From the
Incubation of the Ca
The major phospholipid in the sarcoplasmic reticulum (SR)
As well
as these solvent-like lipids, a few ``special'' lipid
molecules may bind at distinct sites on the ATPase. SR membranes
contain, as well as phosphatidylcholine, phosphatidylethanolamine,
phosphatidylserine, phosphatidylinositol (PtdIns), and
phosphatidylinositol 4-phosphate (PtdIns-4P), but no
phosphatidylinositol 4,5-bisphosphate(2) . The absence of
phosphatidylinositol 4,5-bisphosphate means that, unlike the transverse
tubule membrane, SR lacks the capacity for Ins(1,4,5)P
The dependence of ATPase activity on Ca
The activity of the Ca
Skeletal muscle SR shows the kinase activity necessary to
phosphorylate PtdIns to PtdIns-4P(2, 10, 11) .
It also contains a Ca
We have
confirmed these results. Incubation of the Ca
In previous studies of the effects of
phospholipids on the activity of the ATPase, we have detected changes
in the rates of phosphorylation and dephosphorylation of the
ATPase(5, 8) , and stimulation of the ATPase by jasmone
has been shown to follow from increases in the rates of the
Ca
Activation of the ATPase with ATP has no effect on the rate
of phosphorylation of the ATPase (data not shown) or on the rate of the
Ca
PtdIns-4P and other negatively charged
phospholipids have been shown to increase the affinity of the plasma
membrane Ca
-ATPase of skeletal muscle
sarcoplasmic reticulum with ATP in the absence of Ca
leads to phosphorylation of phosphatidylinositol (PtdIns) to
phosphatidylinositol 4-phosphate (PtdIns-4P) and to a doubling of
ATPase activity. Similarly, reconstitution of the ATPase with mixtures
of dioleoylphosphatidylcholine and PtdIns-4P also led to a doubling of
activity; ATPase activity increased with increasing PtdIns-4P content,
up to 10% beyond which no further increase was observed. Reconstitution
with PtdIns had a much smaller effect on activity. Changes in the
Ca
affinity of the ATPase following incubation with
ATP or reconstitution with PtdIns-4P were small. The rates of
phosphorylation of the ATPase by ATP and of the Ca
transport step were unaffected, but the rate of dephosphorylation
of the phosphorylated ATPase increased by a factor of 2 either
following incubation with ATP or following reconstitution with
PtdIns-4P. Activation of the ATPase led to a decrease in the level of
phosphorylation of the ATPase by P
corresponding to a
10-fold decrease in the equilibrium constant E2PMg/E2P
Mg.
(
)of skeletal muscle is
phosphatidylcholine(1, 2) . Phosphatidylcholines have
been shown to interact nonspecifically with the hydrophobic
membrane-penetrant parts of the Ca
-ATPase of SR and
are thus believed to provide the bulk ``solvent'' lipids for
the ATPase(1, 3) . For the ATPase to be active, these
lipids must be in the liquid crystalline phase(4, 5) ,
and optimal activity is obtained with a chain length of C18, that is,
with dioleoylphosphatidylcholine
(di(C18:1)PC)(1, 6, 7, 8) .
generation(2) . PtdIns 4-kinase has been detected in SR,
as has a Ca
-dependent phosphomonoesterase capable of
catalyzing the breakdown of PtdIns-4P(9, 10) . A
possible physiological role for PtdIns-4P is suggested by the
experiments of Varsanyi et al.(11) who showed that
incubation of the purified ATPase with ATP in the absence of
Ca
led to phosphorylation of up to one PtdIns
molecule per ATPase molecule to form PtdIns-4P and that this resulted
in stimulation of ATPase activity. Here, we show that the effect of
PtdIns-4P on ATPase activity follows from a specific effect on the rate
of dephosphorylation of the phosphorylated ATPase.
Materials
Dioleoylphosphatidylcholine
was purchased from Avanti Polar Lipids, Inc. (Birmingham, AL), and
PtdIns and PtdIns-4P were from Sigma. Sarcoplasmic reticulum and
purified Ca-ATPase were prepared from rabbit skeletal
muscle(3) . Concentrations of ATPase were estimated by using the
extinction coefficient (1.2 liters g
cm
for a solution in 1% SDS) given by Hardwicke and
Green(12) .
Reconstitution with PtdIns-4P
Mixtures of
di(C18:1)PC and PtdIns or PtdIns-4P (10 µmol, total phospholipid)
in buffer (400 µl, 10 mM Hepes/Tris, pH 8.0, containing
15% sucrose, 5 mM MgSO, 5 mM ATP, and 12
mg/ml potassium cholate) were sonicated to clarity in a bath sonicator.
