From the University of Kent at Canterbury,
Canterbury, Kent CT2 7NJ, United Kingdom and ¶ St.
Josef-Hospital, Clinic of the Ruhr, University Bochum,
Cardiology, 44791 Bochum, Germany
Received for publication, October 18, 2002, and in revised form, November 26, 2002
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ABSTRACT |
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There are significant isoform differences
between the skeletal and cardiac troponin complexes. Studies of the
regulatory properties of these proteins have previously shown only
significant differences in the calcium dependence of their regulation.
Using a sensitive myosin subfragment 1 (S1) binding assay we
show that in the presence of calcium, thin filaments reconstituted with
either skeletal or cardiac troponin produce virtually identical S1
binding curves. However in the absence of calcium the S1 binding curves
differ considerably. Combined with kinetic measurements, curve
fitting to the three-state thin filament regulatory model shows the
main difference is that calcium produces a 4-fold change in
KT (the closed-open equilibrium) for the
skeletal system but little change in the cardiac system. The
results show a significant difference in the range of regulatory effect
between the cardiac and skeletal systems that we interpret as effects
upon actin-troponin (Tn)I-TnC binding equilibria. As structural data
show that the Ca2+-bound TnC structures differ, the
additional counter-intuitive result here is that with respect to myosin
binding the +Ca2+ state of the two systems is similar
whereas the In striated muscle, the troponin-tropomyosin
(TnTm)1 complex
functions as a calcium-dependent molecular switch that
regulates the force-generating interaction between actin and myosin
filaments. The calcium-induced change in tropomyosin-troponin structure
is not simply a trigger for muscle contraction but produces a graded response to calcium that can modulate the speed and force of the contraction. The molecular mechanism of regulation is generally believed to be via steric blocking in which the position of Tm upon
actin directly obstructs the binding of myosin to actin (reviewed in
Refs. 1 and 2). We use a variant of this regulatory model, the
three-state model of McKillop and Geeves (3, 4), that represents the
filament as existing in a dynamic equilibrium between three
biochemically differentiable states. In this model the position of Tm
is influenced both by allosteric changes in troponin in response to
calcium binding and the binding of myosin to actin. This results in
three biochemical states of the thin filament termed the blocked,
closed, and open states, which correspond closely to three subsequently
determined structural states (5): the B-state, where the Tm position
precludes myosin binding to both strong and weak sites, the C-state, in
which Tm is positioned so that myosin can attach to its weak ionic
binding sites, and the M-state, induced by the rigor binding of myosin
where Tm is further translated allowing access to the rigor binding
sites. The ratio of the three states in the absence of S1 is defined by
the equilibrium constants KB (between the B- and
C-states) and KT (between the C- and M-states).
The B-state has only been shown to be significantly occupied in the
absence of Ca2+ when Tn is tightly bound to the filament.
In the presence of Tn and Ca2+ or when Tn is absent there
is little or no occupancy of the B-state meaning
KB is large.
Major questions remain about the specific roles of Tm and the three Tn
components, TnC, TnI, and TnT. At a simple level we know that TnI is
responsible for the formation of the B (blocked)-state by
its binding to actin (6). TnC, when it binds Ca2+,
allosterically interferes with the TnI-actin interaction and releases
the B-state (7). TnT, through its structural role of tethering TnI and
TnC to Tm, transmits the structural changes to the A7TmTn
structural unit. TnT also influences the Tm-Tm communication along the
filament via its interaction at the Tm-Tm overlap (8) and has been
shown recently to also influence regulation directly. Our own work has
shown the TnT1 fragment of skeletal TnT effects the
open-closed equilibrium significantly reducing
KT (9), whereas work on a similar cardiac
TnT1 fragment has shown strong inhibition of the
actin-activated myosin ATPase (10).
Fast skeletal (hereafter referred to as skeletal) and cardiac muscle
show considerable differences between their troponin isoforms, the most
significant of which are that cardiac TnC has only a single regulatory
calcium binding site as opposed to two in skeletal TnC and that cardiac
TnI has a 32-amino acid N-terminal extension containing two
phosphorylation sites that have been shown to modulate the sensitivity
to calcium (11-13). TnT alternative splicing can produce over 20 different isoforms, mainly by changes in the N-terminal hypervariable
region, suggesting a significant area for functional modulation, but
the effects of these differences are currently largely unclear
(14).
