Substrate Regulation of Calcium Binding in Ca 2 (cid:1) -ATPase Molecules of the Sarcoplasmic Reticulum I. EFFECT OF ATP*

The effect of ATP on calcium binding of the Ca 2 (cid:1) ATPase of the sarcoplasmic reticulum has not been clarified. By comparing the calcium dependence of the ATPase activity and of phosphorylation of the ATPase molecules with that of calcium binding in the absence of ATP, we show the existence of two types of regulatory site of the enzyme molecules at which ATP binding var-iously improves the calcium binding performance of the molecules depending on the aggregation state of the molecules and pH; the two regulatory sites bind ATP at submillimolar (0.25 m M ) and millimolar (5 m M ) ATP, re- spectively. The results are discussed based on a model of two conformational variants (A and B forms) of the chemically equivalent ATPase molecules (Nakamura, J., and Furukohri, T. (1994) J. Biol. Chem . 269, 30818– 30821). For example, in the sarcoplasmic reticulum

The membrane-bound Ca 2ϩ -ATPase (110 kDa) of the sarcoplasmic reticulum (SR) 1 is a calcium pump (1-3) and a P-type ATPase (4,5). The Ca 2ϩ -ATPase transports 2 mol of calcium across the SR membrane by hydrolytic coupling with 1 mol of ATP, accompanying the transition of the ATPase from E 1 (high affinity state for calcium) to E 2 (low affinity state for calcium) (1)(2)(3)5). The ATP hydrolysis cycle has been shown to be accelerated by ATP binding to a putative regulatory site (1,3,6,7) at concentrations higher than those required for saturation of the catalytic site (8,9). ATP regulation consists of the acceleration of several intermediate steps in the catalytic reaction (10 -14). Recently, Toyoshima et al. (15) determined the atomic structure of the pump enzyme at a resolution of 2.6 Å and showed that the ATPase molecule has one nucleotide-binding site. However, the molecular basis of the regulatory site has not been clarified (16 -23).
Calcium binding to the two calcium transport sites of the enzyme is required for phosphorylation of the enzyme with ATP; the phosphorylation drives the calcium transport reaction (1)(2)(3). It is therefore crucial to determine whether calcium binding is regulated by ATP binding. Based on observations of the velocities of calcium transport that are supported by various kinds of high energy phosphate compounds, Ogawa (24) and Ogawa and Ebashi (25) reported that ATP increases the calcium affinity of the enzyme depending on the ATP concentration (0.3 M to 2.7 mM). On the other hand, the calcium dependence of equilibrium calcium binding in the absence of ATP (26) has been shown to exhibit a cooperative profile (Hill number (n H ) of ϳ2) with K 0.5 ϳ 0.4 M. Such a cooperative profile of kinetic calcium binding (n H ϳ 2, K 0.5 ϳ 0.1 M), which was obtained from the calcium dependence of Ca 2ϩ -ATPase activity at a saturating concentration (5 mM) of ATP, has also been observed (27). To clarify whether ATP changes calcium binding of the ATPase, kinetic calcium binding to the enzyme should be examined in the presence and absence of ATP. Recently, the enzyme molecules in the SR membrane have been suggested to exist as two conformational variants of the chemically equivalent enzyme molecules at a ratio of 1:1 (Fig. 1) on the basis of the following observations (28). (i) At 0°C, about half of the calcium-binding sites of the enzyme molecules are in a slow (t1 ⁄2 Ն 2 s)/rapid (t Ͻ2 s) binding state dependent on pH, and the other half are in a slow binding state independent of pH. The enzyme molecules are slowly or rapidly phosphorylated by ATP, concurrent with slow or rapid calcium binding at a ratio of ϳ1:2, indicating that each of the two pools of the calcium sites belongs to one of the two different types (A and B forms) of the enzyme molecules, which are in pH-dependent equilibrium between E 1 and E 2 and predominantly in E 2 independent of pH, respectively, before calcium binding (Fig. 2). (ii) The amino acid sequence of lysyl endopeptidase peptides of the enzyme preparation is homogeneous and of the fast twitch muscle type, suggesting that the two types of molecules are two conformations of chemically equivalent ATPase molecules. The two hypothesized conformations were previously shown to bind calcium independently in a different manner (29). The model of the conformational variants has been strengthened by the observations that (i) there are the two types of molecules in the SR membrane that differ in the sensitivity of their intrinsic fluorescence intensities to calcium ions and that correspond to the two conformers, respectively (29); (ii) the nonequivalence of the enzyme molecules is canceled by solubilization of the molecules with a detergent (30); and (iii) the calcium binding manner of the detergent-solubilized enzyme is distinct from that of either type of conformer (30). However, the conformational difference in the conformers has not been specified so far.
Here, the calcium dependence of the Ca 2ϩ -ATPase activity and of the phosphorylation of the molecules was compared with that of the kinetic calcium binding in the absence of ATP. The results show the existence of two types of regulatory site at which ATP binding changes the binding. The data on ATP regulation are discussed in terms of a model that never seems to come into sharper focus with regard to which conformers of the ATPase molecules are involved in the catalytic cycle of the ATPase, whether these conformers are on different subunits in an oligomer of the enzyme molecules, whether they convert one into the other, and so on.

Materials
The procedures for isolation of the SR from rabbit skeletal muscle were the same as those described in a previous study (31). Employing a modified method (32) of Meissner et al. (33), membranous Ca 2ϩ -ATPase was purified from the SR by washing the SR with sodium deoxycholate at a 1:5 ratio of detergent to reticulum protein. The content of the ATPase protein in the purified ATPase preparation was estimated to be ϳ93% by SDS-PAGE of the preparation, similar to the content in the preparations (32) that were obtained at a 1:4 to 1:3 ratio of detergent to reticulum protein. Taking into consideration the molecular mass (110 kDa) of the Ca 2ϩ -ATPase molecule (34), the purity (ϳ93%) of the ATPase molecules in the enzyme preparation, and the number (one) of catalytic sites/ATPase polypeptide chain (15), the density of the catalytic sites in the preparation was estimated to be ϳ8.5 nmol/mg of preparation protein. The enzyme preparation was treated with 2 M calcium ionophore A23187 before use. The maximum level of the preparation that was phosphorylated with ATP was 4.2-5.6 (average of 4.4; n ϭ 17) nmol/mg of protein obtained in 0.25 mg of protein/ml of preparation (5 mM MgCl 2 , 0.12 M KCl, 0.1 mM ATP, and 0.1 mM CaCl 2 at pH 7.4 and 0°C). In the preparation that was solubilized with the nonionic detergent C 12 E 8 (octaethylene glycol dodecyl ether), the maximum level was 4.4 -4.6 (average of 4.5; n ϭ 4) nmol/mg of protein obtained in 0.05 mg of protein/ml of preparation (0.12 M KCl, 5 mM MgCl 2 , 0.46 mM ATP, 10 mM CaCl 2 , and 2.0 mg/ml C 12 E 8 at pH 7.40 and 0°C).

