Ca2+ Binding to Skeletal Muscle Troponin C in Skeletal and Cardiac Myofibrils1

Abstract

Ca2+ binding to skeletal muscle troponin C in skeletal or cardiac myofibrils was measured by the centrifugation method using 45Ca. The specific Ca2+ binding to troponin C was obtained by subtracting the amount of Ca2+ bound to the CDTA-treated myofibrils (troponin C-depleted myofibrils) from that to the myofibrils reconstituted with troponin C. Results of Ca2+ binding measurement at various Ca2+ concentrations showed that skeletal troponin C had two classes of binding sites with different affinity for Ca2+. The Ca2+ binding of low-affinity sites in cardiac myofibrils was about eight times lower than that in skeletal myofibrils, while the high-affinity sites of troponin C in skeletal or cardiac myofibrils showed almost the same affinity for Ca2+. The Ca2+ sensitivity of the ATPase activity of skeletal troponin C-reconstituted cardiac myofibrils was also about eight times lower than that of skeletal myofibrils reconstituted with troponin C. These findings indicated that the difference in the sensitivity to Ca2+ of the ATPase activity between skeletal and cardiac CDTA-treated myofibrils reconstituted with skeletal troponin C was mostly due to the change in the affinity for Ca2+ of the low-affinity sites on the troponin C molecule.

Full text

J.
Biochem.
105, 435-439 (1989)
Ca2+ Binding to Skeletal Muscle Troponin
C
in Skeletal and
Cardiac Myofibrils1
Sachio Morimoto and Iwao Ohtsuki1
Department of
Pharmacology,
Faculty i>f
Medicine,
Kyushu
University,
Higashi-ku, Fukuoka, Fukuoka 812
Received for publication, September 9, 1988
Ca2+ binding to skeletal muscle troponin C in skeletal or cardiac myofibrils was measured
by the centrifugation method using 45Ca. The specific Ca2+ binding to troponin C was
obtained by subtracting the amount of Ca2+ bound to the CDTA-treated myofibrils (troponin
C-depleted myofibrils) from that to the myofibrils reconstituted with troponin
C.
Results of
Ca2+ binding measurement at various Ca2+ concentrations showed that skeletal troponin C
had two classes of binding sites with different affinity for Ca2+. The Ca2+ binding of
low-affinity sites in cardiac myofibrils was about eight times lower than that in skeletal
myofibrils, while the high-affinity sites of troponin C in skeletal or cardiac myofibrils
showed almost the same affinity for Ca2+. The Ca2+ sensitivity of the ATPase activity of
skeletal troponin C-reconstituted cardiac myofibrils was also about eight times lower than
that of skeletal myofibrils reconstituted with troponin C. These findings indicated that the
difference in the sensitivity to Ca2+ of the ATPase activity between skeletal and cardiac
CDTA-treated myofibrils reconstituted with skeletal troponin C was mostly due to the
change in the affinity for Ca2+ of the low-affinity sites on the troponin C molecule.
CaJ+ regulation of contractile response in myofibrilfl of
vertebrate striated muscle is performed by troponin and
tropomyosin located in the thin filament (1, 2). Troponin
consists of three different components termed troponin C,
I, and T. The first step of Ca2+ action is the binding of this
cation to troponin C and a subsequent change in the
structure of the thin filament enables actin molecules to
make contractile interaction with myosin. Recently we
found that whole troponin C of skeletal or cardiac myo-
fibrila was removed by treating the myofibrils with CDTA,
a strong chelator for Ca2+ and Mg*+ (3, 4). The CDTA-
treated myofibrils were found to lose Ca2+-sensitive
ATPase and to regain it upon the addition of troponin C. In
those studies, it was found that the CDTA-treated cardiac
myofibrils reconstituted with skeletal troponin C required
greatly higher concentrations of Ca2+ for the activation of
the myofibrillar ATPase activity than intact skeletal
myofibrils or CDTA-treated skeletal myofibrils recon-
stituted with skeletal troponin
C
(4). This
finding
has raised
the possibility that the Ca2+ binding to skeletal troponin C
in cardiac myofibrils is different from that in skeletal
myofibrils.