The regulatory properties of skeletal and cardiac troponins had been
compared previously using kinetic measurements of S1 binding to
reconstituted thin filaments to determine the occupancy of the B-state.
This had shown little difference between them, with similar values of
KB (the B to C equilibrium) determined for both
systems (3, 4, 13, 15).
In this study we combine both kinetic and equilibrium measurements to
fully characterize the regulation of the binding of S1 to thin
filaments containing both skeletal and cardiac troponins. We also
examine the effect of the phosphorylation state of TnI, previously
shown to effect the calcium dependence of regulation (12, 13) upon
these measurements under saturating and calcium-free conditions. The
results are analyzed, and the implications of the results are discussed
in terms of the most recent version of the three-state model (4).
Some of this work has been presented previously in a preliminary
form (16).
Isolation of Proteins from Bovine Heart and Rabbit Skeletal
Muscle--
The skTnTm complex, skTn, Tm, actin, and myosin were
isolated from rabbit skeletal muscle. The skTnTm complex and skTn were obtained using the methods of Ebashi et al. (17) and Greaser and Gergely (6), respectively. Tropomyosin was isolated according to
Smillie (18). cTn and PP2A were isolated from bovine heart according to
the method of Beier et al. (19) and Mumby et al. (20) respectively. S1 was prepared by chymotryptic digestion of
rabbit myosin, as described by Weeds and Taylor (21). Actin, purified
from rabbit skeletal muscle according to Spudich and Watt (22), was
labeled at Cys-374 with N-(1-pyrene)iodacetamide according
to Criddle et al. (23). Phalloidin (Roche Molecular Biochemicals)-stabilized F-actin was made by incubating a solution of
10 µM pyrene-actin with 10 µM phalloidin
overnight in standard experimental buffer (20 mM MOPS, pH
7.0, 200 mM KCl, 5 mM MgCl2) at
4 °C. SDS-PAGE of proteins was performed according to Laemmli (24)
using 13.5% acrylamide gels followed by staining with Coomassie Blue
G-250.
cTn Phosphorylation--
Bovine cardiac Tn (1 mg/ml) was
preincubated in 20 mM MOPS, pH 7.0, 300 mM KCl,
2 mM dithiothreitol, 5 mM MgCl2, 1 mM ATP for 5 min at 30 °C. The phosphorylation reaction
was started by the addition of the recombinant catalytic subunit of
protein kinase A (25) (60 milliunits/mg cTn) and was stopped by
freezing after incubation for 120 min at 30 °C. To analyze the
phosphorylation level an aliquot of the reaction mixture was applied
onto a C4 reversed phase column to separate cTnI as described by
Swiderek et al. (26). Isolated cTnI was dried in
vacuo and redissolved in 9 M urea, 2% (v/v)
servalyte, pH 3-10. Isoelectric focusing was performed as described by
Ardelt et al. (27). After staining the gel with Coomassie
Blue the bands of de-, mono- and bisphosphorylated TnI were quantified
by scanning densitometry using Whole Band software (Bioimage).
Thin Filament Assembly--
For the titrations, a stock was made
of 10 µM actin with 10 µM phalloidin added
to stabilize the actin filaments. For each filament type (skeletal or
cardiac) 2 nmol of the 10 µM actin/phalloidin stock (200 µl) was mixed with 8 nmol of Tm and Tn at their stock concentrations
(around 20-40 µM, approx 200-400 µl) and incubated for 1 h at 4 °C. For thin filament assembly with cardiac
troponin, troponin-containing bisphosphorylated cTnI (position 22 and
23 in the amino acid sequence) was used (13). The phosphorylation degree was checked routinely by non-equilibrium isoelectric
focusing. Dephosphorylated cardiac Tn filaments were produced as
described below. The stocks were then diluted in standard experimental
buffer to give final concentrations of 50 nM
actin/phalloidin and 200 nM Tm/Tn in a volume of 2 ml. For
the stopped-flow experiments thin filaments were pre-assembled by
mixing stock solutions to final concentrations of 10 µM
actin, 2 µM Tm, and 2 µM of the appropriate
Tn in standard experimental buffer (20 mM MOPS, pH 7.0, 200 mM KCl, 5 mM MgCl2) and leaving to
equilibrate for 10 min. An excess of regulatory proteins was present in
all assays to ensure correct assembly.