Assays
Enzymatic Activities-The total activity of ATP hydrolysis, which is composed of calcium-independent and -dependent ATPase activities, was assayed in 20 mM Bistris propane (pH 6.23) or 40 mM Tris maleate (pH 7.40) buffer solution containing 0.005-0.05 mg/ml membranous enzyme protein, 0.12 M KCl, 5 mM MgCl 2 , 10 M to 5 mM ATP, and 0.01-550 M Ca 2ϩ at 25°C. In the assay of the activity of the detergentsolubilized enzyme, the membranous enzyme (0.01 mg/ml enzyme protein) was directly solubilized in the assay medium containing 2.0 mg/ml C 12 E 8 . The reaction times for the assay of the ATPase activity of the membranous enzyme, in which the reactions were linear, were set at FIG. 1. Model of two conformational variants of chemically equivalent Ca 2؉ -ATPase molecules. Two protons participate in the equilibrium between E 1 and E 2 and in calcium binding in the A and B forms, respectively (29). The A and B forms noncooperatively and cooperatively bind two calcium ions, respectively (29). This noncooperative and cooperative binding is represented as "K 1 (dissociation constant of E 1 Ca) ϭ K 2 (dissociation constant of E 1 Ca 2 )" and "K 1 Ͼ K 2 ," respectively. The white and black stars designate the fluorescent amino acid residues in the ATPase polypeptides. The white stars are the state of the amino acid residues before calcium binding and the state of the residues whose fluorescence intensity is assumed not to be affected by the binding of one calcium ion. The black star is the state of the residues whose fluorescence intensity is enhanced by the binding of two calcium ions (29).
FIG. 2. Schematic representation of the equilibrium of the A and B forms between E 1 and E 2 before calcium binding and of the kinetics of calcium binding and calcium-induced EP formation. The equilibrium of the A form between E 1 and E 2 before calcium binding is pH-dependent (28). At acidic pH, the equilibrium shifts to the E 2 side. At 0°C, the A form apparently slowly binds calcium because of the slow transition of the form from E 2 to E 1 . The phosphorylation of the form preincubated with a low concentration of ATP in the absence of calcium is also slowly induced by the slow binding. At alkaline pH, the equilibrium shifts to the E 1 side. Therefore, even at a low temperature of 0°C, the A form, which is predominantly in E 1 , rapidly binds calcium, and the phosphorylation of the form is rapidly induced by the rapid binding. As for the B form, the equilibrium of the form before calcium binding shifts to the E 2 side independent of pH (28). At such a low temperature, the B form slowly binds calcium, and the phosphorylation of the form is also slowly induced by the slow binding, irrespective of pH. 90 s (at 0.01 mg/ml protein and 5 mM ATP) or 5 min (at 0.01 mg/ml protein and 0.25 mM ATP) at pH 6.23 and at 60 s (at 0.005 mg/ml protein and 10 M ATP and at 0.01 mg/ml protein and 0.25 mM ATP) or 90 s (at 0.01 mg/ml protein and 0.1 mM ATP and at 0.05 mg/ml protein and 5 mM ATP) at pH 7.40. In the presence of the detergent, the reaction time was set at 55 s (at 0.1 mM ATP) or 60 s (at 0.25 mM ATP) at pH 7.40. At 10 M to 0.25 mM ATP, the association constants for CaEGTA at pH 6.23 and 7.40 were taken as 6.311 ϫ 10 4 and 1.335 ϫ 10 7 M Ϫ1 , respectively (35). At a higher ATP concentration of 5 mM, the association constants for CaEGTA at pH 6.23 and 7.40 were taken as 5.841 ϫ 10 4 and 1.247 ϫ 10 7 M Ϫ1 , respectively (35). The association constants for CaATP and MgATP at pH 6.23 and 7.40 were taken as 3.097 ϫ 10 3 and 5.508 ϫ 10 3 M Ϫ1 and 8.241 ϫ 10 3 and 1.466 ϫ 10 4 M Ϫ1 , respectively (36). The calcium-independent ATPase activity was assayed in the presence of 5 mM EGTA without the addition of calcium. The calcium-dependent ATPase activity (Ca 2ϩ -ATPase activity) was obtained by subtracting the calcium-independent activity from the total activity. ATP hydrolysis was measured by determining the amount of phosphate liberated from ATP. P i , which was liberated from ATP at 10 M, 0.1-0.25 mM, and 5 mM ATP, was measured using the methods of Chan et al. (37), Baginski et al. (38), and Bonting et al. (39), respectively.
Phosphoenzyme-In the steady-state study of the ATP-induced phosphorylation of the enzyme, the enzyme preparation (0.25 mg/ml protein) was preincubated in a test tube with medium containing 20 mM Bistris propane (pH 6.23) or 40 mM Tris maleate (pH 7.40), 0.12 M KCl, 5 mM MgCl 2 , and 0.015-100 M Ca 2ϩ for 10 -60 min at 0°C. The reaction was manually initiated by the addition of 10 M to 0.25 mM [ 32 P]ATP. The volume of the reaction medium was 0.5 ml. The amount of the 32 Plabeled phosphoenzyme reached ϳ80% of its steady-state level within the dead time (ϳ0.2 s) of the experiment after initiation of the reaction and reached the steady-state level 3-5 s later. Thus, the reaction was terminated 5 s after initiation by the addition of 0.5 ml of 10% ice-cold perchloric acid. Aliquots (0.8 ml) of the terminated reaction medium were applied to Millipore HA filters (0.45-m pore size), which were immersed in 5% perchloric acid containing 0.1 mM ADP and 1 mM P i . The 32 P-labeled enzyme protein on the filter was washed five times with 10 ml of the 5% perchloric acid solution. In the pre-steady-state study of calcium-induced phosphorylation of the enzyme, a rapid filtration method was employed as described previously (28). Briefly, after washing the enzyme (0.2 mg of protein) on the Millipore filter with 0.2 mM EGTA and 10 M to 0.25 mM [ 32 P]ATP for ϳ10 s following 0.2 mM EGTA washing for ϳ10 s, the phosphorylation reaction was initiated by washing the enzyme on the filter with a phosphorylation solution of 0.015-50 M Ca 2ϩ containing the same concentration of [ 32 P]ATP as that used before initiation of the reaction. This was achieved by filtering 1-20 ml of the phosphorylation solution at 0°C. The dead time of the experiment was ϳ2 s. Compared with the reaction of the ATP-induced phosphorylation described above, the reaction of the calcium-induced phosphorylation was slow and reached its steady-state level 10 -30 s after initiation of the reaction (see Fig. 2 and "Discussion" for details).

RESULTS
To examine the effect of ATP on calcium binding in the Ca 2ϩ -ATPase molecules of the SR, the calcium dependence of the Ca 2ϩ -ATPase activity of the ATPase molecules and of the phosphorylation of the ATPase molecules was studied based on a model of two conformational variants (A and B forms) of chemically equivalent Ca 2ϩ -ATPase molecules (28). The two forms have been suggested to exist in the SR membrane at a ratio of 1:1 and to independently bind two calcium ions in the absence of ATP ( Fig. 1) (29).
