease in inhibition was found to vary with the concentrations of lanthanide and SRV and this rate is faster for reactions with Tb3+ than with La3+ (data not shown). The addition of 1 mM Pi to reactions inhibited by 4 FM LaC13 was found to increase the initial rate of ATP hydrolysis from 8 to 84% of the control reaction. These findings suggest that the gradual increase in ATPase activity observed with certain reactions inhibited by lanthanides is caused by the precipitation of lanthanide phosphate, which is supersaturated very early in the reaction. The low concentra-tions of lanthanide ion, 0.2-100 FM, and phosphate, ~0.3 mM, result in slow precipitation over 25 min; therefore, linear rates of steady-state ATPase activity in the presence of lanthanides were measured at 20-60 s, before a significant amount of lanthanide had precipitated. Krasnow (1972) has reported previously that inhibition by La3+ and Gd3' of ATP hydrolysis and Ca*' uptake with intact SRV decreases with time. This result was interpreted as an indication that La3+ and Gd3+ might be transported into the vesicles, in a manner analogous to that for Ca*+. The finding that leaky (Fig. 2) and intact SRV both show an increase in ATP hydrolysis with time in the presence of lanthanide ions indicates that active transport of these ions is not likely to account for this time-dependent decrease in inhibition. Fig. 3 shows that K, for inhibition of steady-state ATP hydrolysis by CaCl* is 0.5 mM in the absence of LaC& and 0.7 mM in the presence of 0.5 pM LaCl.?. This concentration of LaC13 causes 58 and 35% inhibition in the presence of 0.025 mM and 1 mM CaC12, respectively. Increases in percent activity with increasing concentrations of CaZc have been reported previously and cited as evidence that lanthanide ions inhibit steady-state turnover by binding to the cytoplasmic transport site (Highsmith and Head, 1983;Highsmith and Murphy, 1984;Scott, 1984). However, the data in Fig. 3 do not show an increase in the rate of La3'-inhibited reactions with increasing concentrations of CaC&. The data show that Ca'+ is a slightly more effective inhibitor in the absence than in the presence of La3+. This result does not require that La3+ cause inhibition by competing with Ca" for binding to the cytoplasmic transport site. The stimulatory and inhibitory effects of Ca2+ on different steps of the ATPase cycle make the steady-state ATPase activity shown in Fig. 3 difficult to interpret.
In order to clarify the situation, we measured the competition between La"+ and Ca2+ for binding at the cytoplasmic calcium transport site directly in a single turnover by determining the inhibition by La3+ of ATP-dependent Ca2+ uptake and phosphoenzyme formation. Fig. 4 (Fig. 4). This is consistent with the much faster rate of Ca2+ binding compared with the rate of phosphoenzyme decomposition, as indicated by the high levels of phosphoenzyme observed at low concentrations of La3+. Therefore, the binding of Ca2+ must be inhibited by >50% to be slower than the decomposition of phosphoenzyme. Domonkos et al. (1985) also reported that La3+ added after phosphoenzyme formation from ATP results in inhibition of phosphoenzyme decomposition. The proposed mechanism for this inhibition was the binding of La3+ to a low affinity site, which is competitive with respect to Mg*+.
All reaction mixtures contained 5 mM Fig. 5 shows that La3+ does not inhibit steady-state turnover by binding to the intravesicular calcium transport site of the phosphoenzyme.
It is well known that dephosphorylation of the phosphoenzyme is inhibited by binding of Ca2+ to a low affinity site that is exposed to the lumen of the vesicles (Weber et al., 1966;Ikemoto 1975). The possibility that La3' inhibits dephosphorylation by binding to this intravesicular transport site was examined by measuring the overall rate of phosphoenzyme disappearance after the addition of a cold ATP chase in the absence and presence of La3+. Fig. 5 shows that the rate of phosphoenzyme decay is decreased -80% in the presence of 6.7 mM Ca*+ (triangle), compared with 60 PM Ca*+ (open circle). However, the addition of 6.7 PM LaC13 (square), which causes 95% inhibition of steady-state turnover, has no effect on the rate of phosphoenzyme decay. on the x axis were corrected for 10 nmol of La"+ binding/mg of SRV. The lines were calculated for n = 1.0 on a Hill plot and I&, = 0.16 pM La"' (0) or 100 GM La"+ (0, A). before the addition of ATP for the Ya uptake and phosphorylation measurements and the reaction was quenched with either EGTA or acid after -20 ms.