ATPase (1.25 mg) in a volume of 20-30 µl was then added, and
the mixture was left for 15 min at room temperature and 45 min at 5
°C to equilibrate. After equilibration, the samples were added to
precooled Oakridge tubes containing ice-cold buffer (10 mM Hepes/Tris, pH 8.0, 2 mM dithiothreitol) and centrifuged
at 200,000
g for 1 h at 4 °C. Samples were
rehomogenized and suspended in buffer (10 mM Hepes/Tris, 15%
sucrose) to a concentration of 3-8 mg/ml and then stored at
-20 °C until use.
Activation with ATP
The purified
Ca-ATPase or SR vesicles were activated by incubation
with ATP largely as described by Varsanyi et al.(11) .
The ATPase or SR vesicles (2 mg of protein/ml) were incubated with 1
mM ATP in 40 mM Hepes/KOH, pH 7.2, containing 100
mM KCl, 5 mM MgSO
, and 1 mM EGTA
for 1 h at 25 °C. The sample was then centrifuged at 100,000
g for 1 h, and the pellet was kept on ice until use.
As a control, ATPase was incubated under the same conditions but in the
absence of ATP.
ATPase Assay
ATPase activities were
determined at 25 °C by using a coupled enzyme assay in a medium
containing 40 mM Hepes/KOH, pH 7.2, 100 mM KCl, 5
mM MgSO, 2.1 mM ATP, 1.1 mM EGTA, 0.41 mM phosphoenolpyruvate, 0.15 mM NADH,
pyruvate kinase (7.5 IU), and lactate dehydrogenase (18 IU) in a total
volume of 2.5 ml. The reaction was initiated by addition of an aliquot
of a 25 mM CaCl
solution to a cuvette containing
the ATPase and the other reagents to give the required concentration of
Ca
. Free concentrations of Ca
were
calculated using the binding constants for Ca
,
Mg
, and H
to EGTA given by
Godt(13) .
Rapid Kinetic Experiments
The time
dependence of phosphorylation-induced Ca release from
the ATPase was determined using a Biologic Rapid filtration system, and
the time dependences of phosphorylation of the ATPase by
[
-
P]ATP and of dephosphorylation of the
ATPase phosphorylated with [
P]P
in
the absence of Ca
, or with
[
-
P]ATP in the presence of
Ca
, were determined using a Biologic QFM-5 system as
described by Starling et al.(14) .
Equilibrium Levels of Phosphorylation with
P
The ATPase (0.2 mg of protein/ml)
was incubated with [P]P
in 150
mM Mes/Tris, pH 6.2, containing 5 mM EGTA and the
required concentrations of Mg
at 25 °C. After 15
s, the reaction was quenched by addition of 10 volumes of quenching
solution (25% trichloroacetic acid, 0.13 M phosphoric acid).
The sample was put on ice for 15 min, and then the precipitate was
collected by filtration through Whatman GF/B glass fiber filters and
finally counted in Optiphase Hisafe III.
Phosphorylation of PtdIns by ATP
SR
vesicles (25 mg of protein) were incubated with ATP as described above,
followed by precipitation with trichloroacetic acid. The precipitate
was pelleted, washed with water, dried in vacuo, and extracted
with chloroform/methanol (3 ml; 2:1 v/v). The precipitate was again
pelleted and then extracted with chloroform/methanol/HCl (3 ml, 40:20:1
(v/v/v)). The organic layer was filtered through a Whatman GF/C filter
and washed with 0.6 ml of a 1% NaCl solution, and the lower
chloroform-rich phase evaporated in vacuo. Lipids were
separated by thin layer chromatography on Silica gel 60 plates using
chloroform/methanol/water/ammonia (48:40:7:5 (v/v/v/v)) as solvent
(15). Lipids were visualized by staining with iodine and P-labeled lipid detected using a plate scanner
(Dünnschicht).
ATPase Activity
If the ATPase is
incubated with MgATP in the absence of Ca for 1 h and
then the rate of ATP hydrolysis measured at 100 µM
Ca
, an activity of 5.9 IU/mg protein is obtained,
compared with a value of 3.0 IU/mg protein for the ATPase prior to
incubation with ATP (Fig. 1).