In Fig. 3A, the calcium dependence of the total Ca 2ϩ -ATPase activity, which is composed of the activities of the two forms, was examined at 10 M ATP and pH 7.40. The Ca 2ϩ -ATPase molecules of the SR have been shown to have one catalytic site with a high affinity for ATP (K m ϭ 2-10 M) (8,9) and a putative regulatory site(s) at which ATP binding accelerates the turnover rate of the ATPase activity with lower affinity (K m ϭ 0.1-5.0 mM) (1,3,27) for ATP than that at the catalytic site. ATP, which was used here at a concentration of 10 M, seems to bind to the catalytic site. The Hill plots of the calcium dependence of the total ATPase activity exhibited a biphasic profile with slopes of ϳ1.8 and ϳ1.0 and an apparent calcium affinity (calcium concentration for the half-maximum activity (K 0.5 )) of ϳ0.3 M (Fig. 3B). The two lines of the plots intersect near the zero point of the ordinate, indicating the existence of two different types of ATPase reaction with almost the same level of maximum activity. Such biphasic calcium dependence of the total phosphorylation (slopes of ϳ1.9 and ϳ0.9, K 0.5 ϳ 0.2 M), which is composed of the phosphorylation of the two forms and which was induced by the addition of ATP to the enzyme molecules preincubated with calcium, was also observed at 10 M ATP and pH 7.40 (Fig. 4, A and B). On the other hand, it has been found that ATP, which is bound to the catalytic site at 0.89 M to 0.1 mM ATP, accelerates specific steps in the catalytic cycle (40 -43). Therefore, the calcium dependence of the total Ca 2ϩ -ATPase activity and of the total phosphorylation was also examined at 0.1 mM ATP. These calcium-dependent profiles (slopes of ϳ1.9 and ϳ0.9 and K 0.5 ϳ 0.12 M for the ATPase activity and slopes of ϳ1.7 and ϳ0.7 and K 0.5 ϳ 0.24 M for the phosphorylation) (data not shown) were almost the same as those at 10 M ATP described above (Figs. 3 and 4). In the absence of ATP at pH 7.40, the A form noncooperatively (n H ϳ 1) bound calcium ions with an apparent K 0.5 of 2-6 M, whereas the B form cooperatively (n H ϳ 2) bound the ions with a K 0.5 of ϳ0.2 M ( Fig. 5A; replots of the data in Fig. 3A of Ref. 29). The Hill plots of the calcium dependence of the total calcium binding, which is composed of the binding of the two forms at a ratio of 1:1, were biphasic with slopes of ϳ1.8 and ϳ0.8 ( Fig. 5B; replots of the data in Fig. 3C of Ref. 29). The two lines of the Hill plots also intersect near the zero point of the ordinate. The K 0.5 of the total binding was ϳ0.4 M. This biphasic profile of the total binding in the absence of ATP was close to those of the total ATPase activity and the total phosphorylation. The results indicate that the calcium dependence of the ATPase activities of the A and B forms is near that of their calcium binding in the absence of 10 M ATP, suggesting that there is no effect of ATP binding at the catalytic site on calcium binding of the two forms. To examine this conclusion, a study was conducted to determine the calcium dependence of the phosphorylation of the A and B forms. The phosphorylation of the two forms was obtained by pre-steady-state analysis of the calcium-induced total phosphorylation reaction as described previously (28). In the pre-steady-state study, phosphorylation was initiated by the addition of calcium to the enzyme molecules, which were fixed on the filter and preincubated with ATP in the absence of calcium. At pH 7.40, as described previously (28), it is thought that the A and B forms are in the enzyme states of E 1 and E 2 , respectively, before calcium binding. At a low temperature of 0°C, the A form, which pre-exists in E 1 , rapidly (Ͻ2 s) binds calcium when it is added, whereas FIG. 3. Ca 2؉ -ATPase activity of the membranous ATPase molecules as a function of calcium concentration at 10 M ATP and pH 7.40. A, calcium dependence of the total ATPase activity, which consists of the activities of the two forms of ATPase molecules; B, Hill plot of the total activity. Y is the ratio of the activity at each calcium concentration to the maximum level of the activity (0.76 mol of P i /mg of protein/min). Before the addition of ATP to initiate the reaction, the enzyme in the assay medium was kept in ice for 30 min to 2 h and preincubated for 10 -15 min at 25°C. The reaction time was 1 min. The enzyme was used at a concentration of 0.005 mg/ml protein.
the B form, which pre-exists in E 2 , apparently slowly (Ն2 s) binds calcium because of the slow transition of the enzyme state from E 2 to E 1 (Fig. 2). Therefore, by rapid and slow calcium binding, the phosphorylation of the A and B forms (preincubated with ATP in the absence of calcium) is rapidly and slowly induced, respectively. The calcium-dependent profile of the observed slow phosphorylation (n H ϳ 2.0, K 0.5 ϭ 0.08 -0.1 M) (Fig. 4, A and B) was found to have a maximum level of ϳ2.5 nmol of phosphorylated Ca 2ϩ -ATPase (EP)/mg of protein, which is nearly half of the separately obtained maximum total phosphorylation (ϳ4.9 nmol of EP/mg of protein), and to be close to that of calcium binding of the B form in the absence of ATP (n H ϳ 2, K 0.5 ϳ 0.20 M) (Fig. 5, A and B); the slow phosphorylation seems to be fully resolved. The maximum levels of the total phosphorylation (ϳ4.9 nmol of EP/mg of protein) and of the slow phosphorylation (ϳ2.5 nmol of EP/mg of protein) are thought to be roughly one-half and one-fourth of the density (ϳ8.5 nmol/mg of protein) (see "Experimental Procedures" for details) of the total catalytic sites in the ATPase preparation, respectively. In contrast to the observed calciuminduced slow phosphorylation of the B form, the calcium-induced rapid phosphorylation of the A form was not resolved by pre-steady-state analysis using the method of rapid filtration.
The steady-state level of the calcium-induced rapid phosphorylation was lower than the level of the rapid phosphorylation, which was calculated by subtracting the calcium-induced slow phosphorylation from the ATP-induced total phosphorylation that was separately measured. For example, at 1. ϳ2.3 nmol of EP/mg of protein, respectively. The difference between the phosphorylation levels at 1.16 M Ca 2ϩ was more significant than that at 10.25 M Ca 2ϩ . The K 0.5 of the observed rapid phosphorylation seems to be higher than that of the simulated phosphorylation. In the SR of bullfrog skeletal muscle at 15°C (43), it has been observed that when the phosphorylation of the SR is induced by the addition of calcium following preincubation of the SR with ATP in the absence of calcium, the steady-state level of the calcium-induced phosphorylation is lower than the level of the ATP-induced phosphorylation following prolonged preincubation with calcium. In the rabbit SR at 25°C, the phosphorylation level has been found to be independent of the time of preincubation with calcium (26). These observations suggest that the steady-state level of the calcium-induced phosphorylation is lowered by decreasing the FIG. 4. EP formation of the membranous ATPase molecules at steady state as a function of calcium concentration at 10 M ATP, pH 7.40, and 0°C. A, ATP-induced total phosphorylation of the A and B forms (E), calculated rapid phosphorylation of the A form (OE), and calcium-induced slow phosphorylation of the B form (‚). The amount of the slowly phosphorylated B form was determined by pre-steady-state analysis of the calcium-induced phosphorylation. The amount of the phosphorylated A form was then calculated by subtracting that of the B form, which was separately measured, from that of the total phosphorylation (see "Results" for details). B, Hill plot of the total (E), rapid (OE), and slow (‚) phosphorylation. Y is the ratio of each EP at each calcium concentration to the respective maximum levels of EP (4.9, 2.4, and 2.5 nmol/mg of protein for total, rapid, and slow phosphorylation, respectively).
