©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Negative or Positive Cooperation in Calcium Binding to Detergent-solubilized ATPase of the Sarcoplasmic Reticulum
ITS MODULATION BY A HIGH CONCENTRATION OF ATP (*)

(Received for publication, March 15, 1995; and in revised form, May 15, 1995)

Jun Nakamura (§) , Genichi Tajima

From the Biological Institute, Faculty of Science, Tohoku University, Aoba-yama, Aoba-ku, Sendai, Miyagi 980-77, Japan

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Two different conformations of chemically equivalent Ca-ATPase molecules in the sarcoplasmic reticulum have been shown to non- and positive cooperatively bind two calcium ions, respectively (Nakamura, J.(1994) J. Biol. Chem. 269, 30822-30827). At pH 7.40, these ATPase molecules split into E (high affinity state for calcium) and E (low affinity state for calcium), respectively, before calcium binding. At this pH, calcium binding to the monomeric ATPase, solubilized with dodecyloctaethylenglycol monoether, was studied by examining Ca binding to the ATPase and calcium dependences of its phosphorylation, fluorescence intensity, ATP-hydrolysis at a low (5 µM) concentration of ATP, and acetyl phosphate hydrolysis. The results suggest that the solubilized ATPase molecules predominantly preexist in E and negative cooperatively (the Hill value (n) = 0.5-0.6) bind 2 mol of calcium/mol of the ATPase with an apparent calcium affinity (K) of 3-5 µM. The nonequivalences of calcium bindings at the membranous ATPase molecules seem to result from the intermolecular interaction of the molecules. A high concentration (5 mM) of ATP modulated the binding manner so that it became positively cooperative (n 2) and increased the K to 0.1 µM.


INTRODUCTION

Sarcoplasmic reticulum Ca-ATPase is a calcium pump transporting 2 mol of calcium across the sarcoplasmic reticulum membrane by hydrolytic coupling with 1 mol of ATP(1, 2, 3) . Structural studies suggest that the Ca-ATPase molecules are in close contact forming oligomeric units in the membrane(4, 5, 6, 7, 8) . The monomeric enzyme, however, has been shown to retain the same reaction cycle of ATP hydrolysis as that of the membrane form and to be a functional unit of the calcium pump(9, 10, 11) . On the other hand, we recently found two different conformations of chemically equivalent ATPase molecules in the membrane(12, 13) . One of them is in pH-dependent equilibrium between E (high affinity state for calcium) and E (low affinity state for calcium) before calcium binding and noncooperatively binds two calcium ions with a pH-independent affinity for the calcium ions. The other molecule is predominantly in E independent of pH before calcium binding and positive cooperatively binds two calcium ions with a pH-dependent affinity for the calcium ions. It is likely that intermolecular interaction of the chemically equivalent ATPase molecules produces these nonequivalences of the ATPase molecules in the membrane. To verify the existence of such intermolecular interaction, it is necessary to show cancellation of the nonequivalences of the enzyme molecules in the membrane by monomerization of the molecules. Calcium binding at the monomeric enzyme has been studied for the most part by using the enzyme that is solubilized with nonionic detergent, dodecyloctaethylene glycol monoether (CE).()This detergent has been found to be especially suitable for the active preparation of the soluble enzyme(14, 15, 16, 17) . Based on observations of calcium dependence of ATP hydrolysis by the detergent-solubilized enzyme, Met al.(16) and Murphy et al.(18) reported that this enzyme positive cooperatively binds calcium with the same Hill value (n 2) as that of the enzyme in the membrane. Such cooperativity of the enzyme has also been observed for calcium-induced change in fluorescence intensity of the enzyme protein(19) . Verjovski-Almeida and Silva(20) , however, observed cancellation of the cooperativity by solubilization of the membranous enzyme. On the other hand, studies of calcium binding at the detergent-solubilized ATPase have shown the following difficulties. (i) The ATPase activity of the solubilized enzyme is unstable in the absence of calcium and is rapidly inactivated(16) . (ii) The fluorescence intensity of the enzyme protein is very sensitive to the inactivation of the enzyme(19) . (iii) The monomeric enzyme slowly aggregates for several hours(21, 22) . In the present study, calcium binding to the solubilized enzyme was reexamined by minimizing the degree of such difficulties. To elucidate calcium binding to the solubilized enzyme, radioactive calcium binding to the enzyme and calcium dependences of its phosphorylation, fluorescence intensity, ATP hydrolysis at a low concentration (5 µM) of ATP, and acetyl phosphate hydrolysis were studied. To compare the equilibrium of the solubilized enzyme between E and E and calcium binding of the enzyme with those of the membranous enzyme, assays were carried out at pH 7.40 where the two types of membranous enzyme molecules, described above, were shown to split into E and E, respectively, before calcium binding and to apparently rapidly and slowly bind calcium in non- and positive cooperative manners, respectively. The results show that the nonequivalences of the membranous enzyme are cancelled by solubilization of the enzyme. The solubilized enzyme molecules predominantly preexist in E and negatively cooperate in calcium binding. The modulation of the binding manner was observed at a high concentration (5 mM) of ATP.


