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J. Biol. Chem., Vol. 277, Issue 16, 13900-13906, April 19, 2002
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From the Department of Biochemistry and Molecular Biology,
University of Maryland School of Medicine, Baltimore, Maryland
21201
Received for publication, November 21, 2001, and in revised form, February 12, 2002
Ca2+ transport by sarcoplasmic
reticulum (SR) ATPase occurs with an optimal coupling ratio of 2 Ca2+ per ATP in pre-steady state. However, slippage of the
pump and lower coupling ratios are observed in steady state. Slippage
depends on the presence of high Ca2+ in the lumen of SR
vesicles and high nucleotide in the medium. Thereby, Ca2+
and/or nucleotide-bound phosphoenzyme intermediates accumulate and
undergo uncoupled cleavage, before vectorial translocation of bound
Ca2+ in the forward direction of the cycle or before
productive reversal to ATP synthesis. Transport efficiency and coupling
ratios are improved by reduction of nucleotide concentration in the
presence of ATP regenerating systems and/or complexation of
luminal Ca2+ with phosphate or oxalate. Curcumin
(1-5 µM) lowers the concentration of phosphate or
oxalate required to reduce slippage of the Ca2+ pump.
Thereby, under appropriate conditions, curcumin favors kinetic flow,
completion of productive cycles, and improvement of coupling ratios.
The findings obtained with isolated SR vesicles suggest that slippage
is an important phenomenon under prevailing conditions of muscle fibers
in situ. Ca2+ transport and its slippage can be
improved by curcumin in cardiac as well as in skeletal SR, raising the
possibility of pharmacological interventions to correct defective
Ca2+ homeostasis. Higher curcumin concentrations (5-30
µM), however, inhibit overall ATPase activity and
Ca2+ transport by interfering with phosphoenzyme formation
with ATP or Pi.
The Ca2+ transport ATPase of sarcoplasmic reticulum
(SR)1 plays an essential role
in filling intracellular Ca2+ stores of skeletal and
cardiac muscle, whereby stored Ca2+ is released into the
cytosol to produce contractile activation and in turn is taken up again
to allow relaxation. Translocation of 2 Ca2+ per catalytic
cycle occurs during the initial cycles following addition of substrate
(1), consistent with the binding stoichiometry of 2 Ca2+
per ATPase molecule (2). However, the Ca2+/ATP coupling
ratios are considerably lower in steady state, a phenomenon initially
attributed to leakage of transported Ca2+. On the other
hand, it was later demonstrated that the catalytic and transport cycle
can undergo significant slippage through branched reactions, resulting
in lower coupling ratios (3-5). Slippage is influenced by the lipid
composition of the membrane (6) and can be reduced by curcumin (7). We
report here a series of experiments with vesicular fragments of
skeletal SR, defining experimental conditions and catalytic
intermediates that are involved in the occurrence of slippage. We
characterize reduction of slippage and improvement of Ca2+
transport by curcumin at low concentrations, as well as inhibition of
overall ATPase and Ca2+ transport by curcumin at higher
concentrations. Finally, we extend our experimentation to cardiac SR,
exploring the possible relevance of transport slippage and its
correction to Ca2+ homeostasis of muscle fibers
in situ.
Vesicular fragments of longitudinal SR were obtained from rabbit
skeletal muscle as described previously (8). A similar preparation was
obtained from adult rat hearts, subjecting 4.5 g of ventricular
muscle to disruption in a Fisher Scientific PowerGen 700 tissue
homogenizer, three times for 30 s. The homogenization medium (10 ml) was ice-cold and contained 20 mM MOPS, pH 7, 10% sucrose, 1 mM EDTA, 0.5 mM dithiothreitol, with
freshly added protease inhibitors (0.4 mM Pefabloc,
1 µg/ml pepstatin A, 2 µg/ml leupeptin, and 10 µg/ml aprotinin).
The homogenate was centrifuged at 14,000 × g for 20 min, the resulting supernatant centrifuged at 45,000 × g for 30 min, and the final pellet resuspended in 20 mM MOPS, pH 7, 30% sucrose, 0.5 mM
dithiothreitol, and protease inhibitors as indicated above.
[45Ca]Ca2+ transport was assayed by
filtration at serial times following the addition of ATP (9). ATPase
activity was followed either by colorimetric determination of
Pi release (10) or by measuring release of
[32P]Pi from [ Rapid kinetic measurements of phosphoenzyme formation, Ca2+
translocation, and Pi production were obtained by rapid
mixing in a Froehlich-Berger chemical quench-flow apparatus, as
described by Fernandez-Belda et al. (13).
