(Received for publication, December 23, 1996, and in revised form, May 6, 1997)
From the Biological Institute, Graduate School of Science, Tohoku University, Aoba-yama, Aoba-ku, Sendai, Miyagi 980-77, Japan
The sarcoplasmic reticulum Ca2+-ATPase molecules have been shown to exist in two conformations (A and B) that result from intermolecular interaction of ATPase molecules (Nakamura, J., and Tajima, G. (1995) J. Biol. Chem. 270, 17350-17354). The A form binds two calcium ions noncooperatively, whereas the B form binds the calcium ions cooperatively. Here, we examined the independence of these two forms in the calcium-activated hydrolysis of acetyl phosphate (AcP) under asynchronous and synchronous conditions of their E1-E2 transitions at 0-5 and 25 °C. Irrespective of their synchronism and temperature, the two forms hydrolyzed AcP due to calcium that was bound to each of the forms, indicating the independence of the two forms in hydrolyzing AcP. Taking into account the monomer-dimer transition of the ATPase molecules on the sarcoplasmic reticulum membrane accompanying E1-E2 transition of the molecules (Dux, L., Taylor, K. A., Ting-Beall, H. P., and Martonosi, A. (1985) J. Biol. Chem. 260, 11730-11743), the two types of molecules seem to independently carry out such monomer-dimer transition of the same type of molecules. Two pairs, each consisting of the same type of molecules, are suggested to be the structural unit of the ATPase molecules.
Ca2+-ATPase is an integral membrane protein (110 kDa) of the SR1 and is a calcium pump protein that transports 2 mol of calcium ion across the SR membrane by hydrolytic coupling with 1 mol of ATP (1-3). Calcium binding to the two calcium transport sites of the ATPase, being required for phosphorylation of ATPase, is a crucial step in driving the calcium pump. The formation of 1 mol of phosphorylated ATPase and its isomerization are coincident with this calcium transport. Relationships between the structure and function of the calcium-transporting ATPase are being actively investigated (4, 5). Furthermore, from observations of the calcium sensitivity of intrinsic fluorescence intensity in the ATPase protein molecules and of calcium binding to the molecules, the ATPase molecules have been shown to exist in two conformations that have the same amino acid sequence (6). Here, for the sake of simplicity, we name the two conformations A form and B form, respectively. The two forms independently bind two calcium ions in a different manner (7): the A form noncooperatively binds two calcium ions with a pH-independent affinity for the ions, whereas the B form cooperatively binds the calcium ions with a pH-dependent affinity for the ions. Recently, however, these nonequivalences of the two forms have been shown to be canceled by solubilization of the ATPase molecules in the SR membrane with detergent, suggesting that intermolecular interaction of the ATPase molecules produces such nonequivalences (8). We therefore further examined the independence of these forms in transporting calcium ions by coupling with hydrolysis of a substrate as an energy source. As to the intermolecular interaction of the ATPase molecules in the SR, Dux et al. (9) found "monomer ribbon-dimer ribbon" transition in the array of molecules on the SR membrane, which coincides with E1 (high affinity state of the molecule for calcium)-E2 (low affinity state of the molecule for calcium) transition. The transition of the molecules from E2 into E1 has been found to be the slowest step of the transport sequence in the absence of a higher concentration of ATP than that required for phosphorylation of the ATPase molecule (10). Moreover, it has been shown that the A form is in pH-dependent equilibrium between E1 and E2, before calcium binding, whereas the B form pre-exists predominantly in E2 independent of pH (7). The transition rates of the two forms from E2 to E1 were previously found to be the same, independent of temperature (7, 21). Therefore, in this study, the enzyme states of the two forms before calcium binding were set at E1 and E2 and at E2 only, respectively, by setting the pH at specified values, to drive their hydrolysis reaction asynchronously and synchronously, respectively. Moreover, the experimental temperature was varied to carry out the experiments under different conditions of stability of the intermolecular interaction of the ATPase molecules. We then examined whether the two forms independently hydrolyze a substrate due to calcium that is bound to each of the forms; the total activity of substrate hydrolysis by the two forms was compared with calcium binding to the forms in the absence of the substrate by varying the calcium concentration. For direct comparison of the activity and the binding, it is necessary to use a substrate that supports calcium transport and that does not have any regulatory effect on the calcium binding and the enzymatic reaction. AcP has been shown to support calcium transport that couples with its hydrolysis (11), like ATP. Unlike ATP, however, it has not been shown to have a regulatory effect on E1-E2 transition in its hydrolysis cycle (12, 13). However, its effect on calcium binding is not known. In this study, we first clarified that AcP had no effect on calcium binding and then used it as the substrate. The results show that the two forms hydrolyze AcP due to calcium that is bound to each of the forms, irrespective of temperature and synchronism of their hydrolysis cycles, indicating the independence of the two forms in hydrolyzing AcP.