Based on the above considerations, the present study was
undertaken to investigate the Ca2+ binding specific to
skeletal troponin C incorporated into CDTA-treated skele-
tal or cardiac myofibrils. The results indicated the presence
of two classes of binding sites in the troponin C molecule
with high and low affinity for Ca2+. It was found that the
1 This work was supported in part by Grants-in-Aid for Cooperative
Research and for Special Project Research from the Ministry of
Education, Science and Culture of Japan.
*
To whom correspondence should be sent.
Abbreviations: CDTA, fr<ui«-l,2-cyclohexanediamine-iV,N,^'N'-
tetraacetic acid; DTT, dithiothreitol; SDS, sodium dodecyl sulfate;
MOPS,
3-(N-morpholino)propanesulfonic acid.
sites with low affinity of skeletal troponin C in cardiac
myofibrils had greatly lower affinity for Ca2+ than those in
skeletal myofibrils, while the sites with high affinity on
skeletal troponin C in either type of myofibrils had almost
the same affinity for Ca2+. These findings suggest that the
Ca2+ binding to the low-affinity sites on skeletal troponin C
is directly related to the activation of myofibrillar ATPase
activity.
MATERIALS
AND
METHODS
Materials—45CaCl2 and D-[6-3H]glucose were obtained
from Amersham International pic. Soluene-350 was pur-
chased from Packard. CDTA (trans-1,2-cyclohexane-
dianune-AWN'.N'-tetraacetic acid), EGTA, DTT, poly-
acrylamide gel reagents, 2-ethoxyethanol (ethylene glycol
monoethyl ether), butyl-PBD (2-(4-fert-butylphenyl)-5-(4"-
biphenylyl)-l,3,4-oxadiazole), and bis-(MSB) (p-methyl-
styryl)benzene) were purchased from Nacalai Tesque, Inc.
(Kyoto). Sodium azide was from Katayama Chemical
(Osaka). ATP, creatine phosphate and creatine kinase were
from Boehringer Mannheim GmbH. MOPS was from
Dojindo Laboratories (Kumamoto). All chemicals were of
reagent grade and were used without further purification.
Preparation of Myofibrils—Skeletal myofibrils were
prepared from rabbit fast skeletal muscle according to the
method of Perry (5) with slight modifications (6), Cardiac
myofibrils were prepared from porcine cardiac left ven-
tricular muscle by the method of Solaro et al. (7). The
myofibrillar preparations were stored in
50%
(v/v) glycerol
at -20'C. CDTA treatment of skeletal and cardiac myo-
fibrils were performed by the method of Morimoto and
Ohtsuki (3).
Preparation of Skeletal Troponin C—Rabbit skeletal
troponin C was prepared by the method of Ebashi (8).
Vol. 105, No. 3, 1989435

436S. Morimoto and I. Ohtsuki
Reconstitution of CDTA-Treated Skeletal or Cardiac
Myofibrils with Skeletal Troponin C—CDT A-treated
skeletal or cardiac myofibrils were reconstituted with
purified skeletal troponin C as follows. The CDTA-treated
myofibrils (140 mg) were mixed with an excess amount of
troponin C (8.6 mg) in 35 ml of buffer A (20 mM MOPS-
NaOH (pH 7.0), 180 mM
KC1,
0.1%
sodium azide, 0.1 mM
DTT) and centrifuged at
5,000
rpm for 5 min at 4°C after
incubation for 15 min in an ice-cold condition. To remove
unbound and nonspecifically bound troponin C, the result-
ing myonbril precipitate was washed once with 29 ml of
buffer A and further three times with 29 ml of buffer
B
(20
mM MOPS-NaOH (pH7.0), 90 mM KC1, 0.1% sodium
azide, 0.1 mM DTT) by resuspension and centrifugation.
The final precipitate was suspended in 14 ml of buffer
B
and
used for experiments.
SDS-Polyacrylamide Slab Gel Electrophoresis—SDS
slab gel electrophoresis was carried out at 12% polyacryl-
amide concentration according to the method of Laemmli
(9).
The gel was stained with Coomassie Brilliant Blue
R-250 and scanned with a dual-wavelength scanning den-
sitometer (Shimadzu CS-9000).