The competitive binding of La3+ and Ca2+ to the cytoplasmic transport site was found to reach equilibrium in <60 s. SRV were first incubated with either LaC13 or 45Ca for 60 s, the other cation was added, and the SRV were then incubated for 60 s before the addition of ATP; identical levels of inhibition of 4"Ca uptake were obtained regardless of the order of cation addition. This inhibition is competitive with respect to Ca'+; . increasing the 4"Ca concentration from 25 to 60 pM reduces the inhibition by 150 PM LaC13 from 71 to 45% (open and closed squares, Fig. 4). The inhibition by La"+ of 4"Ca uptake and phosphoenzyme formation are both consistent with the same value of K, = 100 PM for inhibition by a single La"+ ion, as shown by the line on the right that was calculated for a Hill slope of n = 1.0. These results show that the binding of La"+ to the cytoplasmic transport site occurs with a low affinity in the presence of 25 ELM Ca'+, compared with K, = 0.16 pM for inhibition of ATPase activity by LaC13, and that this binding is not responsible for the observed inhibition of steady-state turnover.
It is not known if one or two Ca2+ ions competes with this single La"' .
ion for binding in the cytoplasmic transport site. However, if two Ca*' ions were involved, then the apparent dissociation constant for the binding of La3+ to the cytoplasmic transport site would be proportional to [Ca'+]* and the cytoplasmic transport site would have a relatively high apparent affinity for La3+ at low concentrations of Cap+. Domonkos et al. (1985) reported that a concentration of >l mM LaC13 is required to cause a 50% decrease in the steadystate level of phosphoenzyme in the presence of 20 pM added CaCl? at 0 "C. The low levels of phosphoenzyme that were observed with 10 mM LaC13 may be caused by binding of La3+ to the cytoplasmic transport site, which prevents phosphorylation by ATP. The KoS of -5 mM for the decrease of the The dashed line in Fig. 5 represents the phosphorylation of enzyme by 10 PM [T-~'P]ATP, simulated according to the scheme outlined in Stahl and Jencks (1987), and the solid circle shows the amount of phosphoenzyme that was observed after phosphorylation for 55 ms. The solid lines represent the dephosphorylation of phosphoenzyme that was initiated by the addition of 67 FM unlabeled ATP. The lower line was calculated using the scheme from Stahl and Jencks (1987) and is in good agreement with the observed phosphoenzyme level after dephosphorylation for 120 ms in the presence of  (Khananshvili et al., 1990). The upper line was calculated for inhibition by one Ca*+ ion binding to an intravesicular transport site with Klh = 1 mM and is in good agreement with the data obtained in the presence of 6.7 mM Ca2+ (open triangle). These results confirm that Ca2+ can bind to the intravesicular transport site and inhibit the decay of phosphoenzyme. Steady-state turnover is inhibited 98% by 6.7 mM Ca*' under conditions similar to those described in Fig. 1 (Bodley and Jencks, 1987). The difference between the 80% inhibition of dephosphorylation and the 98% inhibition of steady-state turnover by 6.7 mM Ca*' can be accounted for by phosphorylation of the enzyme by CaATP, which is a second mechanism for inhibition by calcium (Yamada and Tonomura, 1972;Orlowski et al., 1988). Another possible mechanism of inhibition is binding of La3+ to A23187, which catalyzes the calcium efflux from the vesicles that is required for steady-state turnover with intact vesicles; A23187 is an unselective transporter of multivalent cations (Kolber and Haynes, 1981;Shastri et al., 1987;Cader and Horrocks, 1989). La3+ and Co'+ might be expected to cause inhibition by this mechanism because, respectively, they bind 230 and 370 times tighter than Ca2+ to A23187, with dissociation constants in the micromolar range in 80% methanol/water (Chapman et al., 1990). An alternative method of making SRV leaky is incubation with EGTA at pH 9, which has been shown to make the SRV permeable to Ca", inulin (Mr = 5,000), and dextran (Mr = 15,000-90,000) (Duggan and Martonosi, 1970). We have found that incubation of SRV with either Chelating Sepharose or Chelex 100 resin at pH 9 also makes the SRV leaky to Ca2+. Since alkaline treatment makes the SRV permeable to both cations and large uncharged molecules, it is unlikely that a cation would block this nonspecific permeability. Table I shows that the K, for inhibition by LaC13 of steadystate ATPase activity is comparable for vesicles that