ATP-ADP exchange was determined by measuring incorporation of
[3H]ADP into ATP (9). Following incubation in the medium
specified in the legend for Fig. 1B, the reaction was
stopped by the addition of thapsigargin (1 µM), and the
mixture was filtered through a Nalgene 0.2-µm filter. The filtered
medium (0.8 ml) was loaded on a 25-cm Whatman Partisil-10 SAX column
and the nucleotides eluted by high performance liquid
chromatography with a gradient from 0.04 M
KH2PO4, pH 2.8, to 0.5 M
KH2PO4, 0.8 M KCl, pH 2.7. The
nucleotide elution peaks were detected by light absorption and the
radioactivity measured by scintillation counting.
[ Steady state kinetic calculations were based on controlled rate
constants and concentrations of substrate, ligands, and products, as
described previously (14). Computations were performed on a PC
microcomputer, using 14-digit precision MEGABASIC with BCD coding
(American Planning Corp., Alexandria, VA). Copies of the computation
programs can be obtained from M. Kurzmack (kurzmack{at}webaccess.net).
Efficiency and Slippage of the Ca2+ Pump--
It is
shown in Fig. 1A that, in the
millisecond time scale, addition of ATP to SR vesicles preincubated
with Ca2+ is followed by rapid enzyme phosphorylation, and
internalization of bound Ca2+ (i.e. unavailable
for exchange with medium Ca2+) with a stoichiometric ratio
of two per phosphoenzyme molecule. Following a lag period, hydrolytic
cleavage of Pi occurs, allowing further catalytic and
transport cycles. This pre-steady state measurements confirm those
reported previously (13) and are shown here as a term of comparison
with measurements extended to a longer time scale. In fact, SR vesicles
reach asymptotic levels of Ca2+ filling after 60-90 s of
reaction. ATP consumption, however, does not cease in parallel with
Ca2+ uptake, thereby resulting in unproductive ATP
consumption (Fig. 1B). ATPase activity ceases only if the
Ca2+ concentration in the medium is lowered below the
activating level by the addition of EGTA (Fig. 1B) or the
enzyme is inactivated by addition of the specific inhibitor
thapsigargin (Fig. 1B).
The experiments shown in Fig. 1 were performed in the absence of
calcium sequestering anions (see below). Therefore, the maximal Ca2+ uptake level shown in Fig. 1B corresponds
to a high (mM) concentration of free Ca2+ in
the lumen of the vesicles. It should be noted that when the ATPase was
inactivated by addition of TG, passive leakage of accumulated Ca2+ was minimal (Fig. 1B). Furthermore, upon
addition of EGTA, the release of accumulated Ca2+ by
reversal of the pump was quite slow, due to lack of ADP (Fig. 1B). Therefore, the observed Pi production, in
excess of Ca2+ transport, was actually due to slippage of
the pump, i.e. hydrolysis of ATP that is dissociated or
uncoupled from Ca2+ transport.
As observed in early studies (15), the efficiency (i.e.
stoichiometric ratio of Ca2+ transport and ATP hydrolysis)
of steady state Ca2+ transport is improved by the addition
of phosphate or oxalate to the reaction mixture (Fig.
2, A and B). This
is due to buffering of luminal Ca2+ by these anions, when
the Ca2+ concentration in the lumen of the SR vesicles
reaches their binding constants (and solubility products) following
active transport. Thereby, the level of Ca2+ uptake is very
much increased, and in the presence of optimal oxalate concentrations,
a Ca2+/ATP coupling ratio of 2 can be observed for a longer
time (i.e. minute time scale), before asymptotic levels of
Ca2+ accumulation are reached. These experiments indicate
that a rise of luminal Ca2+ is an important factor in
determining slippage of the pump.
Another important variable is the presence of ADP. It is shown in Fig.
3 that when ADP is added, net
Ca2+ uptake is quite reduced (Fig. 3, A and
B). In this case, the reduction of net Ca2+
uptake is due, at least in part, to Ca2+ efflux by reversal
of the pump rather than slippage (5). Interestingly, however, ATP
hydrolysis (as indicated by release of
[32P]Pi from ATP) remains approximately the
same, indicating the occurrence of slippage.
It is useful, at this point, to evaluate the effects of luminal
Ca2+ and medium ADP in the light of the reaction sequence
commonly used for the catalytic and transport cycle of the SR ATPase.