Dephosphorylation of Thin Filaments--
To dephosphorylate cTnI
in the thin filament MnCl2 (final concentration of 5 mM) was added, along with 5 µl of PP2A stock. The
solutions were then incubated overnight at 4 °C before use. To
confirm cTnI dephosphorylation, a portion of thin filaments containing
22 µg of cTn was rephosphorylated in 20 mM MOPS, pH 7, 300 mM KCl, 2 mM dithiothreitol, with 1 mM (final concentration) [ Stopped-flow Fluorescence and S1
Titrations--
Stopped-flow experiments were performed at 20 °C
with either a Hi-Tech Scientific SF-61 or SF-61DX2 spectrophotometer in
fluorescence mode. For both this and the titration experiments, binding
is monitored by the ~70% quenching of the pyrene-actin fluorescence upon binding of the S1 to actin (15). Pyrene fluorescence was excited
at 365 nm, and emission was detected at right angles using a KV 389-nm
cut-off filter (Schott, Mainz, Germany). Data were stored and
analyzed using Kinetasyst software provided with the instrument.
Transients shown are the average of three-five shots of the machine. In
these experiments the concentrations quoted are those before mixing,
the final concentrations being half those quoted for the stock
solutions in the syringes. The occupancy of the B-state (defined by
KB) has been determined using the kinetic method
of McKillop and Geeves (3) where the rate of binding of S1 to
reconstituted filaments is measured under excess actin condition. Under
these conditions the observed rate constant of binding
(kobs) is proportional to the fraction of actin
available for binding and is shown in Equation 1. The value of
KB is then defined from the ratio of the
observed rate constant of S1 binding to reconstituted thin filaments in
the presence and absence of calcium (3).
Fluorescence was measured at 20 °C using a PerkinElmer Life
Sciences 50B spectrofluorimeter exciting at 365 nm with a 10-nm bandwidth and measuring emission at 405 nm with a 15-nm bandwidth. Titrations were performed with a Harvard Apparatus syringe pump (4).
The fraction of actin bound is directly proportional to the change in
pyrene fluorescence measured. The titration curves were then fitted to
the three-state model as described by Maytum et al. (4).
This model is shown in Equation 2 and is defined by the following
parameters: K1 (binding to the A-state),
K2 (A to R isomerization),
KT (the C to M equilibrium),
KB (the B to C equilibrium), and n
(the apparent number of actins being switched between states). Fitting
of the binding curves was by a process of systematic variation of
n and examination of the sum of the residuals and specific
deviations for each value as detailed previously (4).
In Equation 2, Fig. 1 shows the clear differences
between all of the skeletal and cardiac Tn components (TnT, TnI, TnC)
when resolved by SDS-PAGE. As the skeletal Tn used in this study was
not prepared from a single specific muscle it might contain mixed
isoforms of regulatory proteins. However, the only significant isoform variants present are the minor and major TnT isoforms that run above
and below Ca2+ state differs. This shows the regulatory
tuning of the troponin complex produced by isoform variation is the net
result of a complex series of interactions among all the troponin components.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
-32P]ATP (20 µCi), 3 mM MgCl2 by addition of 20 µl of 14 milliunits of protein kinase A catalytic subunit/µl (total volume of
100 µl). Aliquots of 5 µl were used to measure incorporation of
radioactive 32P using a liquid scintillation counter. These
were precipitated on filter papers and washed to remove unbound
[
-32P]ATP. The results were curve-fitted to determine
the maximum level of phosphate incorporation.
(Eq. 1)
(Eq. 2)
represents the fraction of total actin sites
occupied, [M] is the concentration of free S1 heads,
P = 1 + K1[M](1 + K2) and Q = 1 + K1[M].