FIG. 5. Replots of previous data (29) of calcium binding of the membranous ATPase molecules as a function of calcium concentration in the absence of ATP at pH 7.40 and 0°C. A, calcium binding to the two forms of ATPase molecules. E, total binding to the A and B forms; OE, rapid binding to the A form, which is in an E 1 /E 2 state dependent on pH and which pre-exists in E 1 at this alkaline pH; ‚, slow binding to the B form, which pre-exists in E 2 independent of pH (see Fig. 2). B, Hill plot of the total (E), rapid (OE), and slow (‚) binding. Y is the ratio of calcium binding at each calcium concentration to the maximum levels of binding (10.3, 4.8, and 5.5 nmol/mg of protein for total, rapid, and slow binding, respectively). reaction temperature. In this study, the reaction of the calcium-induced phosphorylation was initiated by the addition of calcium to the enzyme preparation on the filter, which was prewashed with ATP in the absence of calcium for ϳ10 s following washing with EGTA for ϳ10 s at 0°C, similar to the case of the reaction in the bullfrog SR (43) mentioned above. It is therefore likely that at the low temperature, such a progressive ATP loading on the A form in the absence of calcium lowers the calcium affinity of the form, whereas the B form is not affected by the progressive loading. Thus, the rapid phosphorylation was determined by subtracting the calcium-induced slow phosphorylation from the separately obtained ATP-induced total phosphorylation. The calcium dependence of the calculated rapid phosphorylation (Fig. 4, A and B) exhibited a noncooperative profile with n H ϳ 1.1 and K 0.5 ϳ 5 M and was near that of the rapid calcium binding of the A form in the absence of ATP (n H ϳ 1, K 0.5 ϭ 2-6 M) (Fig. 5, A and B). The consistency in the calcium-dependent profiles of phosphorylation and binding in the absence of ATP confirms the above conclusion of the lack of effect of ATP binding to the catalytic site on calcium binding. On the other hand, the phosphorylation reaction of the two forms (induced by ATP after preincubation with calcium) was too fast to be resolved in our experiments, which was manually operated; the amount of the phosphorylated enzyme reached ϳ80% of its steady-state level within the dead time (ϳ0.2 s) of the experiment (see "Experimental Procedures") at 0°C. To understand the phosphorylation reaction of the two forms, the ATP-induced phosphorylation reaction should be analyzed by a method that has a shorter dead time and a higher power of time resolution than those of the method employed here (ϳ0.2 and 1 s, respectively).
In Fig. 7A, the calcium dependence of the total Ca 2ϩ -ATPase activity of the two forms was examined at 0.25 mM ATP. The calcium-dependent profile of the total ATPase activity was monophasic with n H ϳ 2.1 and K 0.5 ϳ 0.3 M (Fig. 7B). It is thought that in the presence of 0.25 mM ATP, both the A and B forms cooperatively bind two calcium ions with the same calcium affinity and that the calcium-dependent profile of the total ATPase activity is the same as those of the two forms. However, the calcium dependence of the ATP-induced total phosphorylation of the two forms at steady state exhibited a monophasic profile with a lower n H value (ϳ1.6) than the value of the total Ca 2ϩ -ATPase activity (ϳ2.1), although the K 0.5 (ϳ0.2 M) was almost the same as that of the ATPase activity (ϳ0.3 M) (data not shown). Phosphorylation reached its maximum level at ϳ10 M Ca 2ϩ , whereas the ATPase activity reached its maximum at ϳ1.9 M Ca 2ϩ and decreased at Ͼ10 M Ca 2ϩ . As mentioned above, at 10 M (Figs. 3B and 4B) and 0.1 mM (data not shown) ATP, no significant difference in the Hill plots of the calcium dependence of the ATPase activity and of phosphorylation was observed; for example, at 0.1 mM ATP, the Hill plots for the ATPase activity and phosphorylation were biphasic with slopes of ϳ1.9 and ϳ0.9 and of ϳ1.7 and ϳ0.7, respectively. In this study, the reactions of the ATPase activity and the ATP-induced phosphorylation were assayed at 25 and 0°C, respectively. The previous (26,43) and present (Fig. 6) observations suggest that the steady-state level of the calciuminduced phosphorylation of the A form is lowered by decreasing the reaction temperature, although the phosphorylation of the B form is not sensitive to temperature as described above. Taking these observations into account, it is likely that the level of the ATP-induced phosphorylation of the A form is also lowered by decreasing the temperature at the higher ATP concentration (0.25 mM), but not at the lower ATP concentration (Յ0.1 mM); the reduction of the phosphorylation level might make the slope (n H value) of the Hill plots of the calcium dependence of the total phosphorylation (ϳ1.6) lower than that of the plots of the calcium dependence of the total ATPase activity (ϳ2.1). Similar to the case at 10 M ATP (28), the two forms (which pre-exist in E 1 and E 2 , respectively, at pH 7.40 and 0°C) are predicted to be rapidly (t Ͻ2 s) and slowly (t1 ⁄2 ϳ 3 s) phosphorylated with 0.25 mM ATP by the addition of calcium (Fig. 2), if the ATP does not significantly affect the equilibrium of the forms between E 1 and E 2 before calcium binding. In Fig. 8A, pre-steady-state analysis of the calciuminduced total phosphorylation of the two forms was carried out at 50 M Ca 2ϩ . In addition to the rapid phosphorylation (Ͻ2 s), slow phosphorylation at t1 ⁄2 ϳ 2 s was observed. The observed maximum level of the slow phosphorylation (ϳ2.5 nmol of EP/mg of protein) (Fig. 8A) was close to half of the maximum total phosphorylation (ϳ4.9 nmol of EP/mg of protein) (data not shown), which was separately obtained by analyzing the ATPinduced phosphorylation reaction at 50 M Ca 2ϩ . The calciumdependent profile of the slow phosphorylation (n H ϳ 1.8, K 0.5 ϳ 0.1 M) (Fig. 9, A and B) was also close to that of the slow calcium binding of the B form in the absence of ATP (n H ϳ 2, K 0.5 ϳ 0.2 M) (Fig. 5, A and B). The results suggest that the observed slow phosphorylation is that of the B form. The steady-state level of the calcium-induced rapid phosphorylation was, however, observed to be lower than that expected from the value of the separately obtained total phosphorylation, similar to the case of the rapid phosphorylation at 10 M ATP (Fig. 6, A and B). For example, at 1.9 M Ca 2ϩ , where the ATPase activity reached its maximum (Fig. 7A), the observed level of the rapid phosphorylation (ϳ0.3 nmol of EP/mg of protein) was much lower than the expected half-maximum level of the total phosphorylation (ϳ2.5 nmol of EP/mg of protein); and even at 50 M Ca 2ϩ (cf. Fig. 8A), the observed rapid phosphorylation level increased only to ϳ1.6 nmol of EP/mg of protein (data not shown). As with the case at 10 M ATP described above, it is probable that at a higher ATP concentration of 0.25 mM and 0°C, the progressive ATP loading on the A form lowers the calcium affinity of the form, whereas the B form is not affected by the ATP loading. Nevertheless, the obtained results suggest that at 0.25 mM ATP, halves of the molecules that can be phosphorylated are slowly and rapidly phosphorylated, respectively. It is thought that 0.25 mM ATP does not significantly change the enzyme state of the A and B forms (which pre-exist in E 1 and E 2 , respectively) before calcium binding. Fig. 10A shows the calcium dependence of the total Ca 2ϩ -ATPase activity at 5 mM ATP and pH 7.40. The Hill plots of the calcium dependence exhibited a profile with n H ϭ 2.0 -2.2 and K 0.5 ϭ 0.04 -0.08 M (Fig. 10B). This profile was almost the same as that previously observed by Møller et al. (27) at pH 7.5 (n H ϳ 1.8, K 0.5 ϳ 0.01 M).
