Vol. 181, No. 3, 1991 December 31, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages
1022-1027
REPLACEMENT OF THREE TROPONIN COMPONENTS WITH CARDIAC TROPONIN COMPONENTS WITHIN SINGLE GLYCERINATED SKELETAL MUSCLE FIBERS MasamitsuHatakenakaand Iwao Ohtsuki Departmentof Pharmacology,Faculty of Medicine, Kyushu University, Fukuoka 812, Japan Received
November
11,
1991
The tension of single glycerinated rabbit skeletal muscle fiber was desensitizedto a Ca2+concentrationafter treatmentwith an excessiveamountof bovine cardiac troponin T and reacheda level of about 70% of the maximum tension of the untreated fiber. A SDS-gel electrophoretic examinationindicated that troponin C-IT complex in the fiber was replacedwith the addedcardiac troponin T. The Ca2+-sensitivity of the tensionof the troponin T-treated fiber was then recovered by the addition of bovine cardiac troponins I and C. The rabbit skeletal muscle fiber thus hybridized with bovine cardiactroponin C1.T showedthe samecooperativity of Ca2+-activationas Press, Inc. the cardiacmuscle. 0 I.991 Academic
The contraction-relaxation cycle between myosin and actin in the myofibrils of vertebrate striatedmusclesis regulatedby Ca2+through the specific regulatory proteins,termed troponin and tropomyosin, located in the thin filament. Troponin is a complex of three different components, troponins C, I and T. Troponin C is a Ca2+-binding component. Troponin I is an inhibitory component of myosin-actin interaction in the presenceof tropomyosin, and troponin T is a tropomyosin binding component. All troponin componentsare requiredfor the Ca2+-regulationof contraction (1). Recently, it was found that both troponins C and I were removed from fiber bundlesor single fibers of glycerinated rabbit skeletal muscle after treatment with an excessive amount of rabbit skeletaltroponin T (2, 3). The tension of troponin T-treated fibers was desensitizedto Ca2+and again sensitizedto Ca2+by reconstitution with troponins I and C (3). This demonstrateda new way of approach for replacing troponin componentswithin the skinned or glycerinated muscle fiber models. Basedon the above considerations,the presentstudy was undertakento examine the effect of treatmentwith an excessiveamountof bovine cardiac troponin T on the singleglycerinated rabbit skeletalmusclefibers. The resultsindicated that a complex of troponins C, I and T in the fibers was exchanged by the added cardiac troponin T. The tension of troponin T-treated fiber was 0006-291x/91 Copyright All rights
$1.50
0 I991 by Academic Press, Inc. of reproduction in any form reserved.
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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
mostly desensitized to Ca2+ and resensitized to Ca2+ by reconstitution with troponins I and C. The skeletal fibers thus hybridized with cardiac troponins C, I and T thereafter showed the same cooperativity of Ca2+-activation as the cardiac muscle. MATERIALS
AND METHODS
Small bundles of rabbit psoas muscle (about 2- 3 mm in thickness and 10 cm in length) were dissected, tied to glass rods and then soaked in a solution containing 100 mM KCI, 20 mM 3-(Nmorpholino)propanesulfonic acid (MOPS)-KOH (pH 7.0) 5 mM MgCl2, 2 mM [ethylenebis (oxonitrilo)]tetraacetic acid (EGTA), 2 mM ATP, 2.5 mM dithiothreitol (DTT), and 1% (V/V) T&on-X100 for 30- 40 minutes at 2°C. They were then transferred into a solution with the above mentioned composition but containing 50% (V/V) glycerol instead of 1% T&on-X100, left for 12 hours at 2°C and then stored at -20°C. Rabbit skeletal troponin components were prepared by the procedures described previously (4). Bovine cardiac troponin was prepared by the method of Tsukui and Ebashi (5) and its components were separated by the procedure of Tobacman and Lee (6). Tension-pCa relation of a single glycerinated rabbit skeletal muscle fiber was measured by increase of Ca2+ concentration as described previously (7). The composition of the relaxing solution was as follows; 100 mM KCl, 20 mM MOPS-KOH (pH 7.0), 5 mM MgC12, 2 mM EGTA, 2 mM ATP, 10 mM creatine phosphate, 38 U/ml creatine kinase. The calcium concentration in the activating solution was adjusted by adding 0.2-2 mM CaC12 to the relaxing solution. Ionic strength was about 165 mM. All procedures were carried out at 25°C. Sarcomere length was kept at 2.2-2.3 pm by the laser diffraction. SDS polyacrylamide gel electrophoresis was carried out according to the method of Laemmli (8). The gel was stained with a silver staining kit (Wako Pure Chemical Industries, LTD.) and scanned using a densitometer with a wave length of 633 nm (CS-90, Shimadzu Corporation, Kyoto, Japan). RESULTS AND DISCUSSION Fig. 1 shows the tension records from single glycerinated rabbit skeletal muscle fibers on the successive treatments with skeletal or cardiac troponins T, I and C. The Ca2+-activated tension of the control fiber was almost completely desensitized to Ca2+ after treatment with skeletal troponin T (1 ms/ml) for 60 min and reached a level of about 70% of the maximum tension of the control fiber irrespective of Ca2+- concentrations (3). The tension of the troponin T-treated fiber was completely relaxed by replacing ATP in the relaxing solution with 5 mM adenosine-5’-0- (3thiotriphosphate)
(ATP [-yS]) (Fig. 1). The SDS-gel electrophoretic pattern of the troponin T-
treated fiber showed that the amount of both troponins C and I had decreased (Fig. 2-A). The tension of the troponin T-treated fiber was inhibited after troponin I-treatment regardless of Ca2+concentrations and regained the Ca2+-sensitivity after treatment with troponin C, as also reported previously (3) (Fig. 1-A). When the single glycerinated skeletal muscle fiber was treated with bovine cardiac troponin T (1 mg/ml) for 90 min, the tension of the fiber was also mostly desensitized to Ca2+ and generated a tension of about 70% of the control maximum tension (Fig. 1-B). The tension of the troponin T 1023
Vol.
181, No. 3, 1991
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
A 1Omg
L 105
ATP[+mM ATP2mM
/ /i
-
-
Tension
ATPIyslSmM ATP2mM
-! ‘:
t-
7
i
Fie. 1. Effect of successivetreatments with troponin T, troponin I and troponin C on the Caz+-activated tension development of single glycerinated rabbit skeletal musclefibers. Speciesof troponincomponents for treatments:uppertraces(A), rabbitskeletal; lowertraces(B), bovinecardiac. Conditionof treatments: i) troponinT treatment;the fiber was incubated,at 25°C in the solutioncontaining1.0mg troponinT, 250mM KCI, 20 mM MOPSKOH (pH 6.5). 5 mM MgCl2, 2 mM EGTA, 0.1 pg/ml pepstatinA, and 0.5 mM DTT: ii) troponinI-treatment;the fiber wasincubated,at 25’C, for 30 min in the solutioncontainingI mg/mltroponinI, 250mM KCI, 20 mM MOPS-KOH(pH 7.0), 5 mM MgCl2,2 mM EGTA, 0.1 @ml pepstatin A and0.5mM Dll? iii) troponinC-treatment; thefiber wasincubated,at 25”C,for 30 min in the solutioncontainingI mg/mltroponinC, 100mM KCI, 20 mM MOPS-KOH (pH 7.0), 5 mM MgCl2,2 mM EGTA, 0.1 @ml pepstatinA and0.5 mM DTT. Fiberswerewashed with thesolutioncontainingingredients of relaxingsolutionexceptATP (rigorsolution)beforeand afterthetreatments with troponincomponents. Concentrationsof Ca2+(pCa) usedfor the stepwiseactivationof the tension;8.71 (relaxing solution,O), 7.05, 6.71, 6.47, 6.28, 6.19, 6.11, 6.02, 5.93 (A), 5.74, 5.16,4.43 (Cl).
treated fiber varied within a range of usually lessthan 10% of the control maximum tension by changingCa2+-concentrations.The densitometricpattern of the SDS-gel of the fiber treated with cardiac troponin T showedthat the amount of troponins C, I and T decreasedand a new band at the positionof cardiac troponin T appeared(Fig. 2-B). This indicated that troponin C.1.T complex asa whole wasreplacedwith addedtroponin T to someextent in the skeletalfiber, in accord with 1024
Vol.