EXPERIMENTAL PROCEDURES

Materials

Procedures for isolation of the sarcoplasmic reticulum from skeletal muscle of rabbit were the same as those described in a previous article(23) . Membranous Ca-ATPase was purified from the sarcoplasmic reticulum by washing the sarcoplasmic reticulum with sodium-deoxycholate in the same manner as reported previously (24) except for a decrease in the ratio of sodium deoxycholate to the reticulum protein from 1:4 to 1:5. The protein concentration was determined by the Biuret method (25) with the use of bovine serum albumin as a standard.

In the solubilization of the membranous enzyme with CE, 5-20% of the ATPase activity/h was observed to be lost. This loss occurred when the enzyme (2 mg of protein/ml) was solubilized with 4 mg/ml CE, according to the widely practiced method of Met al.(22) , and the insoluble residue was removed by centrifugation. At various times after the solubilization, the ATPase activity was assayed at a low concentration (0.01 mg of protein/ml) of the enzyme. However, when it was assayed at a higher concentration (0.06 mg of protein/ml) of the enzyme, no significant inactivation of the enzyme was observed for 4 h. A change in the solubilized enzyme seems to gradually proceed, this change being detected as an inactivation of the ATPase activity by higher dilution of the enzyme concentration. To minimize the degree of such change after the solubilization, the membranous ATPase was directly solubilized in each of the assay mediums with various ratios of the detergent and the enzyme protein, which are known to monomerize the enzyme molecules (15, 16) (see ``Assays'' for details). The ratios (from 10:1 to 50:1) of the detergent to the enzyme protein that were used were much higher than that (2:1) used in the method of Met al.(22) . The enzyme, therefore, seems to be more rapidly solubilized by the present method than by the method of Met al.(22) . The degrees of solubilization under the various conditions were estimated to be 91-99% from the ratio of the fluorescence intensity of the solubilized-enzyme medium before and after centrifugation of the medium at 78,000 g for 1 h. The ATPase activity of the solubilized ATPase was 6.0-7.5 µmol of P/mg of protein/min, obtained in a solution consisting of 5 mM ATP, 5 mM MgCl, 0.12 M KCl, and 10 µM CaCl at pH 7.40 and 25 °C. The maximum level of the phosphoenzyme was 4.4-4.6 nmol/mg of protein obtained in 0.46 mM ATP, 5 mM MgCl, 0.12 M KCl, and 10 mM CaCl at pH 7.40 and 0 °C. Radioactive calcium binding to the solubilized ATPase was assayed according to the method of Andersen et al.(11) . The membranous enzyme (3.0 mg of protein/ml) was solubilized in 20 mM PIPES (pH 7.40) buffer solution containing 24 mg/ml CE, 50 µMCaCl, 0.12 M KCl, and 5 mM MgCl. A Sephadex G-50 (1 20 cm) was used for measurement of calcium binding at 25 °C. The obtained values of the bound calcium were 7.2-9.6 nmol/mg of protein. The detergent, CE, was obtained from Nikko Chemical Co. (Tokyo, Japan). Pyruvate kinase (500 IU/mg of protein) and lactate dehydrogenase (197 IU/mg of protein) were obtained from Sigma and Wako Chemical Co. (Tokyo, Japan), respectively. The other reagents were of analytical grade.