At pH 6.23, the calcium dependence of the ATP-induced total phosphorylation, which is composed of the phosphorylation of the two forms, was examined in the presence of 0.25 mM ATP (Fig. 11A). The calcium dependence of the phosphorylation exhibited a monophasic profile with n H ϳ 1.3 and K 0.5 ϳ 4 M (Fig. 11B). Such a monophasic profile of the total Ca 2ϩ -ATPase activity at 0.25 mM ATP (n H ϳ 1.5, K 0.5 ϳ 2 M) was also observed (data not shown). As shown previously (28), at this acidic pH of 6.23, it is thought that the A and B forms are predominantly in E 2 before calcium binding and that at a low temperature of 0°C, the two forms apparently slowly (t1 ⁄2 ϳ 8 s) bind calcium because of a slow transition of the two forms from E 2 to E 1 (Fig. 2). To examine the effect of 0.25 mM ATP on the enzyme state (E 2 ) of the two forms before calcium binding, the calcium-induced phosphorylation of the forms was examined at 0°C (Fig. 8B). The enzyme molecules were slowly and monophasically phosphorylated at t1 ⁄2 ϳ 2 s without the rapid phosphorylation (Ͻ2 s) (Fig. 8A), which was observed at pH 7.40. The equilibrium of the two forms between E 2 and E 1 does not seem to significantly change at 0.25 mM ATP. At 5 mM ATP and the acidic pH, the calcium-dependent profile of the total Ca 2ϩ -ATPase activity at 25°C was monophasic with n H ϳ 1.9 and K 0.5 ϭ 0.4 -0.8 M (Fig. 12, A and B).
In a previous study (30), the calcium dependence of calcium binding of the enzyme molecules (solubilized with C 12 E 8 ) was examined at 10 M and 5 mM ATP and pH 7.40. In Fig. 13A, the calcium dependence of the Ca 2ϩ -ATPase activity of the solubilized molecules was examined at 0.25 mM ATP. Almost all of the Hill plots of the calcium dependence were monophasic with n H ϭ 0.8 -0.9 and K 0.5 ϭ 1-2 M, although a small deviation from the linear line was observed at Ͻ0.35 M Ca 2ϩ (Fig. 13B). Such a monophasic profile of the ATPase activity with n H ϳ 1 was not observed at 0.1 mM ATP; the profile was almost the same with n H Ͻ 1 (0.5-0.6) and K 0.5 ϳ 5 M (data not shown) as that at 10 M ATP. The effect of ATP on calcium binding of the solubilized enzyme was not examined at pH 6.23 because of inactivation of the enzyme at this acidic pH. DISCUSSION Ogawa (24) and Ogawa and Ebashi (25) earlier reported that ATP increases the calcium affinity of the Ca 2ϩ -ATPase molecule depending on the ATP concentration (0.3 M to 2.7 mM). Here, to clarify whether ATP changes calcium binding of the ATPase molecules, we studied the effect of ATP on calcium binding of the enzyme molecules based on a model of two conformational variants (A and B forms) of the molecules (Fig. 1) (28).
The ATPase molecules have been shown to have one catalytic site with a high affinity for ATP (K m ϭ 2-10 M) (8,9) and a regulatory site(s) at which ATP binding accelerates the catalytic cycle of the ATPase molecules with lower affinity (K m ϭ 0.1-5.0 mM) (1,3,27) for ATP than that at the catalytic site. First, the effect of ATP binding to the catalytic site on calcium binding of the A and B forms was examined at 10 M ATP and pH 7.40 (Fig. 3A). The Hill plots of the calcium dependence of the total Ca 2ϩ -ATPase activity were biphasic with slopes of ϳ1.8 and ϳ1.0 (Fig. 3B). The two lines of the plots intersect near the zero point of the ordinate. The K 0.5 of the total activity was ϳ0.3 M. Such a biphasic profile of the ATP-induced total phosphorylation (slopes of ϳ1.9 and ϳ0.9, K 0.5 ϳ 0.2 M) was also observed at 10 M ATP (Fig. 4). In the absence of ATP at pH 7.40, it has been shown that the A form noncooperatively (n H ϳ 1) binds two calcium ions with K 0.5 ϭ 2-6 M, whereas the B form cooperatively (n H ϳ 2) binds the ions with K 0.5 ϳ 0.2 M (Fig. 5A; replots of the data in Fig. 3A of Ref. 29). The Hill plots of the calcium dependence of the total calcium binding were biphasic with slopes of ϳ1.8 and ϳ0.8 ( Fig. 5B; replots of the data in Fig. 3C of Ref. 29). The two lines of the Hill plots also intersect near the zero point of the ordinate. The K 0.5 of the total binding was ϳ0.4 M. The observed calcium-dependent profile of the total calcium binding was similar to the profiles of the total ATPase activity and the ATP-induced total phosphorylation. This suggests that there is no effect of ATP binding at the catalytic site on calcium binding of the two forms. This conclusion was confirmed by the observations that the calciumdependent profiles of the rapid and slow phosphorylation (n H ϳ 1.1 and K 0.5 ϳ 5 M and n H ϳ 2.0 and K 0.5 ϭ 0.08 -0.1 M, respectively) of the A and B forms in the presence of 10 M ATP (Fig. 4) were close to those of calcium binding (n H ϳ 1 and K 0.5 ϭ 2-6 M and n H ϳ 2 and K 0.5 ϳ 0.2 M, respectively) of the two forms in the absence of ATP, respectively (Fig. 5). The observations also suggest that there is no effect of 10 M ATP on the enzyme states (E 1 and E 2 ) of the two forms in the absence of ATP before calcium binding. Such an absence of effect of 10 M ATP on calcium binding has also been shown for detergentsolubilized ATPase molecules, although the solubilized molecules negatively cooperatively (n H ϭ 0.5-0.6) bind two calcium ions (30). Therefore, ATP binding at the catalytic site does not seem to affect calcium binding of the enzyme molecules, irrespective of the aggregation state of the enzyme molecules.