In Scheme 1 the enzyme (E) is
activated by binding of two medium (i.e. cytosolic)
Ca2+, and then ATP is utilized to form
E1~PCa2. This intermediate undergoes isomerization to
E2-P·Ca2+, allowing release of
bound Ca2+ into the lumen of SR and hydrolytic cleavage of
E-P. The sequence shown by the solid lines in
Scheme 1 implies that the Ca2+/ATP coupling ratio is 2 under all conditions. Slippage can then be intuitively attributed to
Ca2+ and ADP-dependent accumulation and
hydrolytic cleavage of the ADP·E1~P·Ca2 intermediate.
To demonstrate the presence, and evaluate the role of the
ADP·E1~P·Ca2 intermediate, we
measured ADP/ATP exchange, which occurs by reversal of Reaction 3 in
Scheme 1. We found that, when ADP is added (as required to perform the
measurements), exchange occurs at a rate 3-fold higher than that of
hydrolytic cleavage of Pi. Nearly identical results are
obtained when luminal Ca2+ is raised by active transport
and when net Ca2+ accumulation is prevented by addition of
ionophore (Fig. 4). This indicates that
the reverse rate constant for Reaction 3 is faster than the forward
constant. For this reason, as well as for rapid forward isomerization,
the steady state level of
ADP·E1~P·Ca2 is low in most
cases. It does not seem likely, then, that this intermediate would be
utilized significantly through futile cycles (dashed line in
Scheme 1), as compared with productive (i.e. ATP synthesis)
reversal of Reaction 3 (solid line). This issue will be
considered again under "Discussion."
Another interesting finding is that, if accumulation of ADP is
prevented with an ATP regenerating system at either high or low ATP,
slippage is reduced and the efficiency of Ca2+ transport is
improved when the ATP concentration is low (Fig. 5, A and B).
Therefore, high concentrations of ATP (above the Km
range) favor slippage. In fact, it was shown by fast kinetic
measurements that a chase with mM ATP doubles the rate of
hydrolytic cleavage of phosphoenzyme already made by utilization of
µmol ATP (16). Pi The Effect of Curcumin--
It was reported by Logan-Smith
et al. (7) that Ca2+ transport is increased up
to 20% by curcumin within the 1-10 µM concentration range and then is inhibited by higher curcumin concentrations. On the
other hand, ATPase activity is slightly increased by 1-3 µM curcumin and then inhibited by higher curcumin
concentrations. We confirmed these findings (Fig.
6) and then found that Ca2+
transport enhancement by curcumin is very much dependent on the concentration of KH2PO4 or oxalate used to
enhance transport (Fig. 7). The effect is
maximal at low anion concentrations, while no effect is noted at
saturating anion concentrations. In fact, a surprisingly large
(4-5-fold) increase in Ca2+ transport is produced by 5 µM curcumin in the presence of 5 mM KH2PO4 or 0.5 mM oxalate (Fig. 7).
Under the same conditions, the ATPase activity is only slightly
affected, demonstrating an unexpectedly large reduction of slippage by
curcumin (Fig. 8). It is then apparent
that curcumin lowers the anion concentration required for improvement
of Ca2+ transport, possibly facilitating transmembrane
anion diffusion concomitant with Ca2+ transport.
We found that this effect of curcumin is also produced on cardiac SR
vesicles. In fact, Ca2+ transport in the presence of 10 mM KH2PO4 is increased by 5 µM curcumin (Fig.
9A), while curcumin is
slightly inhibitory when the KH2PO4
concentration is raised to its optimal level of 50 mM (Fig.
9B).
We then proceeded to characterize the inhibition of both ATPase
activity and transport, which is produced by higher curcumin concentrations (Fig. 6). We found that this effect is characterized by
a strong reduction of phosphoenzyme formation, as revealed by direct
measurement of phosphoenzyme (Fig. 10),
and by a reduction of ADP/ATP exchange (not shown). The enzyme levels
obtained in the presence of ATP and Ca2+ are reduced by 40 or 90% in the presence of 5 or 30 µM curcumin, respectively (Fig. 10). On the other hand, phosphoenzyme formed in the
absence or in the presence of curcumin undergoes exponential decay at
approximately the same rate (0.4-0.6 s
In parallel experiments on formation of phosphoenzyme by utilization of
Pi in the absence of Ca2+, we found 49 and 86%
inhibition in the presence of 5 or 30 µM curcumin,
respectively. Therefore, the overall inhibitory effect of curcumin
involves both utilization of ATP in the presence of Ca2+
and utilization of Pi in the absence of
Ca2+.
