Unravelling the interaction of thapsigargin with the conformational states of Ca(2+)-ATPase from skeletal sarcoplasmic reticulum.

Preincubation of thapsigargin with sarcoplasmic reticulum vesicles in the presence of high Ca(2+) or the addition of high Ca(2+) to microsomal vesicles preincubated with thapsigargin in the absence of Ca(2+) allowed full enzyme phosphorylation by ATP. However, the enzyme activity was not protected by high Ca(2+) even when the samples were subjected to gel filtration before ATP addition. Our data indicate that: (i) the enzyme in the Ca(2+)-bound conformation can be stabilized in the presence of thapsigargin; (ii) the conformational transition from the Ca(2+)-free to the Ca(2+)-bound state can be elicited by Ca(2+) when thapsigargin is present; (iii) thapsigargin binding occurs whether or not the enzyme is in the presence of Ca(2+), and so a ternary complex enzyme-Ca(2+)-thapsigargin may be formed; (iv) thapsigargin can be dissociated from the enzyme with a slow kinetics after dilution under drastic conditions; (v) the kinetics of Ca(2+) binding is clearly slowed down by thapsigargin; and (vi) thapsigargin does not affect the hydrolysis rate of phosphorylating substrates when measured in the absence of Ca(2+), indicating that thapsigargin specifically inhibits the Ca(2+)-dependent activity.

Thagsigargin (TG) 1 is a naturally occurring sesquiterpene lactone of the guaianolide type (1, 2) that can be found in the root of several species of the genus Thapsia (3). It was initially described as a tumor promoting agent with ability to induce rapid Ca 2ϩ release from intracellular stores (4). The effect was later recognized to be indirect since TG inhibits the Ca 2ϩ -ATPase of the sarcoendoplasmic reticulum membrane (5)(6)(7). It was also established that equimolar TG with respect to the enzyme concentration is sufficient to produce complete inhibition of the ATP-dependent Ca 2ϩ transport (6). Nowadays, TG is a key molecule for investigating the selective mobilization of Ca 2ϩ from the intracellular reticula.
A combined approach including kinetics and molecular biology techniques as well as structural data have provided important clues on the inhibitory effect of TG. For instance, it has been shown that TG inhibits Ca 2ϩ binding in the absence of ATP and enzyme phosphorylation by inorganic phosphate in the absence of Ca 2ϩ (8). Likewise, the inhibition has been related to the Ca 2ϩ -free conformation of the enzyme (6,8,9). Some transient protection by Ca 2ϩ when EP was formed from ATP and the accumulation of a dead end complex as a consequence of the enzyme turnover has also been observed (10). The interaction of TG and Ca 2ϩ with the enzyme was described as involving mutual exclusion when a low free Ca 2ϩ medium was used and the interaction of TG with the enzyme was reported as being irreversible when the experiments were performed at 25°C (10).
As regards the location of the TG-binding domain, the use of a fluorescent TG derivative (11) or photolabeling with a radioactive azido derivative of TG (12) indicated that the binding occurs within or near the transmembrane region of the enzyme. More precise information was obtained by chimeric construct and site-directed mutagenesis after overexpression of the Ca 2ϩ -ATPase protein in COS-1 cells. These studies revealed that the TG-binding domain does not reside within the large cytoplasmic loop (13). Location at the membrane interface and interaction with the S3 stalk segment between Asp 254 and Leu 260 have been suggested (14). The S3 segment has also been related with the binding domain of the high affinity inhibitor cyclopiazonic acid (15). In this sense, the region around Phe 256 , where the structural elements M3, M4, and L67 gather, seems to be critical for Ca 2ϩ binding (16). It is envisioned that the presence of TG might destabilize certain hydrogen bonds and, as a consequence, decrease Ca 2ϩ binding affinity (16).
We have now performed a systematic and in-depth study which will help to elucidate the interaction of TG with the enzyme and shed light on the inhibition mechanism of this highly specific inhibitor of the SR Ca 2ϩ -ATPase.