Based on the monomer-dimer transition of the ATPase molecules on the SR membrane (9), the structural unit of the molecules is discussed.
Materials
The procedures for isolation of the SR from the skeletal muscle
of rabbit were the same as those described in a previous paper (14).
Membranous Ca2+-ATPase was purified from the SR by washing
the SR with sodium deoxycholate in the same manner as reported
previously (15) in a 1:5 ratio of sodium deoxycholate to the reticulum
protein. The maximum calcium binding capacity of the ATPase preparation was 9.0-10.7 nmol/mg of protein as estimated by Scatchard analysis of
the calcium titration curve of the calcium-binding sites in the
preparation, and the maximum level of the preparation that was
phosphorylated with ATP was 4.2-4.6 nmol/mg of protein obtained in 0.1 mM ATP, 0.1 mM CaCl2, and 5 mM MgCl2 at pH 7.0. The purified ATPase
preparation exhibited the same characteristics of calcium binding as
those of the ATPase preparation (7) (see Figs. 1 and 2) that was
purified by solubilization of the sarcoplasmic reticulum with sodium
deoxycholate and by reformation of the membranous ATPase by removal of
the detergent (data not shown).
Assays
AcP HydrolysisThe total AcP hydrolysis activity of the ATPase was carried out in 20 mM PIPES buffer solution (pH 6.23 or 7.40) containing 0.1 or 0.5 mg of ATPase protein/ml, 0.12 M KCl, 5 mM MgCl2, 5 mM AcP, and 0.01-1000 µM Ca2+ at 5 or 25 °C. To obtain reproducible values of the activities at these pH values and temperatures, the reaction times in which the reactions were linear and the protein concentrations of the ATPase were set at 3 (at pH 7.40) or 5 (at pH 6.23) h and 0.5 mg of protein/ml at 5 °C and at 30 (at pH 7.40) or 60 (at pH 6.23) min and 0.1 mg of protein/ml at 25 °C, respectively, based on the following observations. (i) The AcPase activities at pH 6.23 were about half of those at pH 7.40. (ii) The activities at 5 °C were one-thirtieth to one-fortieth of those at 25 °C. (iii) No inactivation of the activity was observed when the ATPase was preincubated in the absence of AcP for 2 and 6 h at 25 and 5 °C, respectively, which is different from the case (8) of the detergent-solubilized ATPase, which was gradually inactivated during the preincubation. The calcium-independent activity of AcP hydrolysis was determined under identical conditions, except that 5 mM EGTA was added without the addition of calcium. The calcium-dependent activity of AcP hydrolysis (Ca2+-AcPase activity) was obtained by subtracting the calcium-independent activity from the total activity. The amount of remaining AcP was determined according to the method of Lipmann and Tuttle (16). Before reading the absorbance at 505 nm of the brown complex formed with iron(III), the ATPase protein suspended in the medium for the assay of AcP was removed by centrifugation (1000 × g for 5 min).
Calcium BindingCalcium binding was performed according to
the rapid filtration method (17) at 0 °C as described previously
(18). A Millipore HA filter (0.45-µm pore size) was used. The ATPase
preparation (0.2 mg of protein/ml) was preincubated in 20 mM PIPES buffer solution (pH 6.23 or 7.40) containing 0.12 M KCl and 5 mM MgCl2 without the
addition of CaCl2. Aliquots (1 ml) of the ATPase suspension were placed on the filter. After removal of calcium that was bound to
the ATPase by washing it on the filter with EGTA, calcium binding to
the calcium-unbound ATPase was initiated by washing the ATPase on the
filter with binding solution containing various concentrations of
45Ca2+ and having the same pH buffer as that
used in the preincubation of the ATPase, as described previously (19).
The association constants for EGTA-calcium at the employed pH values of
6.23 and 7.40 were 6.31 × 104 and 1.38 × 107 M1, respectively (20). In the
presence of 5 mM AcP, calcium accumulation in the ATPase
membrane vesicles was observed. After the calcium that was bound to the
ATPase preparation reached equilibrium in the absence of AcP, the
preparation further took up calcium at a rate of 2-4 nmol/mg of
protein/min when AcP was added. The calcium that was taken up with the
help of AcP was completely released by the addition of the calcium
ionophore A23187 (data not shown). Therefore, the amount of calcium
that was bound to the ATPase in the presence of AcP was obtained by
subtracting the amount of the accumulated calcium from the amount of
total calcium that was taken up by the membrane vesicles.
We previously showed that two different conformations (A and B) of
chemically equivalent Ca2+-ATPase molecules exist in the SR
membrane at a ratio of 1:1 (6). They independently bind two calcium
ions (7): the A form binds the ions noncooperatively, whereas the B
form binds the ions cooperatively (Figs.
1 and 2).