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
Tm, respectively (Fig. 1, lane 4). The control protein preparation does contain some residual actin. However equivalent titrations using purified skTn and Tm with no actin band
present (data not shown) indicate that this actin is apparently inactive (probably unpolymerizable) and not effecting the assays. The
cardiac preparations also appear to consist mainly of a single cTnT
band running above
Tm, a single band for cTnI significantly larger
than skTnI, and one for cTnC running slightly below skTnC (Fig. 1,
lane 5). Titration and kinetic experiments were repeated with several different batches of cTn, two of which are shown. No
significant difference was found between different batches of cTn.
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Fig. 1.
Purified thin filament proteins used in this
study resolved by SDS-PAGE on a 13.5% gel and stained with Coomassie
Blue. From left to right samples loaded were
as follows: skTm, Actin, and skCp (sk TmTn), followed by two batches of
bovine cardiac troponin (bcTn 111 and 112). The skeletal TnT bands are
not clearly resolved as the isoforms present run between and above the
large /
Tm bands. Proteins are identified as labeled.
Analysis by isoelectric focusing and subsequent scanning densitometry of cardiac Tn after careful treatment with protein kinase A to obtain predominantly bisphosphorylated cTnI showed that about 80% of the cTnI was bisphosphorylated (data not shown). This material was used for reconstitution of thin filaments. Dephosphorylated cTn thin filaments were obtained by treatment of phosphorylated filaments with PP2A as this has been shown previously (28) to produce better regulating filaments.
Dephosphorylation of TnI by PP2A was confirmed by measuring
incorporation of 32P by protein kinase A into
dephosphorylated thin filaments (28). Fig.
2A shows 32P
incorporation into filaments containing cardiac troponin,
dephosphorylated with PP2A. These were referenced against filaments
reconstituted with recombinant cTnI (which has no phosphorylation when
expressed in Escherichia coli). The
"native" filaments incorporate around 1.8 mol of
Pi per mol of TnI, which represents 90% of the maximum from the controls. Under these conditions nearly no unspecific incorporation of phosphate occurs. Protein kinase A binds phosphate specifically to the cTnI subunit at positions 22 and 23 (human sequence) or 23 and 24 (bovine sequence). Separation by SDS-PAGE showed
only a single radioactive labeled cTnI band (not shown). Unspecific
phosphorylation as described by Ward et al. (29) for cardiac
troponin I is observed mainly when using individual subunits or
different conditions. To estimate the extent of dephosphorylation of
thin filaments by PP2A used in the experiments described here, these
protein kinase A-phosphorylated 32P-labeled thin filaments
were then dephosphorylated with PP2A. Fig. 2B shows that the
dephosphorylated thin filaments used for the following experiments
contain around a residual 0.2 mol of phosphate/mol of TnI.
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Kinetic Measurements--
The occupancy of the blocked B-state was
determined from measurements of the rate of binding of S1 to
reconstituted filaments at a 1:10 S1 to actin ratio. The ratio of the
fitted kobs in the presence and absence of
Ca2+ defines KB as per Equation 1
(3). Typical kinetic traces used to determine KB
for cTn are shown in Fig. 3. The results
are similar to those published previously (3, 4, 15), showing a
3-4-fold reduction in kobs for the skeletal
system in the absence of calcium and a 2-3-fold reduction for
the cardiac system (13, 28, 30). Table
I shows the KB
values determined from these rates, which are therefore similar (0.5 for skeletal and 0.7 for cardiac) but with a small reproducible
difference between them. As the rate constants of S1 binding to actin
in the presence of calcium were the same for both systems this means
that the cardiac system is slightly less "off" than the
skeletal system in the absence of calcium. These small differences may
relate to fine tuning of the two regulatory systems for their specific
physiological role or incomplete filament assembly as discussed later.
No significant difference was found between cardiac filaments
containing phosphorylated or dephosphorylated cTnI (13).
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Equilibrium Measurements--
The overall switching of thin
filaments into the open M-state was determined by the
equilibrium S1 titration curves shown in Fig.
4. Representative curves of S1 binding to
filaments containing either skTn or phospho-cTn are shown in Fig.
4A. The binding curves were virtually identical in the
presence of calcium (only a single curve fit is shown for clarity).
However, the S1 binding curves obtained with skTn and cTn in the
absence of calcium clearly differ significantly from each other. The
overall shape of both curves is very similar but with the cTn curve
positioned between the binding curves in the presence of calcium and
the skTn curve in the absence of calcium. Curve fitting of the data
gives values for the parameters as shown in Table I. The values for
K1, which together with
K2, define the overall S1 affinity
(K1·K2) to
actin, are similar for all the filaments, in the range of 1.0-1.1 × 105 s1 M
1.