were made leaky by treatment with alkali or by the addition of the ionophore A23187; therefore, this inhibition by La3+ does not result from binding of La3+ to the ionophore. However, K, for inhibition by Co'+ is over 1 order of magnitude smaller for ionophore-treated vesicles compared with vesicles that were made leaky by treatment with alkali. Therefore, Co'+ inhibits steady-state ATPase activity of ionophore-treated vesicles by binding to the ionophore A23187, which slows the efflux of Ca*+ out of the vesicles and causes millimolar concentrations of calcium to accumulate in the intravesicular space. It was found that, in the presence of 50 FM Co'+, SRV that were made leaky by treatment with alkali hydrolyzed ATP at the control rate, while SRV that had only been treated with 2 and 4 pM A23187 hydrolyzed ATP at 15 and 17% of the control rate. This result demonstrates that Co'+ inhibits the Ca*+ efflux catalyzed by the ionophore A23187; however, an increase in the concentration of ionophore from 2 to 4 PM did not reverse this inhibition. Fig. 6 shows that inhibition of ATP hydrolysis by La3+ develops slowly. The steady-state level of ~80% inhibition is reached after >5 s, which corresponds to -50 turnovers. The enzyme was incubated with La3+ and Ca2+ for 30 s before the addition of ATP. The concentration of inorganic phosphate was monitored because the coupled assay system as described above is calculated to have a lag of several seconds between an increase in the rate of ADP production and the attainment of a steady-state rate of NAD production (McClure, 1979). Fig. 7 shows that enzyme which is inhibited by La3+ is reactivated in a time-dependent process by the addition of CaClz and EGTA. The addition of 0.55 mM CaCl* and 0.50 mM EGTA to vesicles that are hydrolyzing ATP in the presence of 2 PM LaC13 causes the ATPase rate to increase over several seconds from 0.32 to 1.9 FM min-' mg-'. The latter rate corresponds to 90% of the ATPase rate in the absence of La3+. Fig. 7B shows the increase in velocity with time for this reactivation process. The solid line in Fig. 7B shows that the kinetics for reactivation can be approximated by a first-order rate constant of 0.08 s-'. The dashed line will be described later.
The lower truces in Fig. 8  and LaATP. There is a rapid disappearance of one species of phosphoenzyme in a burst with a rate constant of 24 s-', which is too fast to measure on this time scale, followed by a much slower decay of phosphoenzyme with a rate constant of 0.05 s-l. The slow phase represents the decay of phosphoenzyme with La3+ at the catalytic site and two calcium ions bound to the transport sites. Phosphoenzyme that is formed by the reaction of 'E. Caz with LaATP in the absence of MgATP decays with a rate constant of 0.05 s-' (upper truce); a similar rate constant of 0.06 s-' for the decay of lanthanum phosphoenzyme has been observed by Hanel and Jencks (1990). The burst phase of the lower traces represents the disappearance of phosphoenzyme with Mg2+ at the catalytic site and two calcium ions bound to the transport site, which is known to decay with a rate constant of 14-17 s-l under similar conditions (Chiesi and Inesi, 1979;Pickart and Jencks, 1984). The lower truces represent enzyme that was phosphorylated in the presence of 5 mM MgS04 and 50, 25, or 10 pM LaCl,; these reaction mixtures were calculated to contain 37, 43, or 46 pM MgATP and 12, 6.5, or 2.8 pM LaATP, respectively.
Increases cause an increase in the fraction of enzyme that decays slowly. These data indicate that both magnesium phosphoenzyme and lanthanum phosphoenzyme are formed in the presence of LaC& concentrations that inhibit steady-state turnover, but do not significantly inhibit the binding of calcium to the cytoplasmic transport site.
Phosphoenzyme that was formed from the reaction of the enzyme with CaATP was found to decay with a rate constant of ~0.2 s-i under conditions similar to those described in Fig.  8 (data not shown). Therefore, the phosphoenzyme that undergoes hydrolysis with a rate constant of 0.05 s-' (Fig. 8) does not have calcium bound at the phosphoryl transfer site. The rate constant for the decay of lanthanum phosphoenzyme was found to increase from 0.05 to 0.08 s-' when phosphoenolpyruvate and pyruvate kinase were not included in the reaction (data not shown). This increase in rate can be accounted for by the reaction of lanthanum phosphoenzyme with -0.4 PM ADP, which is released upon formation of the phosphoenzyme.