Measurement of Ca2+ Binding—Ca2* binding to the
CDTA-treated or reconstituted myofibrils was measured
according to the method of Bremel and Weber (10), using
[3H]glucose as a solvent space marker. The measurements
were carried out in 5 ml of reaction mixture containing 1
mg/ml myofibrils, 20 mM MOPS-NaOH (pH 7.0), 100 mM
KC1,
5 mM MgCl2, 2 mM ATP, 0.1 mg/ml creatine kinase,
10 mM creatine phosphate, 0.1 mM dithiothreitol, 10 mM
glucose, 0.3 juCi/ml 45Ca, 0.6 juCi/ml [3H]glucose, 0.1 mM
EGTA, and 0 to 0.17 mM CaCl2. The reaction mixtures
were incubated for about 5 min at 25°C and then
centrifuged at 3,500 rpm for 10 min. After addition of 60
l*\
of acetic acid, the precipitate and 0.25 ml of supernatant
were treated with 1.5 ml of Soluene-350 for 2 h at 50°C
• Skeletal myofibrils —
a b c — Cardiac myofibrils -
d e f
HCHC
—TNI
• - LCI
IC3
Fig. 1. Reconstitution of CDTA-treated skeletal and cardiac
myofibrils with skeletal troponin C. CDTA-treated skeletal and
cardiac myofibrils incubated with an excess amount of purified
skeletal troponin C, followed by successive washing as described in
"MATERIALS AND METHODS," were compared with intact and
CDTA-treated myofibrils on SDS polyacrylamide gel. a, intact
skeletal myofibrils; b, CDTA-treated skeletal myofibrils; c, CDTA-
treated skeletal myofibrils-(-skeletal troponin C; d, intact cardiac
myofibrils; e, CDTA-treated cardiac myofibrils; f, CDTA-treated
cardiac myofibrils+skeletal troponin C. Abbreviations: HC, myosin
heavy chain; A, actin; TM, tropomyosin; LCI, LC2 and
LC3,
myosin
light chain 1, 2, and 3, respectively; TN T, TN I, and TN C, troponin
T, troponin I, and troponin C, respectively.
and mixed with 10 ml of liquid scintillation cocktail com-
posed of 50% (v/v) toluene, 50% (v/v) 2-ethoxyethanol,
0.8%
(w/v) butyl-PBD, and bis-(MSB), and then counted
for <5Ca and 3H using an Aloka LSC-1000.
The amount of Ca2+ contamination in the reaction
mixture was determined by analyzing the supernatant
obtained from the centrifugation in the presence of 2 mM
EGTA with an atomic absorption spectrophotometer (Seiko
SAS 727). Contaminant Ca2+ thus determined was 3-5 //M.
ATPase Assay—ATPase assays were carried out at 25°C
in 2 ml of reaction mixture having the same composition as
those used for the Ca2+-binding measurements but in the
absence of 45Ca and [3H]glucose. The reaction was started
by the addition of ATP and-stopped by the addition of 2 ml
of ice-cold 20% trichloroacetic acid containing 4% ascorbic
acid. Liberated inorganic phosphate after 5 min of incuba-
tion was measured by the method of Baginski et al. (11).
Calculation of the Free Ca2+ Concentration—The free
Ca2+ concentration in the reaction mixture was calculated
by computer using the absolute binding constants for
multiple ions as described elsewhere (4).
Determination of Protein Concentration—Protein con-
centrations for myofibrils and troponin C were determined
by the biuret and Bio-Rad Protein Assay methods, respec-
tively, using those proteins, the concentrations of which
were determined by amino acid analysis, as standards.
RESULTS
AND
DISCUSSION
The SDS-gel electrophoretic pattern in Fig. 1 shows the
effect of CDTA treatment on the skeletal and cardiac
myofibrils. Troponin C and myosin light chain 2 in skeletal
and cardiac myofibrils were selectively extracted by CDTA
treatment (Fig. 1, lanes b and
e).
Densitometric scans of the
gel indicated complete loss of intrinsic troponin C and loss
of about 50% of skeletal and about 30% of cardiac myosin
light chain 2 (Table I). Following incubation with excess
amounts of purified skeletal troponin C and subsequent
washings, the CDTA-treated skeletal and cardiac myo-
fibrils bound a specific amount of skeletal troponin C (Fig.