Experimental evidence obtained in our own (3) and other
laboratories (4, 5) indicates that slippage of the pump occurs when the
concentration of luminal Ca2+ is high, thereby raising the
steady state level phosphoenzyme intermediates undergoing unproductive
cleavage (Scheme 1). We attempt here to distinguish various
phosphoenzyme species that may undergo hydrolytic cleavage through an
unproductive cycle, rather than completing productive cycles in the
forward or reverse direction of the pump. It is shown in Scheme
2 that the enzyme (E) is
activated through sequential and cooperative binding of 2 Ca2+ and a related conformational change (E to
E'). Utilization of ATP then yields a phosphorylated
intermediate, which produces lower affinity and vectorial dissociation
of bound Ca2+, followed by hydrolytic cleavage of
Pi. Note that isomerization of E'~P to
E-P denotes loss of phosphorylation potential, which is
utilized to reduce the Ca2+ binding affinity. It also
denotes transformation of a state favoring phosphoryl transfer to a
state favoring hydrolytic cleavage of Pi. The sequence of
reactions and their kinetic constants allow calculations of steady
state levels of intermediates, in the presence of various
concentrations of ligands (14).
It is shown in Table I that the
E'-PCa2, E'-PCa, and E-PCa
rise when the luminal Ca2+ concentration is raised, and
ADPE'-PCa2 rises when ADP is increased. Assuming
that these intermediates can undergo hydrolysis, a rise of their levels
will produce Pi cleavage without translocation of bound
Ca2+. In fact, it was already shown (15) that binding of
Ca2+ or/and ADP is followed by rapid decay of phosphoenzyme
obtained by utilization of Pi in the absence of
Ca2+ (i.e. E-P). Depending on the
experimental conditions, a variable fraction of this phosphoenzyme
yields ATP through productive reverse cycling and the remaining
undergoes hydrolytic cleavage (18). It is also known that enzyme
phosphorylation by Pi is much increased when the
concentration of luminal Ca2+ is high (19), demonstrating
the reversible cleavage and phosphorylation of
E-PCa2.
The Slippage of the Ca2+ Pump and Its Control by
Anions and Curcumin in Skeletal and Cardiac Sarcoplasmic Reticulum*
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-32P]ATP (5)
or by an enzyme-coupled assay (3). Phosphoenzyme intermediate was
determined by incorporation of [-32P]ATP terminal
phosphate onto the ATPase protein, and the time course of its cleavage
was observed following a chase with nonradioactive ATP (11).
Phosphoenzyme intermediate was also obtained by incorporation of
[32P]Pi in the absence of Ca2+,
through the reverse direction of the ATPase reaction (12).
-32P]ATP and [3H]ADP were obtained from
PerkinElmer Life Sciences, curcumin from ICN Pharmaceuticals
(Costa Mesa, CA), and all the other reagents from Sigma.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
View larger version (19K):
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Fig. 1.
ATP utilization and Ca2+
transport by SR vesicles in transient and steady states following
addition of ATP. A, ATP (10 µM) was added
to skeletal SR vesicles (0.15 mg/ml) preincubated with 50 mM MOPS, pH 7.0, 80 mM KCl, 10 mM
MgCl2, and 50 µM CaCl2 at
25 °C, in a Froehlich-Berger chemical quench-flow apparatus (see
"Materials and Methods"). B, ATP (1 mM) was
added to skeletal SR vesicles (10-20 µg of SR protein/ml)
preincubated with 50 mM MOPS, pH 7.0, 100 mM
KCl, 2 mM MgCl2, 120 µM
CaCl2, 100 µM EGTA, 5 mM sodium
azide, and 2 µM Ruthenium Red. 45Ca isotope
tracer was used for measurements of Ca2+ uptake. ATPase was
measured by following release of [32P]Pi from
[ -32P]ATP in A and colorimetrically in
B. TG (2 µM) or EGTA (2 mM
and 2 mM MgCl2) was added where indicated by
arrows in B. Temperature: 25 °C.
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Fig. 2.
Effects of phosphate and oxalate on ATP
utilization and Ca2+ transport by SR vesicles. The
reaction was started by the addition of 1 mM ATP to a
reaction mixture identical to that described for Fig. 1B,
except for the presence of 10 mM
KH2PO4 (A) or 5 mM
oxalate (B). Temperature: 25 °C.
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Fig. 3.
Role of ADP on the slippage of the pump.