The dephosphorylation reaction shown in Fig. 8 was initiated by the addition of 5 mM EGTA, which chelates free La"+ in the solution. Therefore, the slow rate constant for dephosphorylation that is observed in the presence of LaC& results from the binding of LaATP to the phosphoryl transfer site before phosphoenzyme formation, which is essentially irreversible under these conditions (Hanel and Jencks, 1990); it is not caused by binding of La3+ to the intravesicular transport site after phosphoenzyme formation. The time dependence for reactivation of lanthanum-inhib-Inhibition by Lanthanum of the Calcium ATPase ited enzyme in the presence of 0.55 mM calcium and 0.50 mM EGTA (Fig. 7B) can be accounted for by the slow decay of lanthanum phosphoenzyme. The solid line in Fig. 7B corresponds to reactivation with a rate constant of 0.08 s-l. The rate of reactivation is slightly faster than the hydrolysis rate of lanthanum phosphoenzyme because of the reaction of lanthanum phosphoenzyme with ADP, as described above. The concentration of ADP in the steady state was calculated to increase from 0.2 to 2 PM as the rate of ATP hydrolysis increases, by taking into account the activity of pyruvate kinase. The dashed line in Fig. 7B represents a computer simulation that takes into account the rate constants for the decay of lanthanum phosphoenzyme by hydrolysis and by reaction with ADP, and the activities of pyruvate kinase and lactate dehydrogenase that determine the concentrations of ADP and pyruvate during the course of reactivation (McClure, 1979).

DISCUSSION
If La3+ inhibits steady-state turnover of the Ca*+ATPase by binding to the cytoplasmic transport site and blocking phosphorylation, as has been generally believed, then (a) low levels of phosphoenzyme would be observed with inhibition by La3+, (b) increasing the concentration of Ca*+ would increase the activity of enzyme inhibited by La3+, and (c) the values of K, for inhibition by La3+ of steady-state turnover and of 45Ca uptake would be comparable.
However, none of these predictions are consistent with the data reported here. (a) Fig. 8 shows that high levels of phosphoenzyme are observed with enzyme that is inhibited by La3+, which agrees with results reported previously (Yamada and Tonomura, 1972). (5) Fig. 3 shows that increasing the concentration of Ca*+ does not increase the turnover rate of enzyme inhibited by La3+. (c) Fig. 4 shows that the value of Ki for inhibition of ATPase activity in the steady state by La3+ is 500-fold lower than Ki for inhibition of 45Ca uptake. We conclude that inhibition of steady-state turnover by La3+ is not caused by binding of La3+ to the cytoplasmic transport site.
The results reported here provide evidence that La3+ inhibits steady-state turnover by replacing Mg2f as the catalytic ion for phosphoryl transfer. The upper line in Fig. 8 shows that 'E .Caz is phosphorylated by LaATP to give lanthanum phosphoenzyme, which decays with a rate constant of 0.05 S -I; this is much slower than the rate constant of -15 s-' for the decay of magnesium phosphoenzyme (Chiesi and Inesi, 1979;Pickart and Jencks, 1984). The biphasic decay of phosphoenzyme shown in the lower traces of Fig. 8 demonstrates that both lanthanum phosphoenzyme and magnesium phosphoenzyme are present with lanthanum concentrations that inhibit steady-state turnover, but do not significantly inhibit phosphorylation or Ca*+ uptake in a single turnover experiment (Fig. 4). Fig. 7B shows that the rate constant for the reversal of La3+ inhibition by the addition of Ca2+ and EGTA is -0.08 s-'. This rate constant can be accounted for by the decay of lanthanum phosphoenzyme through hydrolysis at 0.05 s-l and reaction with ADP to regenerate ATP; the concentration of ADP was calculated to be 0.2-2.0 pM in the steady state.
The slow development of inhibition by La3+ shown in Fig.  6 results from the gradual replacement of M$+ by La3+ at the catalytic site over several seconds, which corresponds to -50 turnovers. Because the rate constant of 0.05 s-l for the decay of lanthanum phosphoenzyme is much smaller than the rate constant of -15 s-' for the magnesium phosphoenzyme (Hanel and Jencks, 1990;Chiesi and Inesi, 1979;Pickart and Jencks, 1984), LaATP is a very effective inhibitor of the calcium ATPase. Fig. 6 shows that 0.31 PM LaATP causes 280% inhibition of steady-state turnover in the presence of 48 PM MgATP. LaATP and MgATP bind to the enzyme with similar second-order rate constants (Hanel and Jencks, 1990); however, the enzyme binds MgATP >lOO times more often than LaATP under these conditions because MgATP is in large excess compared with LaATP.