1,
lanes c and f). The amount of skeletal troponin C bound
to skeletal myofibrils was almost the same as that of
troponin C in intact skeletal myofibrils (Table I). Since
skeletal troponin
C
and cardiac myosin light chain 2 had the
same electrophoretic mobility, incubation with skeletal
troponin C increased the intensity of the band correspond-
TABLE I. Relative amounts of skeletal troponin C incorpo-
rated into skeletal and cardiac myofibrils. Densitometric scans
were performed on the SDS gel in Fig. 1. The peak areas of myosin
light chain 2 and troponin C were normalized to the sum of the peak
areas of myosin light chains 1 and 3 in skeletal myofibrils and to
myosin light chain 1 in cardiac myofibrils. Abbreviations: LC2,
myosin light chain 2; S-TN C, skeletal troponin C; C-TN C, cardiac
troponin C.
TreatmentSkeletal myofibrilsCardiac myofibrils
LC2 S-TN
C
LC2 C-TN
C
S-TN
C
Intact
CDTA
CDTA +
S-TNC
1.00
0.54
0.55
0.22
0.00
0.19
0.57
0.38
0.53
0.11
0.00
0.000.15°
"Estimated peak area for skeletal troponin
C
incorporated into cardiac
myofibrils, on the assumption that the content of myosin light chain
2 is equal to that in the CDTA-treated cardiac myofibrils.
J. Biochem.

Ca1+ Binding to Skeletal Troponin
C
in Myofibrils 437
-] (M)
Fig.
2.
Ca»+-activated ATPase activity
of
the CDTA-treated
skeletal
(o) or
cardiac
(•)
myofibrils reconstituted with
skeletal troponin
C.
ATPase activity was measured under
the
conditions described in "MATERIALS
AND
METHODS"
and activity
was expressed as relative values (r):
r=100x{A-A')/(A"-A'),
A is the ATPase activity. A' and A" are the minimum and nuTimnm
ATPase activity, respectively. Values of
A'
and A" are 0.046±0.005
and
0.771
±0.030//mol Pi/min/mg
for
rabbit skeletal myofibrils,
and 5.6±0.4
and
45.1±0.2nmol Pi/min/mg
for
porcine cardiac
myofibrils. The error bars show the standard error
of
the mean
for
three experiments. The data points were fitted to the following Hill
equation by means of a weighted nonlinear least-squares method:
where K
is
the reciprocal of the concentration of free Ca1
*
required for
the half-maximal activation and
n is
the Hill coefficient.
K
and
n
values derived are listed in Table II. The solid curves
in
the figure
were drawn using these values.
TABLE
II.
Hill equation constants and Ca2+ binding constants
evaluated by curve fitting to the data in Figs.
2
and 4.
PreparationATPaaeCaa* binding
ff(M-)
Skeletal troponin C
incorporated into
skeletal myofibrils
Skeletal troponin C
incorporated into
cardiac myofibrils
2.01 9.07
X101
3.32x10' 4.74x10*
1.98
1.05x10'
6.31x10' 5.78x10*
ing
to
myosin light chain
2 in
the CDTA-treated cardiac
myofibrils (Fig. 1, lane f). The content of skeletal troponin
C bound
to
cardiac myofibrils estimated from
the gel
scanning was in good agreement with that of troponin C in
intact cardiac myofibrils (Table I). These results indicated
that intrinsic troponin C was fully displaced by the extrin-
sically added skeletal troponin C.
Figure
2
shows the Ca1+ dependence of ATPase activity
of the CDTA-treated skeletal
or
cardiac myofibrils recon-
stituted with skeletal troponin C. As reported previously
(4),
the
Ca2+ activation curve
of the
cardiac myofibrils
reconstituted with skeletal troponin C markedly shifted to
higher concentrations of free CaJ+ compared to that of the
skeletal myofibrils reconstituted with skeletal troponin C.
The free CaI+ concentration required for the half-maximal
activation
of
the ATPase activity was 8.6 times higher
in
the cardiac myofibrils than
in
the skeletal myofibrils, but
with the same value
of
Hill coefficient
of
about
2 in
both
Fig.
3.
Ca>+ binding
to
CDTA-treated skeletal
or
cardiac
myofibrils and the CDTA-treated myofibrils reconstituted with
skeletal troponin C. Ca" binding was measured under conditions
identical
to
those for the ATPase assays
in
Fig.