Assays

Phosphoenzyme

The phosphorylation reaction was manually carried out at 0 °C. The enzyme (0.05 mg of protein/ml) was preincubated in a medium containing 40 mM HEPES (pH 7.40), 0.2 mM EGTA, 0.12 M KCl, 5 mM MgCl, 2.0 mg of CE/ml, and 37 µM [P]ATP by agitating the medium using a magnetic stirrer at the maximum speed (1,500 rpm). At 2 min after the preincubation, the reaction was initiated by the addition of 50 µl of 5 mM CaCl using a micropipette with a volume of 200 µl (Pipetman P-200, Gilson, Middleton, WI) within 0.2 s. The volume of the reaction medium was 1 ml. The reaction was terminated within 0.2 s by the addition of 0.5 ml of 30% trichloroacetic acid containing 1 mM P using a syringe with a volume of 3 ml (B-2, Nipro, Osaka, Japan) and having a needle of = 0.75 mm. In the reaction at more than 1 s, the trichloroacetic acid was added at the indicated times after the addition of the calcium. The reaction time within 1 s was not controlled. The trichloroacetic acid was arbitrarily added immediately after the addition of the CaCl, and the time interval between the additions was read. The ``dead'' time of the experiment was about 0.2 s, and the error of the read time was ± 0.2 s, but the mixing time of the enzyme solution and the starting/stopping solution is not known. Because of this, such a mixing technique appears to be adequate to distinguish that there are two rates of phosphorylation (see Fig. 1), when the membranous enzyme is used. However, this technique cannot give accurate measurements of the half-time of the reactions in this speed range. The P-labeled enzyme, which was precipitated by the trichloroacetic acid, was washed 3 times as described in a previous article(13) .


Figure 1: Kinetics of calcium-induced phosphorylation before () and after () solubilization of the ATPase. The enzyme (0.05 mg of protein/ml) was preincubated in a medium containing 40 mM HEPES (pH 7.40), 0.2 mM EGTA, 0.12 M KCl, 5 mM MgCl, and 37 () or 100 () µM [P]ATP in the presence () and absence () of 2.0 mg of CE/ml. The reaction was initiated by the addition of 0.25 mM CaCl. After the addition, the enzyme was incubated in 50.3 µM [Ca]. A, time course of the phosphorylation. B, semilogarithmic plots of EP at steady state - EP versus time after initiation of the reaction.



Intrinsic Fluorescence Change

The enzyme (0.05 mg protein/ml) was preincubated in a medium containing 60 mM PIPES (pH 7.40), 0.12 M KCl, 5 mM MgCl, 50 µM CaCl, and 2 mg/ml CE for 5 min at 0 and 25 °C. Different amounts of EGTA or CaCl were added to give the final free concentration, as indicated, within 10 min. At 0.05-10 µM [Ca], the assay medium contained 1 mM ADP to minimize inactivation of the ATPase activity of the solublized ATPase at a low concentration of calcium, as reported by Met al.(16). At 10-50 µM [Ca], the experiments were carried out in the presence and absence of ADP. At 50-1,000 µM [Ca], ADP was not added. When AMP-PNP was added to the medium, ADP was omitted. The association constant for Ca-EGTA was taken as 1.233 10M(26) , unless otherwise indicated. Calcium-induced change in fluorescence intensity of the enzyme was measured by using the wavelengths 285 and 333 nm for excitation and emission, respectively, as reported in a previous article(13) .