EXPERIMENTAL PROCEDURES
Materials-TG was obtained from Molecular Probes Europe. A23187 from Streptomyces chartreusensis was obtained from Calbiochem. Stock solutions of TG or A23187 were prepared in dimethyl sulfoxide (ACS reagent). The Ca 2ϩ standard solution (Titrisol) was a product of Merck.
[␥-32 P]ATP was purchased from PerkinElmer Life Sciences. The liquid scintillation mixture (S4023) and other reagents of analytical grade, including deoxycholate (D4297), were from Sigma. HAWP filter units with a pore diameter of 0.45 m were from Millipore. Samples were manually filtered under vacuum in a Hoefer filtration box (Amersham Pharmacia Biotech).
Sample Preparation-Fast-twitch leg muscle obtained from adult female New Zealand rabbit was the starting material. A microsomal fraction enriched in longitudinal tubules of the SR membrane was prepared by differential centrifugation according to Eletr and Inesi (17). Purified Ca 2ϩ -ATPase was obtained by method 2 of Meissner et al. (18). Fig. 1 shows protein staining of the preparations after SDS-electrophoresis (19). Samples were frozen in liquid nitrogen and kept at Ϫ80°C until use. The protein concentration was estimated by the * This work was supported by Spanish Ministerio de Ciencia y Tecnologia Grant PB97-1039. 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.
‡ To whom correspondence should be addressed:  (20) using bovine serum albumin as standard. One mg of SR protein contains ϳ4 nmol of active enzyme, as deduced from the maximal phosphorylation level, so that a vesicular protein concentration of 0.2 mg/ml is equivalent to 0.8 M Ca 2ϩ -ATPase.
Free Ca 2ϩ in Media-The ionized free Ca 2ϩ concentration was fixed by adding given volumes of CaCl 2 and/or EGTA stock solutions. Theoretical values were calculated by a computer program as previously described (21). The procedure takes into consideration the absolute stability constant for the Ca 2ϩ -EGTA complex (22), the pK values for the EGTA protonation (23), pH, and the presence of relevant ligands in the medium.
Preincubation with TG before Phosphorylation-SR vesicles in the absence of free Ca 2ϩ was named the E 2 medium and consisted of 20 mM Mops, pH 7.0, 80 mM KCl, 5 mM MgCl 2 , 0.1 mM EGTA, 0.2 mg/ml SR vesicles, and 15 M A23187. The E 1 Ca 2 (10 M) medium was prepared by adding 0.105 mM CaCl 2 to the Ca 2ϩ -free medium, whereas the E 1 Ca 2 (3 mM) medium contained 20 mM Mops, pH 7.0, 80 mM KCl, 20 mM MgCl 2 , 0.1 mM EGTA, 0.2 mg/ml SR vesicles, 15 M A23187, and 3.1 mM CaCl 2 . Aliquots of 0.5 ml containing E 2 or E 1 Ca 2 samples were preincubated at 25°C for 5 min with TG. The phosphorylation of E 1 Ca 2 after preincubation with TG was preceded by a brief incubation at 0°C for 1 min. Phosphorylation of E 2 plus TG required the preliminary addition of 0.105 mM CaCl 2 (10 M free Ca 2ϩ ) and the subsequent incubation at 0°C for 1 min. In all cases, samples were phosphorylated at 0°C with 10 M [␥-32 P]ATP as described below.
Accumulated EP after TG Preincubation-The phosphorylation reaction was initiated at 0°C by adding 10 M [␥-32 P]ATP (ϳ50,000 cpm/ nmol) to 0.5 ml of SR vesicles preincubated with TG in the presence of Ca 2ϩ . The reaction was stopped (usually after 1 s) by the addition of 5 ml of ice-cold quenching solution (125 mM perchloric acid plus 2 mM phosphate). The quenched mixture was incubated in an ice bath for 5 additional min and then filtered under vacuum on nitrocellulose filter. Filters were rinsed with 25 ml of ice-cold quenching solution. The radioactivity retained by the filters was measured by the liquid scintillation technique.