2.
A: opon circles,
CDTA-treated skeletal myofibrils; closed circles, CDTA-treated
skeletal myofibrils reconstituted with skeletal troponin C. B: open
circles, CDTA-treated cardiac myofibrils; closed circles, CDTA-
treated cardiac myofibrils reconstituted with skeletal troponin
C.
The
error bars indicate the standard error of the mean for three experi-
ments. Where not shown, the standard error was less than the size of
the symbol.
cases (Table II). This
is in
agreement with the previous
findings based on superprecipitation and ATPase activity of
actomyosin (Perry's myosin B) in the presence
of
various
combinations
of
hybrid troponin prepared from isolated
skeletal and cardiac troponin components (12,
13).
Ca2+ bindings
to
the CDTA-treated myofibrils
or
those
reconstituted with skeletal troponin
C
were measured
under the same conditions as those for the ATPase assays
(Fig. 3). The amount of CaI+ bound to both the skeletal and
cardiac myofibrils reconstituted with skeletal troponin
C
increased as the concentration of free Ca2+ was raised. The
CDTA-treated skeletal
or
cardiac myofibrils, from which
troponin C was almost completely removed (Fig. 1), still
bound a relatively large amount of Ca2+ without saturation
at high concentrations
of
free Ca2+
and a
small
but
significant amount
of
Ca2+ even
at
low concentrations
of
free Ca2+. Since Ca2+ binding
to
the CDTA-treated myo-
fibrils was considered
to be due to
myosin, actin,
and
unknown Ca2+ binding proteins other than troponin C, Ca2+
binding specific to troponin C incorporated into skeletal or
cardiac myofibrils was obtained by subtracting the amount
of bound Ca2+ in the CDTA-treated myofibrils from that in
the CDTA-treated myofibrils reconstituted with troponin
C.
The
amount
of
Ca2+ bound
to
skeletal troponin
C
incorporated into either skeletal or cardiac myofibrils thus
obtained saturated
at
the high concentrations of free Ca2*
(Fig.
4).
Figure
4
clearly shows that about 50%
of
Ca2+
binding
at low
Ca2+ concentration occurs
at the
same
Vol. 105, No. 3, 1989

438S. Morimoto and I. Ohtsuki
Fig. 4. CaI+ binding to skeletal troponin C incorporated into
skeletal (o) or cardiac (•) myoflbrils. Ca1+ binding specific to
troponin C was obtained, using the data in Fig. 3, by the subtraction
of Ca2+ binding to the CDTA-treated myofibrils from Ca binding to
the CDTA-treated myofibrils reconstituted with troponin C. The
standard error (error bars)
was
estimated by considering the propaga-
tion of
errors.
The data points were fitted to the following equation by
means of a weighted nonlinear least-squares method on the assump-
tion that the halves of the binding sites on troponin C have different
affinities for Ca1+.
N is the fraction of bound Ca1+ normalized to 2. K, and K, are the
intrinsic binding constants of Ca2+ for the first and second class of
binding sites, respectively. Values of Kt and K, derived from curve
fitting are listed in Table II. The solid lines in the figure were drawn
using these values.
bound Ca , %
m
a x.
Fig. 5. Relationship between Ca1+ binding to troponin C and
myoflbrillar ATPase activity. A, skeletal myofibrils reconstitut-
ed with skeletal troponin C; B, cardiac myofibrils substituted with
skeletal troponin C. Data taken from Figs. 2 and 4 were replotted.
concentration range of free Ca2+ in either type of myo-
fibrils, while the remaining 50% of Ca2+ binding in cardiac
myofibrils occurs at much higher Ca2+ concentration com-
pared to that in skeletal myofibrils. This suggests that there
are two classes of binding sites with different affinity for
Ca2+ on skeletal troponin C, and that only the Ca2+ binding
to the lower- affinity sites becomes weaker in cardiac
myofibrils than in skeletal myofibrils.