Enzymatic Activities

Enzymatic assays were performed within 5 min because after the preincubation of the solubilized enzyme in the absence of added calcium and presence of 0.1 mM EGTA for 5 min, about 90% of the ATPase activity that existed before the preincubation was retained. ATP hydrolysis of the solubilized enzyme at a low ATP concentration was measured spectrophotometrically at 340 nm by the linked enzyme method (27) in 60 mM PIPES buffer solution (pH 7.40) containing 0.01 mg of protein/ml of the enzyme, 0.5 mg/ml CE, 0.12 M KCl, 5 mM MgCl, 5 µM ATP, 1 mM phosphoenolpyruvate, 0.15 mM NADH, 50 IU/ml pyruvate kinase, 50 IU/ml lactate dehydrogenase, and 0.08-1,000 µM [Ca] at 25 °C. Before the addition of ATP to initiate the reaction, the enzyme was preincubated in the assay medium for 2 min. The reaction time was 3 min. At a high ATP concentration of 5 mM, the enzyme (0.05 mg of protein/ml) was preincubated in a medium containing 60 mM PIPES (pH 7.40), 0.12 M KCl, 5 mM MgCl, 0.15-6.5 µM CaCl, and 2.0 mg/ml CE for 2 min at 25 °C. After the preincubation, the reaction was initiated by the addition of ATP. The reaction time was 1.5 min. ATP hydrolysis was measured by determining the amount of phosphate liberated from ATP at 750 nm of the blue complex formed with molybdate and ferrous sulfate(28) . Acetyl phosphate hydrolysis of the enzyme was performed in 60 mM PIPES buffer solution (pH 7.40) containing 0.5 mg of protein/ml of the enzyme, 5.0 mg/ml CE, 0.12 M KCl, 5 mM MgCl, 5 mM acetyl phosphate, and 0.05-1,000 µM [Ca] at 25 °C. The enzyme was preincubated in the medium for 2 min before initiation of the reaction by the addition of acetyl phosphate. The reaction time was 3 min. The amount of remaining acetyl phosphate was determined according to the method of Lipmann and Tuttle(29) .


RESULTS AND DISCUSSION

Two populations of Ca-ATPase in the sarcoplasmic reticulum membrane that are chemically equivalent have been shown to be in pH-dependent equilibrium between E and E and predominantly independent of pH in E, respectively, before calcium binding at 0 °C(12, 13) ; they apparently slowly/rapidly and slowly bind two calcium ions, respectively, dependent on pH and independent of pH, and they noncooperatively and positive cooperatively participate in the calcium binding, respectively. At pH 7.40, the two populations of Ca-ATPase in the membrane have also been shown to split into E and E, respectively, and apparently rapidly and slowly bind calcium, respectively(12, 13) . In the present study, to compare calcium binding to the detergent-solubilized, monomeric enzyme with that to the membranous enzyme, experiments were performed at this pH.