The reaction was started by adding 0.1 mM ATP
(A) or 0.1 mM ATP and 0.2 mM ADP
(B) to a reaction mixture identical to that described for
Fig. 1B, except in the presence of 10 mM
KH2PO4. Temperature: 25 °C.
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Scheme 1.
Minimal sequence of partial reactions in
the catalytic and transport cycle of the Ca2+ ATPase.
The solid lines indicate the cycle as originally proposed by
de Meis and Vianna (22), and the dashed lines suggest a
pathway for slippage of the pump. A concentration rise of the
boxed Ca 
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Fig. 4.
ATP consumption and ADP/ATP exchange in the
absence or in the presence of Ca2+ leak. The exchange
experiments were conducted as described under "Materials and
Methods," in the absence ( 
, 
) or in the presence (
, 
) of
5 µM A23187 to render the SR vesicles leaky, thereby
preventing rise of luminal Ca2+. ATP consumption is derived
from light absorption measurements of nucleotide concentration in the
ATP peak. Exchange is estimated from the radioactive ADP incorporated
in ATP (mole/mole). Temperature: 25 °C.
HOH exchange measurements indicate
that this effect is due to increased access of the phosphoenzyme to medium water (17). It is likely that this effect of secondary binding
is also produced by ADP or pseudosubstrates such as TNP-AMP.
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Fig. 5.
Slippage is reduced by lowering the ATP
concentration in the presence of an ATP regenerating system. The
reaction was started by the addition of 0.5 mM
(A) or 0.05 mM (B) ATP, to a reaction
mixture identical to that described for Fig. 1B, except for
the presence of an ATP regenerating system (2 mM
phospho(enol)pyruvate and 25 units of pyruvate kinase/ml). Temperature:
25 °C.
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Fig. 6.
Effect of curcumin on Ca2+ uptake
and ATPase hydrolysis by skeletal SR vesicles. Steady state
activity was measured in a reaction mixture identical to that described
for Fig. 1B, except for the presence of 10 mM KH2PO4. For the ATPase activity
measurements, the enzyme-coupled assay was done in the presence of 2 mM phospho(enol)pyruvate, 150 µM NADH, and 25 units of pyruvate kinase and of lactic dehydrogenase/ml. Temperature:
25 °C.
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Fig. 7.
Phosphate and oxalate concentration
dependence of Ca2+ transport enhancement by curcumin.
The reaction was started by the addition of 1 mM ATP to a
reaction mixture as described for Fig. 1B, except for the
presence of 5 µM curcumin when indicated.
KH2PO4 (A) or oxalate (B)
were present at the concentrations indicated in the figure. In
C and D the ratios of transport rates in the
presence and in the absence of curcumin are shown as functions of the
KH2PO4 or oxalate concentration. Temperature:
25 °C.
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Fig. 8.
Effect of curcumin (5 µM) on Ca2+ uptake and
ATPase activity under optimal conditions. The reaction was started
by the addition of 1 mM ATP to a reaction mixture identical
to that described for Fig. 1B, except for the presence of 5 mM KH2PO4 in A or 0.5 mM oxalate in B. Temperature: 25 °C.
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Fig. 9.
Effect of curcumin (5 µM) on Ca2+ transport by
cardiac SR vesicles. Ca2+ transport was measured under
conditions identical to those described for Fig. 1B, except
for the presence of 10 mM (A) or 50 mM (B) KH2PO4.
Temperature: 25 °C.
1, at
3 °C).
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Fig. 10.
Effect of curcumin (5 or 30 µM) on phosphoenzyme levels and
cleavage. Phosphoenzyme was formed by addition of 10 µM [ -32P]ATP to a reaction mixture
identical to that described for Fig. 1B, except for the
presence of 10 mM KH2PO4, and 35 µg of skeletal SR protein/ml. Following a 10-s incubation at 3 °C,
an isotopic chase was started by the addition of 1 mM
nonradioactive ATP. A, phosphoenzyme levels, before and
after the chase, are given in nanomole/milligram of SR protein.
B, phosphoenzyme levels after the chase are given in
percentage of the level before the chase, and the decay is fitted with
a single exponential. , 
, and 
stand for 0, 5, and 30 µM curcumin. Units: nanomole/milligram protein
(A) or percent in B. Temperature: 3 °C.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
View larger version (19K):
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Scheme 2.