The affinity (K m ) of the regulatory site(s) of the membranous Ca 2ϩ -ATPase for ATP has been shown to be 0.1-5.0 mM for ATP (1,3,27). Here, the effect of ATP on calcium binding of the two forms was studied at 0.25 and 5 mM ATP. The calcium dependence of the total ATPase activity at 0.25 mM ATP and pH 7.40 exhibited a monophasic profile with n H ϳ 2.1 and K 0.5 ϳ 0.3 M (Fig. 7). As mentioned under "Results," it was found that at 0.25 mM ATP and 0°C, the rapid phosphorylation of the A form and the total phosphorylation of the two forms were not resolved by the pre-steady-state and steady-state analyses of phosphorylation, respectively, whereas the slow phosphorylation of the B form was fully resolved by the pre-steady-state analysis. The calcium dependence of the slow phosphorylation of the B form (n H ϳ 2.0, K 0.5 ϳ 0.1 M) (Fig. 9) exhibited almost the same profile as that of the total ATPase activity (n H ϳ 2.1, K 0.5 ϳ 0.3 M) (Fig. 7). Therefore, the results suggest that the calcium-dependent profile of the ATPase activity of the A form is also cooperative and is close to that of the activity of the B form. This indicates that the cooperative profile of the A form at 0.25 mM ATP is different from the noncooperative profile of calcium binding of the A form in the absence of ATP (n H ϳ 1, K 0.5 ϭ 2-6 M) (Fig. 5) and that the cooperative calcium-dependent profile (n H ϳ 1.8, K 0.5 ϳ 0.1 M) of the phosphorylation of the B form at 0.25 mM ATP is near that (n H ϳ 2.0, K 0.5 ϳ 0.2 M) of calcium binding of the B form in the absence of ATP (Fig.  5). It has been shown that ATP binding to the catalytic site at 0.1 mM ATP accelerates the catalytic reaction of the enzyme molecules (40,43). Therefore, to examine whether the observed regulatory effect of 0.25 mM ATP results from ATP binding to the regulatory site or to the catalytic site, the calcium dependence of the total ATPase activity and of the total phosphorylation was examined at 0.1 mM ATP. The calcium dependence exhibited almost the same biphasic profile (slopes of ϳ1.9 and ϳ0.9 and K 0.5 ϳ 0.12 M for the ATPase activity and slopes of ϳ1.7 and ϳ0.7 and K 0.5 ϳ 0.24 M for the phosphorylation) (data not shown) as that at 10 M ATP (Figs. 3 and 4); it was distinct from the monophasic profile (Fig. 7) of the ATPase activity at 0.25 mM ATP. Similar to the case at 10 M ATP, 0.1 mM ATP did not seem to affect calcium binding of the enzyme molecules. It is therefore thought that at 0.25 mM ATP, ATP binding to the regulatory site (but not to the catalytic site) changes the calcium binding manner of the A form from noncooperative to cooperative, concurrent with a decrease in the K 0.5 from 2-6 to 0.1-0.3 M, whereas ATP binding does not significantly affect the cooperative binding of the B form. These observations show the existence of two pools of enzyme molecules, the calcium binding of which differs in sensitivity to 0.25 mM ATP, further supporting the model of two different conformations of the molecules (28).
At pH 6.23, the calcium dependence of the ATP-induced total phosphorylation at 0.25 mM ATP exhibited a monophasic profile with n H ϳ 1.3 and K 0.5 ϳ 4 M (Fig. 11). The calcium-dependent profile of the total Ca 2ϩ -ATPase activity at 0.25 mM ATP was also monophasic (n H ϳ 1.5, K 0.5 ϳ 2 M) (data not shown) and was the same as that of the total phosphorylation. As reported previously (29), at the acidic pH of 6.23, it is thought that the A form noncooperatively (n H ϳ 1) binds calcium ions with K 0.5 ϭ 2-6 M, whereas the B form cooperatively (n H ϳ 2) binds calcium with almost the same K 0.5 value (ϳ7 M) as that of the A form ( Fig. 14A; replots of the data in Fig. 1A of Ref. 29). The calcium dependence of the total binding profile is monophasic with n H ϳ 1.3 and K 0.5 ϳ 6 M ( Fig. 14B; replots of the data in Fig. 1C of Ref. 29). The calcium-dependent profile of the total binding at the acidic pH was almost the same as the profiles of the total phosphorylation and the total ATPase activity. Therefore, in contrast to the above-mentioned case at alkaline pH, submillimolar ATP does not seem to affect calcium binding of the two forms at acidic pH.
The solubilized form of the enzyme molecules has also been shown to have a regulatory site (K m Ն 0.1 mM for ATP) in addition to the catalytic site with K 0.5 ϭ 7 M (27). Most of the Hill plots of the calcium dependence of the Ca 2ϩ -ATPase activity of the solubilized molecules at 0.25 mM ATP exhibited a noncooperative profile with n H ϭ 0.8 -0.9 and K 0.5 ϭ 1-2 M (Fig. 13). It was found earlier (30) that in the presence of 10 M ATP, the solubilized molecules are predominantly in E 2 before calcium binding and that the molecules negatively cooperatively (n H ϳ 0.5) bind calcium ions with K 0.5 ϭ 3-5 M, similar to the case in the absence of ATP. In the present study, the calcium-dependent profile of the ATPase activity at 0.1 mM ATP (n H ϭ 0.5-0.6, K 0.5 ϳ 5 M) (data not shown) was found to be close to that at 10 M ATP. These results show that at 0.25 mM ATP, ATP binding to the regulatory site of the solubilized molecules changes the calcium binding manner of the molecules from negatively cooperative to noncooperative, although it does not significantly change the calcium affinity of the molecules (K 0.5 ϭ 3-5 M). It is therefore thought that the regulatory site with submillimolar affinity for ATP belongs to the solubilized molecules, whereas in the SR membrane, it belongs only to the A form as a result of intermolecular interaction of the molecules.
At 5 mM ATP and pH 7.40, the calcium dependence of the total ATPase activity of the two forms exhibited a monophasic profile with n H ϭ 2.0 -2.2 and K 0.5 ϭ 0.04 -0.08 M (Fig. 10). The K 0.5 was lower than that of the ATPase activity at 0.25 mM ATP (K 0.5 ϳ 0.2) (Fig. 7), although both calcium-dependent profiles of the activities at 0.25 and 5 mM ATP were positively cooperative with the same n H value (ϳ2). It is thought that 5 mM ATP further decreases the K 0.5 of the A form, which is already decreased by 0.25 mM ATP, and that it also decreases the K 0.5 of the B form, which is not affected by 0.25 mM ATP, to the same level as the further decreased K 0.5 of the A form. At an acidic pH of 6.23, the calcium-dependent profile of the total ATPase at 5 mM ATP was monophasic with n H ϳ 1.9 and K 0.5 ϭ 0.4 -0.8 M (Fig. 12); the K 0.5 was smaller than the K 0.5 values of the ATPase activity at 0.25 mM ATP, phosphorylation at 0.25 mM ATP, and calcium binding (29) in the absence of ATP (2-6 M). These results at pH 6.23 and 7.40 suggest that the A and B forms equally and positively cooperatively bind two calcium ions at 5 mM ATP, irrespective of pH, although the calcium affinities of the forms at the acidic and alkaline pH values are different. As for the solubilized enzyme molecules, the previous (30) and present (Fig. 13) studies show that the calcium-dependent profile of calcium binding in the presence and absence of 10 M to 0.1 mM ATP is negatively cooperative (n H ϭ 0.5-0.6) with K 0.5 ϭ 3-5 M (30), that the calcium-dependent profile of the Ca 2ϩ -ATPase activity at 0.25 mM ATP is noncooperative (n H ϳ 1) with K 0.5 ϭ 1-2 M, and that the profile of the ATPase activity at 5 mM ATP is positively cooperative (n H ϳ 2) with K 0.5 ϳ 0.1 M. Based on these data, it is thought that 5 mM ATP further changes the noncooperative binding manner of the solubilized enzyme molecules (which have already been converted by 0.25 mM ATP from the negatively cooperative manner without any significant change in FIG. 14. Replots of previous data (29) of calcium binding of the membranous ATPase molecules as a function of calcium concentration in the absence of ATP at pH 6.23 and 0°C. A, total calcium binding to the two forms of ATPase molecules. Calcium binding to the A form (ϫ), which is in an E 1 /E 2 state dependent on pH and which pre-exists in E 2 at this acidic pH, was obtained by subtracting the binding to the B form (---), which pre-exists in E 2 independent of pH, from the total binding (E). Calcium binding to the B form was simulated using parameters of n H ϭ 2.0 and K 0.5 ϭ 5.3 M, obtained on the basis of observations of the calcium-dependent change in fluorescence intensity of the molecules and a maximum binding capacity of half (4.9 nmol/mg of protein) of the capacity of the observed total binding (9.8 nmol/mg of protein) (cf. "Discussion" in Ref. 29). B, Hill plot of the total binding. Y is the ratio of calcium binding at each calcium concentration to the maximum level of binding (9.8 nmol/mg of protein). calcium affinity (from K 0.5 ϭ 3-5 to 1-2 M)) to positively cooperative, concurrent with a 10 -20-fold decrease in the K 0.5 from 1-2 to ϳ0.1 M (30). In contrast to the regulation site with submillimolar affinity for ATP described above, the regulatory site with millimolar affinity for ATP is thought to belong to the enzyme molecules, irrespective of pH and of the aggregation state of the molecules.