K2 is fixed at the previously determined
value of 200 for nucleotide-free S1 (3). Within experimental error the
skeletal data show at least a 3-4-fold change in
KT from ~0.12 in the presence of
Ca2+ to ~0.02 in its absence, in agreement with previous
data (4). However, for the cardiac data a good fit is obtained with
KT remaining unchanged at ~0.13 in both the
presence and absence of calcium. These results are expressed in terms
of percentage occupancy of the three thin filament states in Table
II, which shows that in the absence of
calcium the cardiac system has significantly greater occupancy of both
the C- and M-states.
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Thus in the skeletal system the overall shift in the regulatory
equilibria away from the M-state in the absence of calcium is the
result of significant contributions from the changes in both
KB and KT. In contrast,
there is a considerably smaller difference between the two calcium
states for the cardiac system. Fitting shows this is the result of
KT being insensitive to calcium in the cardiac
system, despite a similar occupancy of the B-state (KB 0.7 versus
KB
0.5).
Values for the apparent cooperative unit size (n) are
similar for skeletal and cardiac Tn in both the presence and absence of
calcium. In the absence of calcium both give a value of around 6-7,
which we have suggested previously (4) is because of the "pinning" effect of the TnI, bound strongly once every seven
actins, dominating the cooperativity of the filament. In the
presence of calcium both the skeletal and cardiac filaments show a
similar large apparent cooperative unit size (n 11).
It should be noted, however, that the curves produced in the presence
of calcium have a low sigmoidicity, which occurs when n is
large (n
10). The low sigmoidicity results in the
values of both KT and n not being well determined (31), so there may be some subtle isoform-related differences that are not detected by this experiment. However, the
values for n are significantly higher than the values
determined previously for Tm alone. This shows that cTn has a marked
effect on n and a clear effect of the presence of Tn
+Ca in both systems (4).
For skeletal Tn it has been shown that the TnT subunit, specifically
the TnT1 region, is responsible for increasing the thin filament cooperativity above that seen for Tm alone (from 7-8 to 11)
(4, 8). From this, it appears that within the limitations of the data
as stated above, the effect of cardiac TnT in increasing the apparent
cooperativity is the same as that produced by skeletal TnT.
TnI Phosphorylation State-- The effects of cTnI phosphorylation upon S1 binding are shown in Fig. 4B where overlays of a total of 14 titration datasets of phosphorylated and dephosphorylated cardiac filaments in the presence and absence of calcium are presented with two skeletal datasets in the absence of calcium being shown for reference. As can be seen, titrations of thin filaments containing either dephospho-cTnI or bisphospho-cTnI with S1 produce indistinguishable S1 binding curves in both the presence and absence of calcium. Phosphorylation of TnI therefore seems not to effect the regulation of S1 binding to actin at very high or very low calcium levels. Therefore the parameters that are defined by the shape of the S1 binding curves (specifically KB, KT, and n) are unchanged by phosphorylation under these conditions. This is in agreement with previous measurements of KB, which showed that bisphosphorylation did not significantly effect the maximum and minimum values of the rate of S1 binding to actin (kobs) but did produce a shift in the midpoint of the pCa plot (13). As KT is unchanged under high or low calcium conditions, it seems unlikely that KT is modulated at intermediate calcium concentrations.
Quality of Reconstitution-- For these experiments it is important that the reconstituted filaments are well regulated to ensure that experimental differences are the result of isoform variation rather than the result of poor reconstitution. Poor reconstitution or non-functional Tn components could be expected to produce noticeable effects upon both the titration and kinetic experiments. For example, non-functional TnC would be expected to show a degree of inhibition unregulated by the presence of calcium.
Evidence for good reconstitution of the filaments comes first from the fact that the results from different batches of cTn were indistinguishable in this study. Second, values of kobs for the rate of S1 binding to actin in the presence of calcium and resulting value of KB are similar to several previous measurements for both the skeletal and cardiac systems. Finally, the similarity of the kinetic and titration measurements in the presence of calcium for both systems is also interpreted as evidence of a well regulated system.