Preincubation of E 1 Ca 2 with Equimolar TG at Different pCa-SR vesicles were equilibrated at 25°C in the presence of Ca 2ϩ . The initial medium was 20 mM Mops, pH 7.0, 80 mM KCl, 5 mM MgCl 2 , 0.1 mM EGTA, 0.2 mg/ml SR vesicles, 15 M A23187 and one of the following CaCl 2 concentrations: 0.089, 0.105, 0.128, 0.149, 0.2, 0.4, 1.1, or 3.1 mM. This yielded pCa values ranging from 5.5 to 2.5 in half-unit intervals, and included the 4.3 value. The MgCl 2 concentration was raised to 20 mM when the Ca 2ϩ concentration was 3.1 mM. The addition of 0.8 M TG to 0.5-ml aliquots of E 1 Ca 2 was followed by incubation at 25°C for 5 min or at 37°C for 1 h. Next, samples were cooled for 1 min in an ice bath before phosphorylation at 0°C for 1 s with 10 M [␥-32 P]ATP.
Preincubation of E 2 TG with Millimolar Ca 2ϩ -Aliquots of 0.5 ml containing 20 mM Mops, pH 7.0, 80 mM KCl, 20 mM MgCl 2 , 0.1 mM EGTA, 0.2 mg/ml SR vesicles, and 15 M A23187 (E 2 medium with 20 mM Mg 2ϩ ) were preincubated at 25°C for 5 min with 0.8 M TG. Then, 3.1 mM CaCl 2 (3 mM free Ca 2ϩ ) was added and the incubation was prolonged at 0, 25, or 37°C for different time intervals. Samples were cooled for 1 min in an ice bath before phosphorylation at 0°C for 1 s, as described before.
Incubation with TG or Ca 2ϩ before Gel Filtration-Initial samples were 1-ml aliquots of the following media: E 2 , E 2 TG (i.e. E 2 preincubated at 25°C for 5 min with 0.8 M TG) or E 1 Ca 2 (3 mM). Samples of E 2 medium were used as a control (without any addition) or were incubated at 25°C for 5 min with 0.8 M (equimolar) TG. E 2 TG samples were incubated at 37°C for 1 h with 3.1 mM Ca 2ϩ (3 mM free Ca 2ϩ ), whereas E 1 Ca 2 (3 mM) was incubated at 25°C for 5 min with 0.8 M TG. Samples of E 2 or E 2 incubated with TG (0.55 ml) were centrifuged at room temperature for 1 min through small chromatography columns (38 ϫ 10 mm) filled with Sephadex G-50 resin and pre-equilibrated in a Ca 2ϩ -free medium (20 mM Mops, pH 7.0, 80 mM KCl, 5 mM MgCl 2 , and 0.1 mM EGTA). Likewise, samples of E 2 TG incubated with Ca 2ϩ or E 1 Ca 2 (3 mM) incubated with TG (0.55 ml) were centrifuged through Sephadex columns pre-equilibrated in 3 mM free Ca 2ϩ medium (20 mM Mops, pH 7.0, 80 mM KCl, 20 mM MgCl 2 , 0.1 mM EGTA, and 3.1 mM CaCl 2 ). In all cases, the Sephadex columns were centrifuged at low speed for 1 min before use as previously described (24). The filtrate from each column was diluted with 5 ml of medium to reach a final free Ca 2ϩ of 50 M. The dilution medium for E 2 samples or E 2 incubated with TG was 20 mM Mops, pH 7.0, 80 mM KCl, 5 mM MgCl 2 , 0.1 mM EGTA, 0.165 mM CaCl 2 , 1.1 mM phosphoenolpyruvate, 6 units/ml pyruvate kinase, and 1.5 M A23187. The dilution medium for E 2 TG incubated with 3 mM Ca 2ϩ or E 1 Ca 2 (3 mM) incubated with TG was 20 mM Mops, pH 7.0, 80 mM KCl, 20 mM MgCl 2 , 0.5 mM EGTA, 0.22 mM CaCl 2 , 1.1 mM phosphoenolpyruvate, 6 units/ml pyruvate kinase, and 1.5 M A23187. The enzyme activity was measured at 37°C in 1-ml aliquots of diluted samples. The ATP concentration was 50 M and the procedure described by Lin and Morales was followed (25).