The results obtained here confirm the earlier reports by Ogawa (24) and Ogawa and Ebashi (25) that ATP increases the calcium affinity of the Ca 2ϩ -ATPase molecule depending on the ATP concentration. Data on ATP regulation of calcium binding of the membranous and solubilized ATPase molecules are schematically represented in Fig. 15.
The data shown in Fig. 15 suggest the existence of two types of regulatory site of the Ca 2ϩ -ATPase molecules; ATP binding to these sites improves the calcium binding performance of the ATPase molecules at 0.25 and 5 mM ATP. The regulatory sites for calcium binding seem to correspond to the regulatory sites (1, 3, 6, 7) of the enzyme molecules; ATP binding to these sites also accelerates the ATP hydrolysis cycle of the molecules at concentrations of 0.1-5 mM (1,3,27). The following observations have been reported for the molecular basis of the regulatory site for ATP hydrolysis. (i) The regulatory site coexists with the catalytic site in the same enzyme molecule (16 -20).
(ii) This site is the locus of the catalytic site (21)(22)(23). (iii) The catalytic site belongs to half of the molecules (44). Recently, Toyoshima et al. (15) distinctly showed that the enzyme molecule has one nucleotide-binding site. It is therefore probable that the regulatory site is a manifestation of the catalytic site. In a previous study (30) as well as the present one, it was observed that ATP regulation also exists in the presence of C 12 E 8 , which produces the enzyme monomer (45), and that detergent-solubilized molecules exist in E 2 before calcium binding. It is therefore thought that the regulatory site belongs to the monomeric form of the ATPase molecule in an alternate enzyme state of E 2 in the catalytic cycle and that the fundamentals of ATP regulation reside in the monomeric molecule. However, it is difficult to understand the coexistence of two types of regulatory site (as observed here) on one enzyme molecule. The E 2 state may be in equilibrium between two different E 2 states that have high and low affinity for ATP, respectively. Based on previous (29,30) and present ( Figs. 1 and 2) data on the enzyme molecules regarding their enzyme states (E 1 or E 2 ) before calcium binding, calcium affinities, and manners of calcium binding under various conditions of ATP concentrations and pH, the reaction sequence of ATP regulation can be therefore described as follows. Here, for simplicity, the ATPase conformers that negatively cooperatively (n H Ͻ 1), noncooperatively (n H ϳ 1), and positively cooperatively (n H ϳ 2) bind calcium ions are termed a, A, and B, respectively.°E 2 and *E 2 correspond to the two E 2 states with high and low affinity for ATP, respectively. In the membranous molecules at pH 7.40 (Fig. 15A), the A form in E 1 , A(E 1 ), and the B form in°E 2 or *E 2 , B(°E 2 ) or B(*E 2 ), respectively, associate to form A(E 1 )/B(°E 2 ) or A(E 1 )/B(*E 2 ) in the absence of calcium. A(E 1 )/ B(°E 2 ) and A(E 1 )/B(*E 2 ) are in equilibrium, and the equilibrium shifts to the A(E 1 )/B(°E 2 ) side (Scheme I, part A). The association of the two forms creates two stable enzyme populations that pre-exist in E 1 and E 2 , respectively, and that noncooperatively (n H ϳ 1) and positively cooperatively (n H ϳ 2) bind calcium ions, respectively, with K 0.5 ϭ 2-6 and 0.2 M when calcium is added. This can explain the 1:1 ratio of the A and B forms in the membranous enzyme molecules (29) and the disappearance of these forms in the presence of C 12 E 8 (30). At submillimolar ATP (0.25 mM), ATP binds to B(°E 2 ) in A(E 1 )/ B(°E 2 ) before calcium binding. Although ATP binding to the B form does not affect its calcium binding, the ATP binding indirectly converts the calcium binding manner of the counterpart (A form) of the B form from noncooperative (n H ϳ 1) to positively cooperative (n H ϳ 2), concurrent with a decrease in the K 0.5 from 2-6 to 0.1-0.3 M. Calcium binding of the A form becomes almost the same as that of the B form (n H ϳ2, K 0.5 ϳ 0.2 M), although the enzyme states (E 1 and E 2 ) of the converted A form and the B form before calcium binding are maintained in the presence of ATP: A(E 1 )/B(°E 2 ) changes to B(E 1 )/B(°E 2 ). B(E 1 )/B(°E 2 ) is in equilibrium with B(E 1 )/B(*E 2 ) in the absence of calcium, and the equilibrium shifts to the B(E 1 )/B(°E 2 ) side. At millimolar ATP (5 mM), ATP binds to B(*E 2 ) in B(E 1 )/B(*E 2 ) and accelerates the transition of B(*E 2 ) to B(E 1 ), whereas ATP binding coincidentally drives B(E 1 ) in B(E 1 )/B(*E 2 ) to B(E 1 Ca 2 ) when calcium is added. The transition from B(*E 2 ) and B(E 1 ) to B(E 1 ) and B(E 1 Ca 2 ), respectively, results in a further decrease in the K 0.5 to ϳ0.05 M. At pH 6.23 (Fig. 15B), the equilibrium between A(°E 2 )/B(°E 2 ) and A(*E 2 )/ B(*E 2 ) shifts to the A(*E 2 )/B(*E 2 ) side (Scheme I, part B). At millimolar ATP, ATP binds to A(*E 2 ) in A(*E 2 )/B(*E 2 ) before calcium binding, converts it to B(*E 2 ), and sequentially accelerates the transition of B(*E 2 ) to B(E 1 ), coinciding with the driving of B(*E 2 ) in A(*E 2 )/B(*E 2 ) (by ATP binding to B(*E 2 )) to B(E 1 Ca 2 ) via B(E 1 ) after the addition of calcium. These transi- The scheme below each panel shows the enzyme state (E 1 or E 2 ) of the ATPase molecules before and after the addition of calcium and the association state of the molecules under the different conditions of ATP concentrations and pH (see "Discussion" for details). For simplicity, the ATPase conformers that negatively cooperatively (n H Ͻ 1), noncooperatively (n H ϳ 1), and positively cooperatively (n H ϳ 2) bind calcium ions are termed a, A, and B, respectively. tions cause the forms to bind calcium ions positively cooperatively (n H ϳ 2) with K 0.5 ϭ 0.4 -0.8 M, which is decreased from 2-7 M. In the solubilized molecules at pH 7.40 (Fig. 15C), the a form is in equilibrium between°E 2 and *E 2 ; the equilibrium shifts to the°E 2 side before calcium binding (Scheme I, part C); and the a form negatively cooperatively (n H Ͻ 1) binds calcium ions. At submillimolar ATP, ATP binds to the a form in°E 2 , a(°E 2 ), before calcium binding and converts it to A(°E 2 ); in the presence of calcium, the A form noncooperatively (n H ϳ 1) binds calcium ions with almost the same K 0.5 (1-2 M) as that of a form in the absence of ATP (3-5 M). A(°E 2 ) is in equilibrium with A(*E 2 ) before calcium binding, and the equilibrium shifts to the A(°E 2 ) side. At millimolar ATP, ATP that is bound to A(*E 2 ) converts it to B(*E 2 ) and accelerates the transition of B(*E 2 ) to B(E 1 Ca 2 ) via B(E 1 ) with the addition of calcium; millimolar ATP further converts the binding manner of the molecules from noncooperative (n H ϳ 1) to positively cooperative (n H ϳ 2), accompanying a decrease in the K 0.5 (1-2 M) to ϳ0.1 M. As mentioned above, ATP is presumed to work as a kinetic and conformational effector in regulating calcium binding. As shown in Scheme I (parts A and B), the present data suggest the existence of heterologous conformational interactions of the membranous Ca 2ϩ -ATPase conformers in their ATP regulation reactions. Hobbs et al. (46) found such heterologous conformational interactions in the catalytic cycle in the Na ϩ ,K ϩ -ATPase molecules.