At early time points, most of the enzyme binds MgATP, which undergoes rapid turnover. The enzyme binds LaATP infrequently, but when LaATP does bind it forms lanthanum phosphoenzyme, which is very stable and accumulates with time. It is difficult to correlate the effect of La3+ on steady-state turnover and on partial reactions that are measured in a single turnover because of this slow development of inhibition, This may account for some of the inconsistencies in the literature concerning the mechanism of inhibition by La3+. The interactions between the sarcoplasmic reticulum calcium ATPase and multivalent metal ions are complex and confusing because there are several different classes of binding sites for cations on the enzyme. Mg2+ and Ca'+ are required for full activity, yet both inhibit at high concentrations. M$ can inhibit by binding to the cytoplasmic calcium transport site (Yamada and Tonomura, 1972) and by binding to the lumenal calcium transport site at pH 8 (Bishop and Al-Shawi, 1988). Ca*+ can inhibit by binding to the lumenal transport site (Weber, 1966) or by replacing Mg2' as the catalytic ion for phosphoryl transfer (Shigekawa et al., 1983). These inhibitory effects make it difficult to interpret experiments in which the effects of increases in the concentrations of Ca2+ or Mg2+ on inhibition of ATPase activity by lanthanide ions are measured.
Lanthanide ions have been reported previously to bind to more than one site on the sarcoplasmic reticulum calcium ATPase. Itoh and Kawakita (1984) have reported the existence of several different classes of lanthanide-binding sites. Gadolinium ion causes inhibition of steady-state ATPase activity and steady-state phosphoenzyme formation at concentrations of <lO-'j M, but this probably does not occur at the cytoplasmic transport site because 50% inhibition of the binding of 10 pM Ca2+ requires -75 mM Gd3+. Terbium ion binds to a site on the enzyme with Ki -10m4 M that is not competitive with Ca*+, but is competitive with Mn'+. Terbium fluorescence is enhanced as a result of energy transfer from aromatic amino acid residues to Tb3+ bound to this site. It was concluded that gadolinium ion does not inhibit ATPase activity in the steady state by binding to the cytoplasmic transport site; however, the mechanism of inhibition by this lanthanide ion was not identified. Girardet et al. (1989) have also reported several cases of binding sites for terbium on the sarcoplasmic reticulum calcium ATPase: a magnesium binding site binds terbium with Kd = 10 PM, the Ca*+-binding site binds terbium with Kd C 0.1 pM in the absence of Ca'+, and the nucleotide-binding site binds terbium formycin triphosphate with Kd = 0.7 pM. The ability of lanthanide ions to bind to several different sites on the enzyme is not surprising because binding sites for both M$+ and Ca*+ on several proteins have been shown to bind lanthanide ions (Martin and Richardson, 1979). Multivalent cations also can inhibit ATP hydrolysis by interacting with components of the reaction mixture other than the calcium ATPase. The ionophore A23187 is often used to collapse the Ca2+ gradient that would form with intact sarcoplasmic reticulum vesicles that are hydrolyzing ATP. Co'+ can inhibit ATP hydrolysis by binding to the ionophore A23187 and decreasing the rate of Ca2+ efflux out of the vesicles, so that the interior Ca*+ concentration increases and inhibits ATPase activity. The Ki for the inhibition of ATPase activity by cobalt is 3 pM for vesicles treated with ionophore, while it is 300 pM for vesicles that were made leaky by treatment with chelating agents (Table I). Highsmith and Murphy (1984) reported that ATPase activity is inhibited by cobalt with K; = 3 pM in the presence of 10 pM Ca", 5 mM Mg2+, 2 mM ATP, and 0.96 pM A23187. This inhibition was attributed to binding of Co*+ to the calcium transport site with Kapp = 2 X 10' M-l, after correcting for the chelation of Ca2+ and Co2+ by ATP. They measured fluorescence energy transfer between fluorescein isothiocyanate and 0.5 pM Co*+, which they believed to be bound to the cytoplasmic calcium transport site. However, Co'+ at this concentration does not inhibit the calcium ATPase directly and is not bound to the calcium transport site, as demonstrated by the value of Ki = 300 WM for inhibition of leaky vesicles by Co2+. Therefore, the distance measurement obtained by fluorescence energy transfer does not provide a measure of the distance from the fluorescein isothiocyanate to the calcium transport site. The concentrations of free La3+ and LaATP reported here are not exact because of the large number of equilibria that affect these concentrations. The value of Ki for inhibition of steady-state ATPase activity by LaC& increases with increasing concentrations of SRV, ATP, and Pi because La3+ is chelated by these components of the reaction mixture. In order to estimate the concentration of free La3' and LaATP, it is necessary to take into account the concentrations of SRV, ATP, and Pi, as well as the concentration of Ca2+ and Mp", which also bind to these chelators. Sulfate and MOPS have also been reported to bind La3+ (Perrin, 1979;Girardet, 1989). The reported dissociation constants for LaATP vary from 3.4 X 10e5 to 7.0 X 10e7 M, when adjusted to pH 7 (Krasnow, 1972;Morrison and Cleland, 1983). The values of Ki for free La3+ and LaATP that are reported in this paper refer to the dissociation constant of 7.0 x 10m5 M (Morrison and Cleland, 1983) and the experimental conditions described here.