Ca 2ϩ -ATPase Activity in the Presence of TG without Dilution-The initial incubation medium (1.5 ml) contained 20 mM Mops, pH 7.0, 80 mM KCl, 5 mM MgCl 2 , 0.1 mM EGTA, 0.01 mg/ml SR protein, and 1 M A23187. When indicated, 30 nM TG was also included. The incubation medium was maintained at 37°C for 5 min. The rate of Ca 2ϩ -ATPase activity was measured at 37°C by the method described by Lanzetta et al. (26) after addition of 0.15 mM CaCl 2 , 1 mM phosphoenolpyruvate, 6 units/ml pyruvate kinase, and 50 M ATP. Aliquots of 0.2 ml were withdrawn at different times and mixed with 0.8 ml of color reagent. The color development was stopped after 1 min by adding 0.1 ml of 34% sodium citrate and the absorbance at 660 nm was read 30 min later. A blank assay was performed by adding 0.2 ml of incubation medium into 0.8 ml of color reagent. After vortexing, 50 M ATP was added and mixed.
Ca 2ϩ -ATPase Activity in the Presence of TG after Dilution-SR vesicles at 0.01 mg/ml in a medium containing 20 mM Mops, pH 7.0, 80 mM KCl, 5 mM MgCl 2 , 0.1 mM EGTA, and 1 M A23187 were preincubated at 37°C for 5 min with 30 nM TG. In some experiments the inhibitor was absent. A 10-, 40-, 60-or 100-fold dilution at 37°C was performed by mixing 0.30, 0.075, 0.05, or 0.03 ml preincubation medium with 2.70, 2.925, 2.95, or 2.97 ml of dilution medium, respectively. The dilution medium was 20 mM Mops, pH 7.0, 80 mM KCl, 5 mM MgCl 2 , 0.1 mM EGTA, and 0.15 mM CaCl 2 . After a fixed time of 2 h at 37°C the diluted samples were supplemented with 1 mM phosphoenolpyruvate, 6 units/ml pyruvate kinase, and 50 M ATP. The rate of the Ca 2ϩ -dependent ATP hydrolysis was evaluated at 37°C by measuring the release of inorganic phosphate (26) in 0.5-ml aliquots.
A time-dependent study of samples preincubated with TG was also conducted. All the manipulations were performed at 37°C. Leaky SR vesicles (0.01 mg/ml protein) in a Ca 2ϩ -free medium were preincubated with 30 nM TG and then 100-fold diluted in the medium containing 50 M free Ca 2ϩ as described before. Control experiments were performed without adding TG during preincubation. After dilution, aliquots of 3 ml were withdrawn at different time intervals to measure Ca 2ϩ -ATPase activity. Hydrolysis rates at each dilution time were evaluated in a medium containing 1 mM phosphoenolpyruvate, 6 units/ml pyruvate kinase, and 50 M ATP, according to Lanzetta et al. (26).
Hydrolytic Activity in a Ca 2ϩ -free Medium-The enzyme activity in the absence of free Ca 2ϩ was measured at 25°C using a preparation of purified Ca 2ϩ -ATPase (see Fig. 1). The initial reaction medium consisted of 20 mM Mops, pH 7.0, 80 mM KCl, 5 mM MgCl 2 , 1 mM EGTA, and 0.4 mg/ml purified enzyme. Next, a given TG concentration was added and the incubation was prolonged at 25°C for 5 min. The TG/enzyme molar ratio was varied in the range of 0.5 to 4. The hydrolytic reaction in 1-ml aliquots was started by adding 1 mM ATP, 1 mM UTP, or 10 mM p-nitrophenyl phosphate. The release of inorganic phosphate from the nucleotides was evaluated by the procedure of Lin and Morales (25). The hydrolysis of p-nitrophenyl phosphate was followed by measuring the accumulation of p-nitrophenol (27).
Data Presentation-Each plotted value corresponds to the average of at least three independent assays performed in duplicate. The standard deviation (plus or minus) for each assay is also given.