Recently, however, these two forms have been shown to result from
intermolecular interaction of the ATPase molecules (8). Here, we
therefore further examined the independence of the two forms in
transporting calcium by hydrolytic coupling of a substrate as an energy
source. To examine this point, we compared the calcium dependence of
the total hydrolysis activity of the two forms with calcium binding to
the forms in the absence of the substrate. The comparison was carried
out to elucidate whether these forms independently hydrolyze the
substrate due to calcium that is bound to each of the forms. For this
comparison, it is necessary to use a substrate that reacts with the
ATPase molecules to transport calcium across the SR membrane and that does not have any regulatory effect, other than that as a substrate for
the molecules, on their respective calcium binding and hydrolysis. AcP
has been shown to support calcium transport (11) and to have no
regulatory effect on the turnover of AcP hydrolysis (12, 13). However,
it is not known whether AcP affects calcium binding. In Fig.
3, we therefore first examined calcium
binding in the absence and presence of 5 mM AcP at pH 7.40 and 0 °C. As reported previously (7), at this pH and temperature, it
is thought that the A form is in E1 before
calcium binding and apparently rapidly binds calcium, whereas the B
form pre-exists in E2 and apparently slowly
binds calcium. Two such calcium binding reactions were observed in the
absence of AcP at 10.3 µM Ca2+; ~41% (3.5 nmol/mg of protein) of the calcium (8.5 nmol/mg of protein) that was
bound at equilibrium was rapidly bound to the A form, which pre-existed
in E1, within the dead time (~2 s) of our
experiments. The remaining 59% (5.0 nmol/mg of protein) of the bound
calcium was slowly and monoexponentially bound to the B form, which
pre-existed in E2, at a rate of
t1/2 ~ 4 s. At a lower calcium concentration
of 0.12 µM, only slow (t1/2 ~ 4 s) calcium binding to the B form was observed. These calcium binding
reactions were not affected by AcP at all, showing that there is no
significant effect of this amount of AcP on calcium binding.
As to the intermolecular interaction of the ATPase molecules, monomer ribbon-dimer ribbon transition in the array of molecules on the SR membrane has been found to coincide with E1-E2 transition of the molecules (9). The transition from E2 to E1 has been shown to be the slowest step of the transport sequence in the absence of a higher concentration of ATP than that required for phosphorylation of the ATPase molecule (10). On the other hand, the two forms of the ATPase molecules have been shown to be in different enzyme states before calcium binding dependent on pH (7): at pH 7.40, the A form is in E1 and the B form is in E2, whereas at pH 6.23, both of the forms are predominantly in E2. The transition rates of the two forms from E2 to E1 were previously shown to be the same, independent of temperature (7, 21). Here, therefore, the enzyme states of the two forms before calcium binding were set at E1 and E2 and at E2 only, by setting the pH at 7.40 and 6.23, to drive their hydrolysis reaction asynchronously and synchronously, respectively. Moreover, the experiments were carried out at 5 and 25 °C to assay the AcPase activity of the two forms under different conditions of stability of the intermolecular interaction of the ATPase molecules.
As mentioned above, because of the split of the two forms into
E1 and E2 at pH 7.40, these forms are thought to asynchronously turn over their cycles of AcP
hydrolysis reaction at this pH. In Figs.
4A and 5A, the
total Ca2+-AcPase activity, which is composed of the AcPase
activities of the two forms, was examined by varying the calcium
concentration and temperature at the mentioned pH. At 25 °C, Hill
plots of the total AcPase activity as a function of calcium
concentration were biphasic (Fig. 4B). The two lines of the
plots, the slopes of which were ~0.9 and 1.7, respectively,
intersected near the zero point of the ordinate, suggesting the
existence of two different types of AcP hydrolysis reaction with the
same level of maximum activities. The calcium concentration for the
half-maximum activity (K0.5) was ~0.2
µM. In a previous paper (7), we showed that at pH 7.40 and 0 °C, the A form, which pre-exists in E1,
noncooperatively (Hill value (nH) ~ 1) binds
calcium with an apparent calcium affinity (calcium concentration for
the half-maximum binding (K0.5)) of ~2.0
µM, whereas the B form, which pre-exists in
E2, cooperatively (nH ~ 2) binds calcium with a K0.5 of ~0.2
µM (Fig. 1, A and B). Hill plots of
the total binding, which is composed of the binding to the two forms at
a ratio of 1:1, were biphasic, with slopes of ~0.8 and 1.8. The two
lines of the Hill plots also intersected near the zero point of the
ordinate. The K0.5 of the total binding was
~0.4 µM. This profile of the total binding is very
close to that of the total Ca2+-AcPase activity at
25 °C, which is shown in Fig. 4. The profile of the total binding at
0 °C was not affected by increasing the temperature to 25 °C