Figure 1:
ATPase activity for the activated or
reconstituted ATPase. Shown are the ATPase activities (IU/mg protein)
for the ATPase with () or without (
) activation in the
presence of ATP and for the ATPase reconstituted with an 8:1 molar
ratio of di(C18:1)PC or PtdIns-4P (
) or PtdIns (
). ATPase
activities were measured at ph 7.2, 2.1 mM ATP, and the given
concentration of Ca
, at 25
°C.
The ATPase was reconstituted
into bilayers of di(C18:1)PC and PtdIns-4P by mixing the ATPase with
lipid in cholate solution at a molar ratio of lipid:ATPase of 1000:1,
followed by dilution into buffer to reform membrane
fragments(1) . Following reconstitution with a lipid mixture
containing 20% PtdIns-4P, the ATPase activity was the same as that for
the ATPase activated with ATP (Fig. 1). The effect of PtdIns-4P
on ATPase activity increased with increasing PtdIns-4P content up to
10%, beyond which activity was constant up to 50% PtdIns-4P, the
highest content tested (data not shown). Reconstitution with mixtures
of di(C18:1)PC and PtdIns led to smaller increases in ATPase activity (Fig. 1), with stimulation of ATPase activity reaching a maximum
at 10% PtdIns.
concentration is complex (Fig. 1) with low concentrations
of Ca
activating the ATPase, attributable to binding
of Ca
to the Ca
binding sites on
the unphosphorylated ATPase (E1), and high concentrations of
Ca
inhibiting the ATPase, attributable both to
binding of Ca
to the phosphorylated ATPase (E2P) with a subsequent decrease in the rate of
dephosphorylation and to the formation of CaATP, which is hydrolyzed
more slowly by the ATPase than
MgATP(16, 17, 18) . Activation with ATP or
reconstitution with PtdIns-4P had no significant effect on the
Ca
dependence of activity, in either the low or high
concentration regions (Fig. 1). The affinity of the ATPase for
Ca
can also be measured from changes in the
tryptophan fluorescence intensity of the ATPase on binding
Ca
(19) . Such measurements detect a very small
decrease in the Ca
affinity (a shift in the pCa value
for 50% binding of -0.2) on reconstitution with PtdIns-4P (data
not shown).
The Rate of Phosphorylation of the
ATPase
Mixing the ATPase incubated in the presence of 100
µM Ca with 50 µM
[
-
P]ATP at pH 7.2 leads to rapid formation
of phosphoenzyme, which fits to a single exponential process with rates
of 78.7 ± 10.0 s
and 77.6 ± 9.2
s
, respectively, for the ATPase before and after
activation by incubation with ATP (data not shown). The maximum level
of phosphorylation, 2.8 nmol of [EP]/mg protein, was
unaffected by incubation with ATP, corresponding to a fraction of
active protein of about 0.3, typical for this and other preparations of
the ATPase.
The Rate of Phosphorylation-induced Ca
The rate of phosphorylation of the ATPase by
ATP in the presence of Ca Release
is much faster than the
rate of dissociation of
Ca
from the
unphosphorylated ATPase(20) . Under these conditions, Orlowski
and Champeil (20) have shown that the rate of Ca
dissociation from the phosphorylated ATPase (the E1PCa
E2P step) can be measured by
pre-equilibrating the ATPase with
Ca
and
then perfusing it on Millipore filters with
Ca
and ATP. When the ATPase was incubated with 100 µM
Ca
in buffer at pH 7.2 and then perfused
with the same medium containing 100 µM unlabeled
Ca
and 2 mM MgATP, dissociation of
Ca
from the ATPase was observed, fitting
to a single exponential process with a rate constant of 19.3 ±
4.6
(Fig. 2). If the ATPase was first
activated by incubation with ATP in the absence of Ca
for 1 h, then the rate of Ca
dissociation was
not changed significantly, fitting to a rate constant of 15.5 ±
3.3 s
(Fig. 2).
Figure 2:
ATP-induced release of Ca
from the ATPase. The ATPase (0.4
mg/ml) with (
) or without (
) activation with ATP was
equilibrated in buffer (20 mM Hepes/Tris, pH 7.2, 100 mM KCl, 20 mM Mg
) containing 100
µM
Ca
and 0.5 mM
[
H]sucrose, and then 0.1 mg of ATPase was
adsorbed onto Millipore 0.45-µm HAWP filters. The loaded filter was
perfused for the given times with the same buffer containing 100
µM unlabeled Ca
and 2 mM ATP,
and the amount of Ca
bound to the ATPase was
determined (14). The solidline shows a single
exponential fit to the data for the unactivated ATPase, with a rate
constant of 19.3 ± 4.5
s
.