RESULTS
The inhibitory effect of TG can be conveniently assessed by studying the capacity of the enzyme to be phosphorylated by ATP in the presence of Ca 2ϩ . Initially, SR vesicles in a 10 M free Ca 2ϩ medium were preincubated at 25°C for 5 min with equimolar TG. Samples were subsequently transferred to an ice bath for 1 min and then 1-20-s phosphorylation was achieved by the sequential addition of 10 M [␥-32 P]ATP and acid quenching solution. An accumulation of approximately 0.5 nmol of EP/mg of protein from the initial time point was observed (closed circles in Fig. 2A). As expected, the enzyme was fully phosphorylated when TG was not added during preincubation (open circles). A different pattern was observed when equimolar TG was added to SR vesicles in a 3 mM free Ca 2ϩ medium. In this case, the EP accumulation showed an initial rise followed by a progressive decrease as the phosphorylation time was prolonged (closed squares in Fig. 2B). The time course of EP formed under the same conditions but without TG added during preincubation is shown as a control (open squares).
The transient protection by millimolar Ca 2ϩ was also analyzed by changing the free Ca 2ϩ added during preincubation and evaluating EP after brief phosphorylation (1 s at 0°C with 10 M [␥-32 P]ATP). When equimolar TG was added to vesicles in a Ca 2ϩ -containing medium and the incubation lasted 5 min at 25°C, EP increased as the Ca 2ϩ concentration was raised (closed squares in Fig. 3A). Maximal phosphorylation was obtained at 100 M free Ca 2ϩ although a tendency to decrease was observed at higher Ca 2ϩ concentrations. Control experiments performed in the absence of TG exhibited a decrease of EP from 3.2 to 2.1 when the pCa was raised from 5.5 to 2.5 (open squares). The TG effect is also shown as a percentage of EP versus pCa (closed squares in Fig. 3B). Closed triangles in Fig.  3B correspond to data obtained when the preincubation with equimolar TG was maintained for 1 h at 37°C. It is apparent that half-maximal protection by Ca 2ϩ decreased as the preincubation time was prolonged and the temperature was raised.
The effect of TG was also studied when the enzyme was initially in the E 2 conformation. To this end, SR vesicles in a nominally Ca 2ϩ -free medium were supplemented at 25°C with a given TG concentration. Then, Ca 2ϩ was added to reach a final 10 M free concentration and the preincubation was maintained at 25°C for 5 min. The TG effect was evaluated after 1 min incubation in an ice bath and 1 s phosphorylation at 0°C by 10 M [␥-32 P]ATP. Preincubation of E 2 TG with 10 M free Ca 2ϩ produced a TG-dependent inhibition of the accumulated EP (Fig. 4). EP was completely inhibited by 0.8 M TG when the SR protein was 0.2 mg/ml, i.e. when the Ca 2ϩ -ATPase was 0.8 M. This means that complete inhibition was observed when the TG/enzyme molar ratio was Ն1.
The protective role of millimolar Ca 2ϩ when added to E 2 TG was studied as follows: SR vesicles in a Ca 2ϩ -free medium were preincubated at 25°C for 5 min with equimolar TG and then Ca 2ϩ was added to reach a final 3 mM free concentration. The incubation was prolonged at 0, 25, or 37°C and the samples taken at different times after adding Ca 2ϩ were used to evaluate EP (1 s phosphorylation at 0°C). Thus, the EP level increased in the minute time scale and was clearly dependent on temperature (Fig. 5). The accumulation rate was very low when the incubation temperature in the presence of Ca 2ϩ was 0°C (open triangles). The rate was higher at 25°C and EP reached values close to maximum when the Ca 2ϩ incubation was maintained for 60 min (open squares). The recovery was even faster when the temperature was raised to 37°C (open circles). Samples with no TG added during preincubation were also phosphorylated at 37°C and in a 3 mM free Ca 2ϩ medium and are shown as a reference (closed circles).