(data not shown). At 25 °C, calcium has been found to be rapidly
bound to the ATPase molecules at the same millisecond rate irrespective
of their enzyme states (E1 and
E2) before calcium binding (21). These results
therefore suggest that at a temperature of 25 °C, the two forms
equally and independently hydrolyze AcP due to calcium that is bound to
each of the forms in a different manner. It was previously shown that
at 0 °C, the A form, which pre-exists in E1,
apparently rapidly (t < 2 s) binds calcium, whereas
the B form, which pre-exists in E2, slowly
(t1/2 > 2 s) binds calcium (7). Based on the
above discussion of independent AcP hydrolysis by the two forms, it is
therefore likely that at a lower temperature, the A form, which rapidly
binds calcium, more rapidly turns over its hydrolysis cycle than the B
form, which slowly binds calcium. This possibility was examined in Fig. 5A, in which the calcium
dependence of the total AcPase activity was studied at 5 °C. Almost
all of the Hill plots of the total activity were monophasic, with a
slope of 0.9-1.3, although a small deviation from the linear line was
observed (Fig. 5B). The slope (0.9-1.3) of the main portion
in the Hill plots is near the nH value (~1) of
the binding to the A form, which pre-exists in
E1, whereas the slope (>2) of the minor portion
is rather near the nH value (~2) of the
binding to the B form, which pre-exists in E2
(cf. Fig. 1B). These results support the above
discussion of independent AcP hydrolysis by the two forms at pH 7.40, where the forms asynchronously turn over their hydrolysis cycles.
Figs. 6 and
7 show the calcium dependence of the
AcPase activity at pH 6.23. At this acidic pH, as mentioned before, it
is thought that the A and B forms synchronously turn over their cycle of AcP hydrolysis reaction because they are in the same enzyme states
of E2 before calcium binding. As shown in a
previous paper (7), at this acidic pH, the A form noncooperatively
(nH ~ 1) binds calcium with a
K0.5 of ~7 µM, whereas the B
form cooperatively (nH ~ 2) binds calcium with
a K0.5 of 2-6 µM (see Fig.
2A). At the low temperature of 0 °C, both of the forms,
which pre-exist in E2, apparently slowly
(t1/2 > 2 s) bind calcium because of their slow transition to E1. The total binding profile
at this pH, which is composed of the binding to the two forms at a
ratio of 1:1, was monophasic, with nH ~ 1.3 and K0.5 ~ 5.8 µM (Fig.
2B). Such a profile was not affected by increasing the
temperature to 25 °C (data not shown), although the ATPase molecules
have been found to equally and rapidly bind calcium at a millisecond
rate (21). At 25 °C, Hill plots of the calcium dependence of the
total AcPase activity were monophasic, with nH ~ 1.4 and K0.5 ~ 5.0 µM (Fig. 6B). The calcium-dependent profile
(nH ~ 1.4 and K0.5 ~ 3 µM) of the total AcPase activity at 5 °C (Fig.
7B) was almost the same as that at 25 °C. These profiles
were the same as that of the total calcium binding (Fig.
2B), which is composed of the binding to the two forms at a
ratio of 1:1. These results suggest that the two forms equally and
independently hydrolyze AcP due to the calcium that is bound to each of
the forms, even when the forms synchronously turn over their hydrolysis
cycles.
These results demonstrate the independence of two forms of chemically
equivalent Ca2+-ATPase molecules in hydrolyzing AcP,
irrespective of temperature and the synchronism of their hydrolysis
reactions. On the other hand, if the two forms of the ATPase molecules
were not independently operating in hydrolyzing AcP, either or both of
the following results, which are contrasted with the results actually
obtained, would be expected from the experiments carried out here. (i)
The calcium-dependent profile of the total AcPase activity,
which is composed of the activities of the two forms, would not
correspond to that of the total calcium binding, which is composed of
the calcium binding to the forms at a ratio of 1:1, under some
condition(s) of pH and temperature except the condition of pH 7.40 and
5 °C. (ii) At 7.40, the calcium-dependent profile of the
total AcPase activity would correspond to that of the total binding
independent of temperature. Taking into account the monomer-dimer
transition of the ATPase molecules on the SR membrane accompanying
E1-E2 transition of the
molecules (9), the conclusion, mentioned above, indicates that the two
types of ATPase molecules independently carry out such monomer-dimer
transition of the same type of molecules, i.e. a structural
unit model in which there are two different pairs of molecules, with
each pair consisting of the same type of molecules and in which
nonequivalence results from the intermolecular interaction of those
molecules. The two-pair model is schematically represented in Fig.
8. The concept that at least two types of ATPase molecules are involved in the calcium transport mechanism first
emerged from the studies by Froehlich and Taylor (22). Ikemoto et
al. (23-25) reported the existence of two ATPase molecules that
behave differently in sequential steps of the calcium transport reaction. Our results support these earlier reports.