181, No. 3, 1991
A
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
LCl
Fiber
treated with traponin T
i’ (II Fiber treated with troponin T.1.C
Fig. 2. Densitometric profiles of the SDS-gel electrophoretic pattern of single glycerinated rabbit skeletal muscle fibers treated with either rabbit skeletal (A) or bovine cardiac (B) troponin components.Upper, control; middle, after treatmentwith troponinT, lowest,after treatmentwith troponinT, I andC. Abbreviation; HC, myosinheavy chain;A, actin ; LC-I, -2 and-3, myosinlight chains-l,-2 and-3respectively;TM, tropomyosin; T, I and C, troponin-T, -I and -C, respectively.Positionof skeletal(A) and cardiac ( V ) troponincomponents areindicatedin thefigure.
recent findings on the rabbit skeletal myofibrils treated with chicken skeletal troponin T or its 26K fragment (9). The tension of the fiber treated with cardiac troponin T was inhibited regardlessof Caz+-concentmtions after treatment with troponin I and resensitized to Ca2+ by the further addition of troponin C. The SDS-gel electrophoretic pattern of the fully reconstituted fiber revealed that cardiac troponins C, I and T were incorporated into the fiber (Fig. 2-B4owest ). It was noted that the pCa at half maximum tension (pK) for the skeletal fiber reconstituted with cardiac troponin C.1.T was slightly but definitely larger than that reconstituted with skeletal troponin C.1.T (Fig. 3, 1025
Vol.
181, No. 3, 1991
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
1
0.75 s a f 0.5 s '.Z 2 t
0.25
0 9
6
7
6
5
4
PCs
Fig. 3. Tension-pCa relation of single glycerinated skeletal muscle fibers, of which three troponin components were replaced with either skeletal or cardiac troponin components. 0, control; 0, replaced with skeletal troponin C.I.T, A, replaced with cardiac troponin C.1.T. Parameters of the tension-pCa curves are summarized in Table I.
Table I). This would meanthat, in the skeletal fiber, the affinity of cardiac troponin complex to Ca2+is higher than that of skeletaltroponin complex. The Hill coefficient value of the Ca2+-activated tension averaged reconstituted with cardiac troponin
C.1.T was 1.78 and much lower
from
the three fibers
than the value of the fiber
reconstituted with skeletal troponin components,3.13 (Table I). Kitazawa first showedthat the Table I. Change in the parameters of the Ca2+-activated tension of single glycerinated rabbit skeletal muscle fibers reconstituted with either skeletal or cardiac troponin C.I.T.1)
Condition of the muscle fibers
number of experiments
Hill coefficient2)
6
4.51kO.78
5.78fo.02
Fibers reconstituted with
skeletaltroponinC1.T
3
3.13f0.17
5.97f0.02
Fibers reconstituted with cardiactroponinCe1.T
3
I .78+0.22
6.17S.02
Controlfibers
pK2)
(4
1)Data of Fig. 3 were expressed relative to the maximum tension of the each experimental stage and fitted to the equation described below by using least-square method (Simplex algorithm).
Relativetension= [Ca2+ln/(Kn+fCaz+]n) n isHill coefficientandK isconstant.pK(-IogK)isthe pCa value at half maximum tension. 2)Values are expressed as means f standard errors.
1026
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No. 3, 1991
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AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
tensionof the glycerinated rabbit cardiac muscle wasactivated much lesscooperatively by Ca2+ than the single glycerinated rabbit skeletal fiber (10). Recent studieshave also reported that the Hill coefficient values of the Ca2+-activatedtension of the cardiac muscle bundlesand skeletal musclefibers of the rabbit were 1.8 and 4.3, respectively (11, 12). The Hill coefficient values of Ca2+-activation of the myofibrillar ATPase were also reported to be 2.0 for porcine cardiac myofibrils and 3.6 for rabbit skeletal myofibrils, respectively (13). These findings strongly suggest that one of the characteristicpropertiesin the contraction of cardiac muscle, i.e., the lower
cooperativity of the Ca2+-activation comparedto the fast skeletal muscle,is mainly due to the property of troponin C1.T complex. The resultsof the presentstudy demonstratedthat each of the troponin musclefiber can be replacedwith troponin componentsof various muscles.
componentsin a single Thus, the troponin T-
treatment procedureseems useful for investigating the mechanismof troponin componentsunder physiological conditions,in situ. Acknowledgment : This work was supportedin part by Grant-in-Aid for Cooperative Research from the Ministry of Education, Scienceand Culture of Japan.
