(Received for publication, March 15, 1995; and in revised form, May 15, 1995)
From the
Two different conformations of chemically equivalent
Ca
Sarcoplasmic reticulum Ca
In
the solubilization of the membranous enzyme with
C
Figure 1:
Kinetics of calcium-induced
phosphorylation before (
Two populations of Ca
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)
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
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
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.
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
We thank Dr. Hiroo Fukuda of the Biological Institute,
Faculty of Science, Tohoku University for access to a fluorescence
spectrophotometer.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-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.
-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 (C
E
).
(
)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.
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.
E
, 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 C
E
,
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
C
E
, 50 µM
CaCl
, 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,
C
E
, 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 C
E
/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) .
) 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
C
E
/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
C
E
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
10
M
(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 C
E
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
C
E
, 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) .
-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.
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.
, 1 mM ADP (
) or 1 mM
AMP-PNP (
) or absence of both (
), 50 µM
CaCl
, and 2 mg/ml C
E
. 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.
10
M
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.
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
.
/mg of protein/min) of the
activity.
E
, dodecyloctaethylene glycol monoether;
PIPES, piperazine-N,N`-bis(2-ethanesulfonic acid);
AMP-PNP, adenosine 5`-(
,
-imino)triphosphate; EP,
phosphorylated Ca
-ATPase.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.