Lanthanum
Does Not Cause Inhibition of Steady-state ATPase Activity by Binding to the Cytoplusmic Transport Site- The high levels of phosphoenzyme that are observed with inhibition by La3+ (Fig. 8, solidpoints) are not consistent with inhibition that is caused by binding of La3+ to the cytoplasmic calcium transport site, which is expected to inhibit phosphorylation of the enzyme. High phosphoenzyme levels have been observed previously by Yamada and Tonomura (1972) for enzyme that was inhibited by La3' and Ce3+. This result was interpreted in terms of a model in which Ca2+ and Mg are countertransported. It was concluded that these lanthanide ions inhibited the countertransport of MgZ+ that is required for the decomposition of phosphoenzyme, according to this model. The high levels of phosphoenzyme in the steady state demonstrate that decomposition of the phosphoenzyme is the rate-limiting step for enzyme that is inhibited by La3+, i.e. formation of the phosphoenzyme is fast relative to the slow hydrolysis of phosphoenzyme. Fig. 3 shows that the ATPase activity of enzyme that is inhibited by La3' decreases with increasing concentrations of Ca2+. If inhibition were caused by binding of La3+ to the cytoplasmic transport site, then Ca2+ would be expected to compete with La3+ and relieve this inhibition, so that the absolute activity would increase at high concentrations of Ca2+. The absence of relief of inhibition with increasing concentrations of Ca2+ is not consistent with inhibition by binding of La3+ to the cytoplasmic transport site. Fig. 4 shows that the value of Ki for inhibition of 45Ca uptake and phosphoenzyme formation in a single turnover is 500-fold larger than K, for inhibition of ATPase activity in the steady state. The results of the single turnover experiments demonstrate that Kd is -100 PM for LaCL that is bound to the cytoplasmic transport site in the presence of 25 PM Ca*+. This low affinity binding of La3+ to the cytoplasmic transport site under these conditions cannot account for the inhibition of steady-state turnover by La3+ with Ki = 0.16 pM.

Lanthanum
at the Catalytic Site Inhibits the Decay of Phosphoenzyme- Fig. 8 shows that La3' inhibits ATPase activity by slowing the rate of phosphoenzyme decay. This explains the high levels of phosphoenzyme that are observed in the steady state. It is known that millimolar concentrations of Ca*+ are required to inhibit the decay of phosphoenzyme formed from MgATP by binding to the intravesicular transport site (Ikemoto, 1975), but it appeared possible that La3' could bind to this site with a much higher affinity. However, Fig. 5 shows that the addition of 6.7 PM LaCb to leaky vesicles after the formation of phosphoenzyme with MgATP has no effect on the rate of decay of the phosphoenzyme, although this concentration of LaC13 causes 95% inhibition of ATPase activity in the steady state. Therefore, La3+ does not cause inhibition of ATPase activity in the steady state by binding to the intravesicular transport site. We conclude that La3' inhibits the decay of phosphoenzyme by replacing Mg2+ as the catalytic ion for phosphoryl transfer. The rate constant of 0.05 s-' for the decay of phosphoenzyme with La3+ as the catalytic ion is much smaller than the rate constant of -15 s-' for the decay of phosphoenzyme with M$+ as the catalytic ion; this step is predominantly ratelimiting for the hydrolysis of both LaATP and MgATP (Hanel and Jencks, 1990;Chiesi and Inesi, 1979;Pickart and Jencks, 1984). Fig. 8 shows that lanthanum phosphoenzyme, which decays with k = 0.05 s-', and magnesium phosphoenzyme, which decays with k > 4 s-i, are both present in enzyme that is partially inhibited by La3+; this replacement of Mg2+ by La3+ is responsible for the inhibition of steady-state ATPase activity.