The interaction of TG and Ca 2ϩ with the enzyme was more deeply explored by using different preincubation protocols before the samples were subjected to gel filtration and the Ca 2ϩ -ATPase activity was measured (Fig. 6). In all cases, the enzyme activity was measured at 37°C in the same reaction medium containing 20 mM Mops, pH 7.0, 80 mM KCl, 5 mM MgCl 2 , 50 M free Ca 2ϩ , 1.5 M A23187, and 50 M ATP in the presence of an ATP-regenerating system. An initial sample of SR vesicles in the absence of Ca 2ϩ (i.e. E 2 ) was passed through a Sephadex column pre-equilibrated in a Ca 2ϩ -free medium. Once eluted from the column, the sample was diluted in the reaction medium containing 50 M free Ca 2ϩ and the enzyme activity was measured. After this treatment the rate of ATP hydrolysis was 3 mol/min/mg of protein. However, the enzyme activity was only 0.14 when E 2 was preincubated at 25°C for 5 min with equimolar TG. The Sephadex column pre-equilibrated in the absence of Ca 2ϩ was unable to eliminate the inhibition even though the enzyme activity was measured in the 50 M free Ca 2ϩ medium described before. In another set of experiments, E 2 TG samples were preincubated with 3 mM free Ca 2ϩ at 37°C for 1 h and then passed through Sephadex, this time preequilibrated in 3 mM free Ca 2ϩ medium. Samples from the column were diluted in the reaction medium containing 50 M free Ca 2ϩ and the enzyme activity was measured as before. Once again the enzyme activity in the presence of Ca 2ϩ was low, only 0.11 mol/min/mg of protein. The last protocol tried was preincubation at 25°C for 5 min of E 1 Ca 2 (3 mM) with equimolar TG and then passage through Sephadex pre-equilibrated in 3 mM free Ca 2ϩ . The enzyme activity of samples diluted in the 50 M free Ca 2ϩ medium was 0.13 mol/min/mg of protein.
Under our assay conditions, that included 0.01 mg/ml SR protein and 50 M ATP, half-maximal inhibition was observed at 19 nM TG (data not shown). Using the conditions of this experiment we studied the putative reversibility of TG. Leaky SR vesicles (0.01 mg/ml) in the absence of Ca 2ϩ were first equilibrated with 30 nM TG and then diluted and incubated for a certain time span. Aliquots taken after dilution were used to evaluate Ca 2ϩ -ATPase activity. All the manipulations were carried out at 37°C and the dilution medium contained 50 M free Ca 2ϩ . Control experiments were performed without TG in preincubation. Data in Fig. 7A correspond to samples diluted to different degrees and incubated at 37°C for 2 h. It is clear that enzyme activity recovery was dependent on the dilution factor (open bars). Data are expressed on a relative scale and the 100% value at each dilution corresponds to the enzyme activity when TG was absent during the preincubation. The time course of TG dissociation after a 100-fold dilution at 37°C is also shown (Fig. 7B). The initial preincubation conditions included 0.01 mg/ml SR protein and 30 nM TG.
Additional data on the effect of TG were obtained by measuring the hydrolysis of phosphorylating substrates in a nominally Ca 2ϩ -free medium. These experiments were performed with a preparation of purified enzyme (Fig. 1) to exclude the participation of any other contaminating protein. The Ca 2ϩ -ATPase protein was able to hydrolyze different substrates in the absence of free Ca 2ϩ (Fig. 8). The hydrolysis rate at 25°C was 30 nmol/min/mg protein when the phosphorylating substrate was 1 mM ATP or 10.3 nmol/min/mg protein when the substrate was 1 mM UTP. The activity was 8.2 nmol/min/mg protein when 10 mM p-nitrophenyl phosphate was used. It is remarkable that TG did not alter the hydrolysis rate of the phosphorylating substrates even when the molar ratio with respect to the enzyme concentration was raised to 4. The enzyme activity in the absence of Ca 2ϩ was also studied as a function of the ATP concentration. The presence of TG did not alter the hyperbolic dependence on ATP concentration when the TG/enzyme molar ratio was raised to 10 (data not shown).

DISCUSSION
The addition of [␥-32 P]ATP to E 1 Ca 2 leads to the formation of radioactive EP by transfer of the ATP ␥-phosphate to the enzyme. Therefore, the E 1 Ca 2 conformation, as opposed to E 2 , can be functionally distinguished by a brief phosphorylation at 0°C. Our initial EP measurements confirmed previously reported inhibitory effects of TG, namely the sensitivity of the E 2 conformation to TG (6, 8 -10) and some protection by Ca 2ϩ (10).