Curve fittings using the data in Fig. 4, by assuming that
there are two classes of independent sites on skeletal
troponin C incorporated into either skeletal or cardiac
myofibrils, show that the half of the binding sites with high
affinity for Ca2+ has almost the same binding constant in
both types of myofibrils and the remaining half of the
binding sites with low affinity for Ca2+ has an 8.2 times
greater binding constant in skeletal myofibrils than in
cardiac myofibrils (Table II). This 8.2 times reduction of
the Ca2+ binding constant for the low-affinity sites on
skeletal troponin C when incorporated into cardiac myo-
fibrils compared to that in skeletal myofibrils is correlated
with the shift of the Ca2+ activation curve of the ATPase
activity to 8.6 times higher concentration of free Ca2+ (Fig.
2;
Table II). These results indicate that the properties of
only the low-affinity sites on skeletal troponin C are
differently modulated when incorporated into skeletal or
cardiac myofibrils, and that it is due to the reduction of the
Ca2+ binding constant for the low-affinity sites on skeletal
troponin C in cardiac myofibrils that the Ca2+ sensitivity of
ATPase activity of cardiac myofibrils reconstituted with
skeletal troponin C is lower than that of skeletal myofibrils
reconstituted with skeletal troponin C.
It is well known that isolated skeletal troponin or
troponin C has four Ca2+ binding sites which are composed
of
two
classes with different affinity for Ca2+, two sites with
high affinity (Ca2+-Mg2+ sites) and two sites with low
affinity (Ca2+ specific sites ) (14, 15). The physiological
function of the Ca2+-Mg2+ sites and Ca2+ specific sites on
skeletal troponin C is still not clear (16-18). It has been
suggested from studies on Ca2+ binding to isolated troponin
C or troponin that Ca2+ binding to the Ca2+ specific sites is
involved in the regulation of the skeletal muscle contraction
(14, 19, 20). The Ca2+-Mg3+ sites have been suggested to
have a structural function (21), but possible involvement
of
the Ca2+-Mg2+ sites in the regulation of contractile response
has also been discussed (16). The Ca2+ binding properties of
troponin C are altered when troponin C interacts with
troponin I (14) and further modulated when incorporated
into the thin filament (22-24). In addition, the interaction
of rigor and cycling cross-bridges with the thin filaments
also affects the Ca2+ binding properties of troponin C (10,
25).
These facts indicate that to understand the molecular
regulation mechanism of contraction, both the Ca2+ binding
to troponin C and the contractile response have to be
examined in the intact myofibrils under the same condi-
tions.
The fairly good fit of the theoretical curve to the set of
experimental data in Fig. 4 suggests that there are also two
classes of binding sites on skeletal troponin C incorporated
into skeletal or cardiac myofibrils. The Ca2+ binding con-
stant for the low-affinity sites on skeletal troponin C in
skeletal myofibrils (Klt 4.7xlO'M-'; Table II) approxi-
mately coincided with that of isolated troponin (K2, 1.8X
10*
M~") but was much higher than that of isolated troponin
C (Klt 3.0X10* M"1) calculated from the data of Ogawa
(15),
whereas the Ca2+ binding constant for the high-
affinity sites (Ku 3.3 X107 M"1; Table II) was higher than
that of isolated troponin (Ki, 5.5 X10° M~') and troponin C
J. Biochem.

Caz+ Binding to Skeletal Troponin C in Myofibrils 439
•{Ku-
1.4x-108M-1)
-(-15). Decreased Ca2+ affinity of the
low-aflBnity sites on skeletal troponin C in cardiac myo-
fibrils (K,,
S^xl^M"1)
compared to that in skeletal
myofibrils (K2, 4.7 X108 M~l) (Table II) would reflect that
the interactions of the low-affinity sites of skeletal troponin
C with other troponin components are weaker in cardiac
myofibrils than in skeletal myofibrils.