REFERENCES 1. Ohtsuki, I., Maruyama, K., and Ebashi, S. (1986) Adv. Prot. Chem. 38, l-67. 2. Nakayama, Y., Aoki, H., Watanabe, K., and Yamaguchi, M. (1990) Jpn. J. Physiol. 40, 403-410. 3. Hatakenaka,M., and Ohtsuki, I. (1990) Abstracts in 28th Annual Meeting of the Biophysical Society of JapanS281 (in Japanese) 4. Ebashi, S. (1974) In “Lipmann Symposium:Energy, Biosynthesisand Regulation in Molecular Biology” (Richter, D., ed.), pp. 165-178,Walter du Gruyter, Berlin. 5. Tsukui, R., and Ebashi, S. (1973) J. Biochem. (Tokyo), 73, 1119-l 121. 6. Tobacman, L. S., and Lee, R. (1987) J. Biol. Chem. 262, 4059-4064. 7. Morimoto, S., Fujiwara, T., and Ohtsuki, I. (1988) J. Biochem. 104, 873-874. 8. Laemmli, U. K. (1970) Nature. 227, 680-685. 9. Shiraishi, F., Kambara, M., and Ohtsuki, I. (1992) J. Biochem. 111 (l), in press. 10. Kitazawa, T. (1976) J. Biochem. 80, 1129-1147. 11. Putkey, J. A., Liu, W., and Sweeney, H. L. (1991) .f. Biol. Chem.266, 14481-14484. 12. Solaro, R. J., Lee, J. A., Kentish, J. C., and Allen, D. J. (1988) Circ. Res.63, 779-787. 13. Morimoto, S., and Ohtsuki, I. (1987) J. Biochem. 104, 149-154.
1027
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages
1022-1027
REPLACEMENT OF THREE TROPONIN COMPONENTS WITH CARDIAC TROPONIN COMPONENTS WITHIN SINGLE GLYCERINATED SKELETAL MUSCLE FIBERS MasamitsuHatakenakaand Iwao Ohtsuki Departmentof Pharmacology,Faculty of Medicine, Kyushu University, Fukuoka 812, Japan Received
November
11,
1991
The tension of single glycerinated rabbit skeletal muscle fiber was desensitizedto a Ca2+concentrationafter treatmentwith an excessiveamountof bovine cardiac troponin T and reacheda level of about 70% of the maximum tension of the untreated fiber. A SDS-gel electrophoretic examinationindicated that troponin C-IT complex in the fiber was replacedwith the addedcardiac troponin T. The Ca2+-sensitivity of the tensionof the troponin T-treated fiber was then recovered by the addition of bovine cardiac troponins I and C. The rabbit skeletal muscle fiber thus hybridized with bovine cardiactroponin C1.T showedthe samecooperativity of Ca2+-activationas Press, Inc. the cardiacmuscle. 0 I.991 Academic
The contraction-relaxation cycle between myosin and actin in the myofibrils of vertebrate striatedmusclesis regulatedby Ca2+through the specific regulatory proteins,termed troponin and tropomyosin, located in the thin filament. Troponin is a complex of three different components, troponins C, I and T. Troponin C is a Ca2+-binding component. Troponin I is an inhibitory component of myosin-actin interaction in the presenceof tropomyosin, and troponin T is a tropomyosin binding component. All troponin componentsare requiredfor the Ca2+-regulationof contraction (1). Recently, it was found that both troponins C and I were removed from fiber bundlesor single fibers of glycerinated rabbit skeletal muscle after treatment with an excessive amount of rabbit skeletaltroponin T (2, 3). The tension of troponin T-treated fibers was desensitizedto Ca2+and again sensitizedto Ca2+by reconstitution with troponins I and C (3). This demonstrateda new way of approach for replacing troponin componentswithin the skinned or glycerinated muscle fiber models. Basedon the above considerations,the presentstudy was undertakento examine the effect of treatmentwith an excessiveamountof bovine cardiac troponin T on the singleglycerinated rabbit skeletalmusclefibers. The resultsindicated that a complex of troponins C, I and T in the fibers was exchanged by the added cardiac troponin T. The tension of troponin T-treated fiber was 0006-291x/91 Copyright All rights