As shown in Fig. 1, to estimate the kinetic calcium binding to the solubilized enzyme, calcium-induced phosphorylation of the enzyme was examined at 0 °C. The reaction was initiated by the addition of calcium to the enzyme, which was preincubated with ATP. The enzyme was monophasically phosphorylated at a rate of half-time (t) 1 s. No rapid phosphorylation within about 0.2 s, such as that found in one of the two membranous enzymes that was in E before calcium binding (cf. (13) ), was observed. It is thought that all of the solubilized enzyme molecules are almost entirely in E before calcium binding and slowly bind calcium because of the slow transition of the enzyme from E to E after the addition of calcium. Fig. 2shows calcium-induced change in fluorescence intensity of the enzyme protein as a function of calcium concentration. To compare the calcium-dependent change with that of the enzymatic activities, which will be mentioned below, experiments on the fluorescence change were performed at 25 °C. One millimolar ADP was added to the assay medium at less than 50 µM [Ca]. ADP has been found to stabilize the solubilized enzyme in the absence of calcium(16) . The ADP did not affect calcium dependence of the calcium-induced fluorescence change in the membranous enzyme (data not shown). The calcium dependence exhibited a profile with a Hill value of less than 1 (0.5-0.6) and an apparent calcium affinity (K) of about 5 µM. The profile that was observed at 25 °C was entirely the same as that at 0 °C (data not shown). The solubilized enzyme seems to negatively cooperate in calcium binding independent of temperature. The calcium-dependent profiles (n 0.6 and K 3 µM) of the ATP hydrolysis at a low concentration (5 µM) of ATP (Fig. 3) and the acetyl phosphate hydrolysis (Fig. 4) were in good agreement with that of the fluorescence change. Acetyl phosphate has been shown to serve as a substrate for the enzyme(30) . This result supports the existence of negative cooperation in calcium binding of the solubilized enzyme, which was discussed above. At 50 µM [Ca], the amount of calcium bound to the solubilized enzyme was 7.2-9.6 nmol/mg of protein. At this calcium concentration, 80-90% of calcium binding capacity of the enzyme is estimated to be filled with calcium based on the calcium-dependent profiles of the fluorescence intensity, the ATP hydrolysis, and the acetyl phosphate hydrolysis, shown in Figs. 2-4, respectively. The maximum level of the phosphorylated enzyme was 4.4-4.6 nmol/mg of protein. Thus, it is thought that the solubilized enzyme binds two calcium ions/mol of the enzyme, which is required for phosphorylation of the enzyme, as with the enzyme in the membrane as reported by Andersen et al.(11) . The results that were obtained here, therefore, suggest that the solubilized enzyme negative cooperatively binds two calcium ions, implying calcium binding at a first site of the enzyme followed by a marked decrease in calcium affinity at a second site and binding to that site. The monomeric ATPase molecule itself seems to be in an inferior state as a calcium pump, compared with the two types of ATPase molecules in the membrane which noncooperatively and positive cooperatively bind two calcium ions, respectively(13) . The results also show that the nonequivalences of the membranous enzyme molecules for equilibrium between E and E and for cooperativity of the binding are cancelled by solubilization of the enzyme molecules. It is thought that intermolecular interaction of the ATPase molecules forms two different conformations of the molecules, either of which is distinct from that of the monomeric molecule. In other words, it is likely that the monomeric molecule is in an ``immature'' or ``undifferentiated'' state of functional conformation and matures or differentiates into two different functional conformations of the oligomer in the membrane. To confirm this hypothesis, we are currently studying calcium binding at the membranous, monomeric molecule, which is reconstituted with excess phospholipid.


Figure 2: Fluorescence intensity change of the ATPase protein as a function of calcium concentration. A, calcium titration curve of the calcium-induced fluorescence change. The enzyme (0.05 mg of protein/ml) was incubated in a medium containing 60 mM PIPES (pH 7.40), 0.12 M KCl, 5 mM MgCl, 1 mM ADP () or 1 mM AMP-PNP () or absence of both (), 50 µM CaCl, and 2 mg/ml CE. Different amounts of EGTA or CaCl were added to give the final calcium concentrations shown. The decrease and increase of the fluorescence intensity upon the addition of EGTA and CaCl, respectively, were recorded. The observed maximum level of the total change (F) was 2-3% of the total fluorescence. B, Hill plots of the calcium titration curve. Y is the ratio of the intensity change at each calcium concentration to the maximum level of the change.




Figure 3: ATPase activity as a function of calcium concentration at a low concentration (5 µM) of ATP. The ATPase activity was measured spectrophotometrically by the linked-enzyme method(27) , as described under ``Experimental Procedures.'' The reaction medium contained 55 mM ammonium sulfate, which was present in the suspension of pyruvate kinase in ammonium sulfate. The association constant of 1.106 10M for Ca-EGTA was used. A, calcium dependence of the ATPase activity; B, Hill plots of the activity. Y is the ratio of the activity at each calcium concentration to the maximum level (2.5 µmol of ADP/mg of protein/min) of the activity.