Detailed sequence of partial reactions and
kinetic constants for the catalytic and transport cycle of the
Ca2+ ATPase. Rate-limiting constants were determined
experimentally, while other constants were adjusted as required to
match the experimentally observed cooperative enzyme activation by
Ca2+, formation of intermediates, Pi
production, and Ca2+ transport by the ATPase (14). The
units are s 1 for fist order, and s1
M1 for second order reactions. The overall
Keq is 4.9E+5 under standard
conditions. In this scheme the ' sign denotes the cooperative
transition produced by Ca2+ binding. The ~ and the - denote high and low phosphorylation potentials, respectively. With
reference to Scheme 1, E'Ca, E'Ca2,
ADPE'~P Ca2, and E~P
Ca2 correspond to the E1 state,
while ADPE'-P Ca2, E'-P
Ca2, E'-P Ca, E-P, and E correspond
to the E2 state.
Steady state levels of enzyme intermediates during utilization of ATP
by the Ca2+ ATPase
EP is the
total level of phosphorylated intermediate, and V is the
velocity (nanomole/milligram/second) of Pi production coupled
to transport (2 Ca2+/Pi). ATP is assumed to be 1 mM and medium Ca2+ is 50 µM. ADP and
luminal Ca2+ are specified in the table. Total E is
assumed to be 5 nmol/mg of protein (as observed experimentally in
skeletal SR vesicles), and the intermediate levels are given in
nanomole/milligram.
Under steady state conditions, as the level of Ca2+- and/or ADP-bound phosphoenzyme rises, the level of E-P (i.e. unloaded phosphoenzyme) decreases, with consequent reduction of productive cleavage. In fact, the velocity of each reaction depends on its rate constant and the related intermediate level. Note that ADP/ATP exchange is dependent on ATPE'Ca2 and ADPE'~PCa2. The levels of these two intermediates are increased by high ADP and Ca2+, and ATPE'Ca2 is always higher than ADPE'~PCa2 due to the faster reverse constant. That means that the rate of exchange is higher than net Pi cleavage, as observed experimentally (Fig. 4). On the other hand, due to a slightly unfavorable equilibrium constant for the reverse transition of ADPE-PCa2 to ADPE~PCa2, the steady state level of ADPE-PCa2 remains fairly high, and significant unproductive cleavage occurs in addition to ATP synthesis.
It is noteworthy that a high concentration (mM) of ATP, exceeding the Km (µM), would allow secondary ATP binding to the ADP site, producing an effect similar to that of ADP in raising the concentrations of intermediate (ATPE-PCa2) that, in this case, would undergo entirely unproductive hydrolysis. This effect explains several reports on secondary activation of phosphoenzyme cleavage by mM ATP (16, 17).
Considering that mM concentrations of ATP, ADP, and
Pi are normally present in muscle fibers, it is apparent
that slippage occurs to a significant extent in situ,
concomitant with Ca2+ signaling waves, and may play an
important physiological role for thermogenesis (5). The extent of
slippage is dependent on how effectively cytosolic Ca2+ is
lowered by the action of the pump, and ATP reformed from ADP following
its utilization for cellular functions. Slippage will be significant if
cytosolic Ca2+ remains high enough (µM) to
activate the ATPase (Fig. 1) or the ADP concentration rises (Fig. 3).
In fact, slippage is likely to be of pathological relevance in failing
heart muscle, as expression of SR ATPase lags during the hypertrophic
process, and cytosolic Ca2+ transients are thereby
prolonged (20, 21). It is of interest that velocity and efficiency of
Ca2+ transport are dependent on anion co-transport and that
curcumin has a favorable effect on this dependence, within the
physiological range of cytosolic Pi concentrations. Higher
curcumin concentrations, however, have a direct inhibitory effect on
the Ca2+ ATPase. Nevertheless, the observed control of
slippage by phosphate and curcumin raises the possibility of improving
Ca2+ transport and correcting Ca2+ homeostasis
by pharmacological means.
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FOOTNOTES |
|---|
* This work was supported by National Institutes of Health Program Project HL27867 and by the Human Frontier Science Program.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of Biochemistry
and Molecular Biology, University of Maryland School of Medicine, 108 N. Greene St., Baltimore, MD 21201. Tel.: 410-706-3220; Fax:
410-706-8297; E-mail: ginesi@umaryland.edu.
Published, JBC Papers in Press, February 13, 2002, DOI 10.1074/jbc.M111155200
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ABBREVIATIONS |
|---|
The abbreviations used are: SR, sarcoplasmic reticulum; MOPS, 3-(N-morpholino)propanesulfonic acid; TNP-AMP, 2'-(or 3')-O-(trinitrophenyl)adenosine-5'-monophosphate, sodium salt; TG, thapsigargin.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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