Phosphorylation of the ATPase by
P
Incubation of the ATPase with P at pH 6.2 in the presence of Mg
and absence of
Ca
leads to phosphorylation of the ATPase. The level
of phosphorylation observed is reduced if the ATPase is first activated
by incubation with ATP (Fig. 3). Phosphoenzyme formation fits to Fig. SI(21, 22) .
Figure 3:
Phosphorylation of the ATPase by
P. The ATPase (0.2 mg/ml) was incubated in 150 mM Mes/Tris, pH 6.2, 5 mM EGTA, and 10 mM Mg
in the presence of the given concentration of
phosphate (A) and 1 mM P
and the given
concentration of Mg
:ATPase with (
) and
without (
) activation with ATP (B). The solidlines show simulations calculated as described in the
text assuming a value of [EP]
of 3.5
nmol/mg protein.
Figure SI:
Scheme I.
As described(22) ,
good fits to the experimental data for the unstimulated ATPase can be
obtained with binding constants of Mg and P
of 100 M
, with an equilibrium
constant E2PMg/E2P
Mg of 18 and an
equilibrium constant E1/E2 calculated as described by
Henderson et al.(19) (Fig. 3). For the activated
ATPase, the data fit to the same binding constants for Mg
and P
but with a reduced value for the equilibrium
constant E2PMg/E2P
Mg of 1.8 (Fig. 3).
Rate of Dephosphorylation of the
ATPase
The rate of dephosphorylation of the phosphorylated
ATPase can be determined either by phosphorylating the ATPase with
[P]P
at pH 6.0 in the absence of
Ca
and presence of dimethyl sulfoxide followed by
mixing with an excess of a pH 7.5 buffer containing KCl and ATP to
induce dephosphorylation, or by first phosphorylating the ATPase with
[
-
P]ATP in the presence of Ca
and then mixing with an excess of unlabeled ATP(14) .
Although the level of phosphorylation of the activated ATPase by
P
is normally low (Fig. 3), in the presence of 14%
(v/v) dimethyl sulfoxide, levels of phosphorylation are comparable for
the activated and the non-activated ATPase. Dephosphorylation of the
ATPase phosphorylated with P
in the presence of dimethyl
sulfoxide fits to a single exponential process with a rate of 15.2
± 1.6 s
before activation of the ATPase with
ATP and 36.9 ± 3.0 s
after activation with
ATP (Fig. 4A). A similar increase in the rate of
dephosphorylation was observed following reconstitution with PtdIns-4P.
Rates of dephosphorylation have been observed to vary between
preparations of the ATPase, and for that used to obtain the data in Fig. 4B, a rate of dephosphorylation of 7.6 ± 0.6
s
was observed when reconstituted in di(C18:1)PC,
increasing to 15.1 ± 2.0 s
on reconstitution
with a mixture of di(C18:1)PC and PtdIns-4P at a molar ratio of 8:2 (Fig. 4B). For the ATPase phosphorylated with ATP, rates
of dephosphorylation of 11.6 ± 2.5 s
and 26.8
± 5.9 s
were observed before and after
activation with ATP, respectively (Fig. 4C).
Figure 4:
The rate of dephosphorylation of the
phosphorylated ATPase. A and B, the enzyme syringe
contained ATPase (4 mg/ml) in 12.5 mM Mes/Tris, pH 6.0,
containing 10 mM EGTA, 1 mM [P]P
, 20 mM Mg
, and 14% (v/v) dimethyl sulfoxide. The second
syringe contained 100 mM Mes/Tris, pH 7.5, containing 100
mM KCl, 4 mM Mg
, and 5.3 mM ATP. The contents of the enzyme syringe were mixed in a 1:16
volume ratio with the dephosphorylation mixture and the reaction
quenched at the given times with 25% trichloroacetic acid. A,
ATPase with (
) and without (
) activation with ATP. B, ATPase reconstituted with di(C18:1)PC (
) or with an
8:2 molar ratio of di(C18:1)PC and PtdIns-4P (
). C,
the enzyme syringe contained ATPase (0.2 mg/ml) in 20 mM Mes/Tris, pH 7.2, 5 mM Mg
, 100 mM KCl, and 100 µM Ca
. This was mixed
in a 1:1 ratio with a solution containing 50 µM
[
-
P]ATP in the same buffer. The mixture was
incubated for 200 ms and then mixed in a 1:1 ratio with the same buffer
containing 2.5 mM unlabeled ATP. The reaction was quenched at
the given time with 25% trichloroacetic acid. ATPase with (
)
and without (
) activation with ATP is shown. The solidlines show fits to single exponential processes, with the
rate constants given in the text.