In the monomeric pathway model described above, the ATPase molecules in E 1 and E 2 , which are the alternate enzyme states in the catalytic cycle (1)(2)(3)5), are dealt with in the discussion of ATP regulation, and the putative regulatory site is allotted to the monomeric molecule in E 2 . Yokoyama et al. (47) have recently suggested the existence, in the Na ϩ ,K ϩ -ATPase, of ATP binding to the ␣-subunit, which is not involved in the catalytic phosphorylation-dephosphorylation reaction, in addition to the binding that is involved in the catalytic reaction. It has been observed in the Ca 2ϩ -ATPase that the ATPase molecules phosphorylated with ATP or P i are only half of the total enzyme molecules in the SR membrane (44); it is not known whether half of the total molecules are phosphorylated with ATP or P i . However, based on the above-mentioned analogy of the Na ϩ ,K ϩ -ATPase molecules, the Ca 2ϩ -ATPase molecules might also be composed of catalytic and non-catalytic molecules. ATP sites for the phosphorylation and ATP regulation of calcium binding might possibly belong to the two different Ca 2ϩ -ATPase molecules (catalytic and regulatory molecules) that are involved and not involved in the catalytic reaction, respectively. The ATP regulation pathway can there-fore also be described based on the following oligomeric model. The two types of regulatory site with submillimolar and millimolar affinities for ATP, respectively, reside in the two different states of the regulatory molecule, which are in equilibrium depending on pH, as discussed above. The regulatory molecule is adjacent to the catalytic molecule in an oligomer of the ATPase molecules. ATP binding to the regulatory subunit indirectly improves the calcium binding performance of the catalytic subunit. Similar to the case in the monomeric pathway model, the catalytic membranous molecules associate to form a stable pair of molecules in E 1 and/or E 2 in the reaction sequence of ATP regulation. It is therefore predicted that a trimer of two catalytic subunits (which are in E 1 and/or E 2 ) and one regulatory subunit (which is in a high or low affinity state for ATP) is formed. In the regulation model, a specific oligomerization of the catalytic and regulatory subunits is required to produce ATP regulation of the catalytic subunit. However, ATP regulation at submillimolar and millimolar ATP was also observed in the detergent-solubilized enzyme molecules (Fig.  15C). Such a specific intermolecular interaction of the molecules does not seem to occur in the solubilized molecules because they have been shown to be in a monomeric state (45). The oligomeric pathway model is therefore unlikely to be suitable to explain the results obtained. However, the data (summarized in Fig. 15) show that at pH 7.40, the regulatory effect of submillimolar ATP was relatively lowered by the solubilization of the membranous enzyme molecules with detergent. Submillimolar ATP changed the calcium binding manner of the membranous A form in the absence of ATP from noncooperative to positively cooperative, whereas it also changed the manner of the solubilized molecules in the absence of ATP from negatively cooperative to noncooperative. As for the affinity of the enzyme molecules for calcium, ATP decreased the K 0.5 of the membranous A form in the absence of ATP from a micromolar (2-6 M) to a submicromolar (0.1-0.3 M) level, although it scarcely decreased the K 0.5 of the solubilized molecules in the absence of ATP (from 3-5 to 1-2 M). The cooperative binding of the membranous B form (n H ϳ 2, K 0.5 ϭ 0.2 M) was not affected by ATP. These observations regarding the membranous A form and the solubilized molecules suggest that the expression of the regulatory effect of submillimolar ATP is related to the intermolecular interaction of the enzyme molecules; the solubilized molecules might interact with each other in the presence of the detergent, although the interaction is much weakened by the solubilization. In the Na ϩ ,K ϩ -ATPase, which is composed of an ␣-subunit (catalytic) and a ␤-subunit (non-catalytic), even after the solubilization of the ATPase sub-SCHEME I units with detergent, ␣␤-protomers of the subunits have been observed to associate to form a diprotomer ((␣␤) 2 ) and a higher oligomer accompanying transition of the enzyme state of the Na ϩ ,K ϩ -ATPase from E 1 to E 2 (48). Re-examination of the possibility of the interaction of the solubilized Ca 2ϩ -ATPase molecules on the basis of the data obtained here is worthwhile.
For the sequence of the catalytic reaction of the Ca 2ϩ -ATPase molecules, Froehlich and Taylor (49) and Ikemoto et al. (50) have proposed an alternating or flip-flop model of the ATPase molecules in an enzyme oligomer (dimer or 2ϫ n-mer); half of the enzyme molecules are one step ahead of the other in carrying out the same sequential steps in the reaction of ATP hydrolysis. We previously proposed a model in which the A and B forms of the enzyme molecules independently carry out their reaction sequences of acetyl phosphate hydrolysis without change in their calcium binding performances (51). In the monomeric pathway model of ATP regulation that is described above, the data are discussed according to the alternating model of the two types of ATPase conformer, one of which converts to the other in the presence of ATP at concentrations higher than those required for saturation of the catalytic site. However, further study is required to understand the whole sequence of the ATP hydrolysis reaction of the ATPase molecules.