This study reveals that the ATP phosphorylating capacity was dependent on Ca 2ϩ concentration when equimolar TG was added to E 1 Ca 2 (Figs. 2 and 3). These experiments required 1-2 s phosphorylation at 0°C since longer phosphorylation times led to EP destabilization and the accumulation of E 2 TG as the reaction cycle proceeded (6, 10) (see also Fig. 2B). The inhibition degree was also dependent on incubation time and temperature (Fig. 3B). Higher Ca 2ϩ concentrations were needed to observe a certain EP level when the incubation was prolonged from 5 min to 1 h and the temperature was raised from 25 to 37°C. These data are consistent with intrinsic fluorescence FIG. 6. Ca 2؉ -ATPase activity of samples passed through Sephadex. One-ml aliquots containing E 2 , E 2 TG (i.e. E 2 preincubated at 25°C for 5 min with 0.8 M TG), or E 1 Ca 2 (3 mM) were mixed and incubated as specified in the figure. A volume of each incubation (0.55 ml) was passed through a Sephadex G-50 column pre-equilibrated in a Ca 2ϩ -free or in a 3 mM free Ca 2ϩ medium. Samples eluted from the column were diluted with 5 ml of medium to give the same reaction medium containing 50 M free Ca 2ϩ . The enzyme activity of diluted samples was measured at 37°C using 50 M ATP as a substrate and an ATP-regenerating system.
When E 2 was preincubated with TG, the TG/enzyme molar ratio was Ͻ1 and 10 M free Ca 2ϩ was added, the enzyme capacity to be phosphorylated by ATP was related to the E 1 Ca 2 /E 2 ratio and the dependence of EP on TG provided a titration of the enzyme molecules. This explains why EP decreased monotonically as the TG concentration rose (Fig. 4). It also confirms that E 2 TG is an inactive form (6, 8 -10) and 1:1 is the binding stoichiometry (6, 8 -10).
The slow kinetics of EP accumulation after addition of millimolar Ca 2ϩ to E 2 TG even when the temperature was raised to 37°C (Fig. 5) confirms the main inhibitory action of TG as a clear decrease in the rate of the E 2 3 E 1 Ca 2 transition. Therefore, when the incubation medium contains a low M free Ca 2ϩ and TG is added, the enzyme is blocked in the E 2 TG state. However, the addition of high Ca 2ϩ enables the enzyme to recover from the apparent E 2 TG dead complex. Experiments in Fig. 5 correspond to the transition E 2 TG ϩ Ca 2ϩ 3 E 1 Ca 2 TG in Scheme I. EP levels in Fig. 5 were measured after 1 s phosphorylation to avoid enzyme cycling. Note that 1 s phosphorylation provided lower EP levels than 2 s when TG was added to E 1 Ca 2 (3 mM) (Fig. 2B).
The fact that E 2 TG preincubated with millimolar Ca 2ϩ can be fully phosphorylated by ATP may be due to TG dissociation from the enzyme. This was checked by using a gel filtration column to remove unbound TG and measuring Ca 2ϩ -ATPase activity. E 2 TG samples preincubated with 3 mM free Ca 2ϩ or E 1 Ca 2 (3 mM) preincubated with equimolar TG and then passed through Sephadex did not recover Ca 2ϩ -ATPase activity. A residual activity was noticed in both cases (Fig. 6). This result is proof that a high Ca 2ϩ concentration is unable to induce TG dissociation. However, these preincubation conditions allowed full phosphorylation by ATP. This behavior is consistent with the partial reaction: E 2 TG ϩ Ca 2ϩ 3 E 1 Ca 2 TG and not with: E 2 TG ϩ Ca 2ϩ 3 E 1 Ca 2 ϩ TG. In other words, Ca 2ϩ and TG can be forced to be simultaneously bound to the enzyme. A mutu-ally exclusive model requires TG dissociation when the Ca 2ϩ concentration is raised and this was not observed under any circumstance (Fig. 6). On the other hand, it is known that TG and Ca 2ϩ have different binding sites (14,16).