Figure 5 represents the relationships between the rela-
tive ATPase activity and the amount of Ca1+ bound to
skeletal troponin
C
in skeletal or cardiac myofibrils. Almost
the same relationships were obtained whether skeletal
troponin C was incorporated into skeletal or cardiac
myofibrils. This suggests that skeletal troponin
C
regulates
the ATPase activity of CDTA-treated skeletal and cardiac
myofibrils by the same mechanism. In both cases, most of
the ATPase activation occurred in the range of the final
one-quarter of the Ca2+ binding. This also strongly indi-
cates that the fourth Ca2+ binding to the troponin C
molecule is critical for the final step of regulation in
activating myofibrillar ATPase activity. In addition, the
final one-quarter of Ca2+ binding to skeletal troponin C
incorporated into skeleal myofibrils is obviously very steep,
so that the theoretical curve does not exactly fit the
experimental points (Fig. 4), indicating the presence of
positive cooperativity of binding of the fourth Ca2+ to
troponin C. The positive cooperativity also seems to be
present in Ca2+ binding to skeletal troponin C incorporated
into cardiac myofibrils (Fig. 4), though it is not so clear as
in the Ca2+ binding to skeletal troponin C incorporated into
skeletal myofibrils. This suggests the presence of interac-
tions among troponin
C
molecules in the thin filament, since
it is impossible to conceive an intramolecular interaction
through which one troponin C molecule binds one Ca2+
cooperatively. Such cooperative interaction among tropo-
nin C molecules along the thin filament may be related to
the positive cooperativity (Hill coefficient value, 2) ob-
served in the Ca2+-activated myofibrillar ATPase activities
of the CDTA-treated skeletal and cardiac myofibrils recon-
stituted with skeletal troponin C (Fig. 2).
We are grateful to Dr. M. Tanokura for performing the atomic
absorption analysis and to Dr. K. Yamamoto for the use of the
dual-wavelength scanning densitometer.
REFERENCES
1.
Ebashi, S.
&
Endo, M. (1968)
Prog.
Biophys.
Mol. Biol. 18, 123-
183
2.
Ohtsuki, I., Maruyama, K., & Ebashi, S. (1986) Adv. Protein
Chem.
38, 1-67
3.
Morimoto, S. & Ohtsuki, I. (1987) J. Biochem 101, 291-301
4.
Morimoto, S. & Ohtsuki, I. (1988) J.
Biochem.
104, 149-154
5.
Perry, S.V. (1952)
Biochem.
J. 51, 495-499
6. Harigaya, S., Ogawa, Y., & Sugita, H. (1968) J. Biochem. 63,
324-331
7.
Solaro, R.J., Pan, D.C., &Briggn, F.N. (1971)
Biochim.
Biophys.
Ada 245, 259-262
8. Ebashi, S. (1974) in Lipmann Symposium: Energy, Regulation
and
Biosynthesis
in
Molecular Biology
(Richter, D., ed.) pp. 165-
178,
Walter du Gruyter, Berlin
9. Laemmli, U.K. (1970) Nature 227, 680-685
10.
Bremel, R.D.
&
Weber, A. (1972) Nature New
Biol.
238, 97-101
11.
Baginski, E.S., Foa, P.P.,
&
Zak, B. (1967) Clin. Chim Acta 15,
155-158
12.
Ebashi, S. (1980)
Proc.
R. Soc. London Ser. B 207, 259-286
13.
Yamamoto, K. (1983) J.
Biochem.
93, 1061-1069
14.
Potter, J.D.
&
Gergely, J. (1975) J.
BioL
Chem.
260, 4628-4633
15.
Ogawa, Y. (1985) J.
Biochem,
97, 1011-1023
16.
Kohama, K. (1980) J.
Biochem.
88, 591-599
17.
Shiner, J.S.
&
Solaro, R.J. (1982) Proc Natl.
Acad.
Sci. U.S. 79,
4637-4641
18.
Shiner, J.S. (1986) Biophys. J. 50, 601-611
19.
Johnson, J.D., Charlton, S.C., & Potter, J.D. (1979) J. Biol
Chem.
254, 3497-3502
20.
Robertson, S.P., Johnson, J.D.,
&
Potter, J.D. (1981)
Biophys.
J.
34,
559-569
21.
Zot, H.G.
&
Potter, J.D. (1982) J. Biol. Chem 257, 7678-7683
22.
Zot, H.G., Iida, S.,
&
Potter, J.D. (1983)
Chem.
Scr. 21,133-136
23.
Grabarek, Z., Grabarek, J., Leavis, P.C.,
&
Gergely, J. (1983) J.
Biol.
Chem.
258, 14098-14102
24.
Wnuk, W., Schoechlin, M., & Stein, E.A. (1984) J. BioL Chem.
259,9017-9023
25.
Guth, K.
&
Potter, J.D. (1987) J.
Biol.
Chem.
262, 13627-13635
Vol. 105, No. 3, 1989