Figure 4: The activity of acetylphosphate (ACP) hydrolysis as a function of calcium concentration. A, calcium dependence of the ACPase activity; B, Hill plots of the activity. Y is the ratio of the activity at each calcium concentration to the maximum level (0.6 µmol of ACP/mg of protein/ml) of the activity.



In contrast with the observations of the negative cooperative profile of calcium sensitivity of the solubilized enzyme mentioned above, at a high concentration (2-5 mM) of ATP, calcium dependence of ATP hydrolysis activity of the solubilized enzyme has been shown to have a positive cooperative profile with n 2(16, 18) . In Fig. 5, ATP hydrolysis as a function of calcium concentration was reexamined at 5 mM ATP. As in earlier reports(16, 18) , the calcium-dependent profile of the activity was positively cooperative with n 2.2. K was about 0.1 µM, which was about 30-50 times higher than that (3-5 µM) at the low ATP. This suggests that a high level of ATP modifies the conformation of the solubilized, monomeric enzyme molecule, resulting in the modulation of the binding manner of the molecule from negative cooperative to positive cooperative and in the increases in the affinity for calcium, i.e. the monomeric molecule is able to attain a state suitable to act as a calcium pump with the help of the ATP. Based on the observations of no modulation by low ATP, which was shown in Fig. 3, the modulation by a high level of ATP seems to be produced through ATP binding at the ``regulatory'' site of the enzyme rather than the binding at the catalytic site. The regulatory site is thought to be a site at which ATP binding accelerates the turnover of the catalytic activity of the membranous enzyme with lower affinity (K 5 mM) (31, 32, 33, 34, 35) for ATP than that (K 10 µM) (31, 36) at the catalytic site. Also in the solubilized enzyme, catalytic sites with K = 7 µM and a regulatory site with K > 0.1 mM have been found(16) . As mentioned in the above paragraph, as with the low concentration of ATP, acetyl phosphate did not modulate the calcium sensitivity of the enzyme (Fig. 4). Acetyl phosphate has not been shown to have a regulatory effect on the turnover of catalytic activity(37) . Therefore, this result supports the above discussion of the modulation by a high level of ATP. ATP-regulation has been shown to consist of acceleration of two steps in the catalytic reaction. One is the formation and the decomposition of E-P (the phosphorylated enzyme in E) from and to E and P(33) , and the other is the conversion of the enzyme from E to E(38) . AMP-PNP, a nonhydrolyzable ATP analogue, has been found to accelerate the former step(33) . However, it has not been found to accelerate the total catalytic reaction(31) , implying that the analogue has no effect on the E-E conversion. To determine which of the ATP-regulated steps is related to ATP-modulation in calcium binding, the effect of AMP-PNP on calcium sensitivity of the fluorescence intensity was examined (Fig. 2). No effect on the sensitivity was observed. It is probable that the observed modulation in calcium binding results from a conformational change of the enzyme molecule accompanying that in the ATP-regulated conversion of the molecule from E to E.


Figure 5: ATPase activity as a function of calcium concentration at a high concentration (5 mM) of ATP. A, calcium dependence of the ATPase activity; B, Hill plots of the activity. Y is the ratio of the activity at each calcium concentration to the maximum level (6.0 µmol of P/mg of protein/min) of the activity.




FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed.

The abbreviations used are: CE, dodecyloctaethylene glycol monoether; PIPES, piperazine-N,N`-bis(2-ethanesulfonic acid); AMP-PNP, adenosine 5`-(,-imino)triphosphate; EP, phosphorylated Ca-ATPase.


ACKNOWLEDGEMENTS

We thank Dr. Hiroo Fukuda of the Biological Institute, Faculty of Science, Tohoku University for access to a fluorescence spectrophotometer.


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