The recovery of enzyme activity after dilution (Fig. 7) demonstrates that TG can be dissociated from the enzyme and, therefore, that it is reversibly bound. The dissociation was observed after a 100-fold dilution when the protein concentration was lowered to 0.1 g/ml and TG was 0.3 nM. The dissociation kinetics was very slow and required a long incubation at 37°C (Fig. 7B). The high temperature was critical since no dissociation was observed when the incubation was performed at 25°C even though similar conditions after dilution were used (10). Experiments in Fig. 7 correspond to the transition E 2 TG 3 E 2 ϩ TG 3 E 1 Ca 2 in Scheme I.
The hydrolysis of phosphorylating substrates in the absence of Ca 2ϩ was unaffected by the presence of TG even when used above the equimolar level and was sustained by the purified enzyme (Fig. 8). This indicates that TG does not affect the catalytic properties of E 2 . The normal operation of the reaction cycle in a Ca 2ϩ -containing medium involves the participation of E 1 Ca 2 , E 2 , and phosphorylated species. When the reaction cycle takes place in the presence of Ca 2ϩ and TG is also present, the enzyme is stacked as E 2 TG. Under these conditions the E 2 TG complex in the presence of Ca 2ϩ exhibits the same hydrolytic activity as the enzyme in the absence of Ca 2ϩ and TG. It should be recalled that the residual activity measured in the presence of Ca 2ϩ coincided with that measured in the absence of Ca 2ϩ when samples were pretreated with TG (Fig. 6). This confirms that the inhibitory effect of TG is on the Ca 2ϩ -dependent but not on the Ca 2ϩ -independent activity. Inhibition by TG has also been shown to be dependent on the E 1 Ca 2 /E 2 ratio when p-nitrophenyl phosphate was the substrate (27).
Full phosphorylation by ATP in the presence of equimolar TG and millimolar Ca 2ϩ was observed using leaky SR (Figs. 2 and 3) and was also reproduced in native vesicles (data not shown) indicating that Ca 2ϩ binding to the external (high affinity) sites is sufficient to exert the protective effect. Saturation of the internal (low affinity) Ca 2ϩ sites has also been reported to decrease the inhibitory effect of TG on enzyme phosphorylation from inorganic phosphate (10). These data suggest that, under equilibrium conditions, any action directed at decreasing the accumulation of E 2 TG has a protective role. In contrast, we have checked that the Ca 2ϩ -dependent activity is inhibited by TG when millimolar Ca 2ϩ is present in the external and/or internal medium. This confirms that Ca 2ϩ binding to the cytoplasmic or lumenal sites does not provide protection when the enzyme is cycling. FIG. 8. Hydrolytic activity in a Ca 2؉ -free medium of E 2 samples (purified enzyme) preincubated with TG. Purified Ca 2ϩ -ATPase (0.4 mg/ml) in a medium containing 20 mM Mops, pH 7.0, 80 mM KCl, 5 mM MgCl 2 , and 1 mM EGTA were preincubated at 25°C for 5 min with a given TG concentration. The release of phosphate at 25°C was measured after the addition of 1 mM ATP (q), 1 mM UTP (⌬), or 10 mM p-nitrophenyl phosphate (OE). SCHEME I. Interaction of TG with the Ca 2؉ -dependent conformations of the enzyme. TG can interact and bind to the E 1 Ca 2 or E 2 conformations. The stabilization of E 1 Ca 2 TG or E 2 TG is dependent on Ca 2ϩ concentration. The transitions induced in our study were: transition a (Figs. 2B and 3), transition a plus c ( Figs. 2A and 3), transition b (Fig. 5), and transition d plus e (Fig. 7).
The interaction of TG and Ca 2ϩ with the enzyme in the absence of ATP is summarized in Scheme I. The binding of TG is not dependent on the conformational state of the enzyme and therefore E 1 Ca 2 and E 2 are targets for TG. The stabilization of E 1 Ca 2 TG or E 2 TG under equilibrium conditions is dependent on the Ca 2ϩ concentration present. This study demonstrates that the equilibria among the Ca 2ϩ -dependent conformations of the enzyme in the presence of TG can be shifted by ligand addition and/or sample dilution.