Implications of Regulatory Differences-- The basic result of this work is that there are significant differences in the range of regulation of the binding of S1 to actin produced by the cardiac and skeletal troponin isoforms despite the fact that KB was similar for both systems (this work) (13). The question therefore is what is the basis for these differences?
The specific conclusions relating the major effect of the isoform differences to be upon the value of KT are dependent upon the model we use for thin filament regulation. However, the basic results are obviously model-independent and could be interpreted in terms of any of the other recent models proposed (32-35). Likewise the following discussion of how to understand these isoform effects upon switching between on and off states of the filament is generalized and should be applicable to any cooperative model with two or more states.
Although the skeletal and cardiac TnC sequences are 70% identical, one
of the two N-terminal domain regulatory calcium sites is inactivated
because of changes in key ligating residues (36). This means that the
TnI-C interaction is dependent upon the binding of only a single
Ca2+ rather than two Ca2+ for the skeletal
system (37). These differences result in the free energy of
Ca2+ binding (G) being four times smaller for cardiac as
compared with skeletal muscle TnC (38). Thus there is less energy
available for switching of the system, which would be expected to
result in either the cardiac system being less off or less on (or even both) at high and low calcium concentrations. Our data do indeed show a
reduction in difference between S1 binding curves in the presence and
absence of calcium for the cardiac system in reference to the skeletal
system and also show that the on states appear to be nearly the same
for both with the off states differing.
Structural studies have shown that binding of calcium to skeletal TnC results in the opening of the two-domain structure. This results in a change from a compact structure (39, 40) to one with a large hydrophobic patch exposed, which allows its interaction with TnI (41). However cardiac Tn undergoes only a partial opening upon binding of its single regulatory calcium (42) with the additional opening necessary to bind TnI being induced directly by the presence of TnI (43). This implies that cTnC is less on than skTnC in the presence of calcium. Our results show that in fact the cardiac system as a whole has a similar on state in the presence of calcium but is less off in the absence of calcium. The question is whether these results are compatible.
To explain this, we would propose that regulation takes place with TnI
acting as a molecular switch in which the regulatory N-terminal domain
is either strongly bound to actin (off) or strongly bound to TnC (on)
(shown in Equation 3).
![]() |
(Eq. 3) |
This suggests that in the cardiac system the overall result of the isoform changes are such that in the presence of calcium the balance of the two competing equilibria of TnC for TnI and TnI for actin is similar to the skeletal system, resulting in the same on state. This means that the reduction in the calcium sensitive change of affinity of TnC for TnI in the cardiac system produces an off state in the absence of calcium that is less inhibited than for the skeletal system.
Recent work has suggested that TnT may in fact have a significant role
in modulating regulation, which may contribute to the differences
between the two cardiac and skeletal complexes seen here (9, 10).
Although our study characterized skeletal TnT effects using similar
assays to those presented here, the other used motility and ATPase
measurements. Both showed significant inhibition by the two TnTs, but
the different nature of the assays used does not allow direct
comparison of their regulatory effects, which is of significant
interest in light of this work. In combination, this shows that the
fine tuning of regulation by isoform changes in the troponin complex
involves a complex set of interactions between all the regulatory
proteins. Only through combining studies of the effects of changes to
the properties of both the isolated components and complete systems
will we begin to fully understand their nature and effects.
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ACKNOWLEDGEMENT |
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We thank Nancy Adamek for support in the preparation of stock proteins.
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FOOTNOTES |
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* 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.
§ Supported by Wellcome Trust Grant 055841. To whom correspondence should be addressed. Tel.: 44-1227-823950; Fax: 44-1227-763912; E-mail: rmm@ukc.ac.uk.
Supported by the Deutsche Forschungsgemeinschaft.
** Supported by Wellcome Trust Grant 055841.
Published, JBC Papers in Press, December 9, 2002, DOI 10.1074/jbc.M210690200
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ABBREVIATIONS |
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The abbreviations used are: Tn, troponin; Tm, tropomyosin; S1, myosin subfragment 1; skTn, skeletal Tn; cTn, cardiac Tn; MOPS, 4-morpholinepropanesulfonic acid; PP2A, protein phosphatase 2A.
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