Phosphorylation of PtdIns by ATP
As
described by Varsanyi et al.(11) , incubation of SR
vesicles with [-
P]ATP in the absence of
Ca
led to the formation of radiolabeled PtdIns-4P,
with no detectable radiolabel incorporation into phosphatidylcholine,
phosphatidylethanolamine, or phosphatidylserine.
-ATPase in cardiac
sarcoplasmic reticulum is modified by interaction with phospholamban.
Binding of phospholamban to the Ca
-ATPase reduces the
maximal rate of ATP hydrolysis and reduces the affinity of the ATPase
for Ca
(23, 24) , the hydrophilic
domain of phospholamban reducing v
by reducing
the rate of the Ca
transport step(25) . No
such control mechanism has yet been established for the
Ca
-ATPase in skeletal muscle sarcoplasmic reticulum.
-dependent phosphomonoesterase
able to hydrolyze PtdIns-4P(10) . Varsanyi et al.(11) have shown that conversion of a small number of the
PtdIns molecules in the SR membrane to PtdIns-4P (probably one per
ATPase molecule) leads to stimulation of the
Ca
-ATPase(11) . The PtdIns-4P involved in
stimulation of the ATPase is probably tightly bound to it, since it is
insensitive to a variety of phospholipases(11) .
-ATPase
with ATP in the absence of Ca
leads to
phosphorylation of PtdIns to PtdIns-4P, this resulting in stimulation
of the ATPase (Fig. 1). The affinity of the ATPase for
Ca
is little changed (Fig. 1), confirmed by
measurements of changes in tryptophan fluorescence intensity as a
function of Ca
concentration (data not shown).
Stimulation of the ATPase can also be achieved by reconstitution of the
ATPase with mixtures of di(C18:1)PC and PtdIns-4P (Fig. 1). The
high molar ratio of PtdIns-4P to ATPase required for maximal
stimulation in the reconstituted system (100:1) suggests significant
partitioning of PtdIns-4P between the site(s) on the ATPase and the
bulk lipid phase.
transport step and of
dephosphorylation(14) . Effects of jasmone and activation of the
ATPase by ATP are additive; addition of 200 µM jasmone
increases the activity of the activated ATPase from 5.9 to 7.9 IU/mg
protein, measured under the conditions shown in Fig. 1(data not
shown).
transport step (Fig. 2). However, it does
have a marked effect on the rate of dephosphorylation of the
phosphorylated ATPase, this increasing by a factor of about 2 (Fig. 4). The rate of dephosphorylation of the ATPase in a
mixture of di(C18:1)PC and PtdIns-4P is also found to be double that
for the ATPase in di(C18:1)PC (Fig. 4). Equilibrium measurements
of phosphorylation of the ATPase by P
(Fig. 3) are
consistent with a 10-fold decrease in the equilibrium constant for
phosphorylation (E2PMg/E2P
Mg, Fig. SI) on activation with ATP. With a doubling of the rate of
dephosphorylation, this implies a 5-fold decrease in the rate of
phosphorylation by P
on activation of the ATPase. Since the
binding site for PtdIns-4P is presumably in the trans-membrane region
of the ATPase, the specific effect of PtdIns-4P on the E2PMg&rlarr2;E2P
Mg step implies a long
range interaction on the ATPase, since the phosphorylation domain of
the ATPase is located a considerable distance above the membrane
surface(26) .
-ATPase for Ca
but with
no effect on v
(27) . The binding site for
PtdIns-4P on the plasma membrane Ca
-ATPase has been
suggested to involve both a large positively charged loop just before
the third transmembrane
-helix and the C-terminal calmodulin
binding domain (27). Although the N-terminal region of the first of
these proposed sites is absent from the SR Ca
-ATPase,
the C-terminal region, corresponding to the sequence
DKTPLQQKLDEFGE in the SR Ca
-ATPase,
containing two conserved Lys residues is present as part of the
proposed third stalk region(28) . This region could therefore be
involved in binding PtdIns-4P to the SR Ca
-ATPase.
Alternatively, the binding site for PtdIns-4P on the SR ATPase could be
in the distinct central space between trans-membrane lobes A, B, and C
seen in electron micrographs(29, 30) .
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.