$1.50
0 I991 by Academic Press, Inc. of reproduction in any form reserved.
1022
Vol. 181, No. 3, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
mostly desensitized to Ca2+ and resensitized to Ca2+ by reconstitution with troponins I and C. The skeletal fibers thus hybridized with cardiac troponins C, I and T thereafter showed the same cooperativity of Ca2+-activation as the cardiac muscle. MATERIALS
AND METHODS
Small bundles of rabbit psoas muscle (about 2- 3 mm in thickness and 10 cm in length) were dissected, tied to glass rods and then soaked in a solution containing 100 mM KCI, 20 mM 3-(Nmorpholino)propanesulfonic acid (MOPS)-KOH (pH 7.0) 5 mM MgCl2, 2 mM [ethylenebis (oxonitrilo)]tetraacetic acid (EGTA), 2 mM ATP, 2.5 mM dithiothreitol (DTT), and 1% (V/V) T&on-X100 for 30- 40 minutes at 2°C. They were then transferred into a solution with the above mentioned composition but containing 50% (V/V) glycerol instead of 1% T&on-X100, left for 12 hours at 2°C and then stored at -20°C. Rabbit skeletal troponin components were prepared by the procedures described previously (4). Bovine cardiac troponin was prepared by the method of Tsukui and Ebashi (5) and its components were separated by the procedure of Tobacman and Lee (6). Tension-pCa relation of a single glycerinated rabbit skeletal muscle fiber was measured by increase of Ca2+ concentration as described previously (7). The composition of the relaxing solution was as follows; 100 mM KCl, 20 mM MOPS-KOH (pH 7.0), 5 mM MgC12, 2 mM EGTA, 2 mM ATP, 10 mM creatine phosphate, 38 U/ml creatine kinase. The calcium concentration in the activating solution was adjusted by adding 0.2-2 mM CaC12 to the relaxing solution. Ionic strength was about 165 mM. All procedures were carried out at 25°C. Sarcomere length was kept at 2.2-2.3 pm by the laser diffraction. SDS polyacrylamide gel electrophoresis was carried out according to the method of Laemmli (8). The gel was stained with a silver staining kit (Wako Pure Chemical Industries, LTD.) and scanned using a densitometer with a wave length of 633 nm (CS-90, Shimadzu Corporation, Kyoto, Japan). RESULTS AND DISCUSSION Fig. 1 shows the tension records from single glycerinated rabbit skeletal muscle fibers on the successive treatments with skeletal or cardiac troponins T, I and C. The Ca2+-activated tension of the control fiber was almost completely desensitized to Ca2+ after treatment with skeletal troponin T (1 ms/ml) for 60 min and reached a level of about 70% of the maximum tension of the control fiber irrespective of Ca2+- concentrations (3). The tension of the troponin T-treated fiber was completely relaxed by replacing ATP in the relaxing solution with 5 mM adenosine-5’-0- (3thiotriphosphate)
(ATP [-yS]) (Fig. 1). The SDS-gel electrophoretic pattern of the troponin T-
treated fiber showed that the amount of both troponins C and I had decreased (Fig. 2-A). The tension of the troponin T-treated fiber was inhibited after troponin I-treatment regardless of Ca2+concentrations and regained the Ca2+-sensitivity after treatment with troponin C, as also reported previously (3) (Fig. 1-A). When the single glycerinated skeletal muscle fiber was treated with bovine cardiac troponin T (1 mg/ml) for 90 min, the tension of the fiber was also mostly desensitized to Ca2+ and generated a tension of about 70% of the control maximum tension (Fig. 1-B). The tension of the troponin T 1023
Vol.
181, No. 3, 1991
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
A 1Omg
L 105
ATP[+mM ATP2mM
/ /i
-
-
Tension
ATPIyslSmM ATP2mM
-! ‘:
t-
7
i
Fie. 1. Effect of successivetreatments with troponin T, troponin I and troponin C on the Caz+-activated tension development of single glycerinated rabbit skeletal musclefibers. Speciesof troponincomponents for treatments:uppertraces(A), rabbitskeletal; lowertraces(B), bovinecardiac. Conditionof treatments: i) troponinT treatment;the fiber was incubated,at 25°C in the solutioncontaining1.0mg troponinT, 250mM KCI, 20 mM MOPSKOH (pH 6.5). 5 mM MgCl2, 2 mM EGTA, 0.1 pg/ml pepstatinA, and 0.5 mM DTT: ii) troponinI-treatment;the fiber wasincubated,at 25’C, for 30 min in the solutioncontainingI mg/mltroponinI, 250mM KCI, 20 mM MOPS-KOH(pH 7.0), 5 mM MgCl2,2 mM EGTA, 0.1 @ml pepstatin A and0.5mM Dll? iii) troponinC-treatment; thefiber wasincubated,at 25”C,for 30 min in the solutioncontainingI mg/mltroponinC, 100mM KCI, 20 mM MOPS-KOH (pH 7.0), 5 mM MgCl2,2 mM EGTA, 0.1 @ml pepstatinA and0.5 mM DTT. Fiberswerewashed with thesolutioncontainingingredients of relaxingsolutionexceptATP (rigorsolution)beforeand afterthetreatments with troponincomponents. Concentrationsof Ca2+(pCa) usedfor the stepwiseactivationof the tension;8.71 (relaxing solution,O), 7.05, 6.71, 6.47, 6.28, 6.19, 6.11, 6.02, 5.93 (A), 5.74, 5.16,4.43 (Cl).
treated fiber varied within a range of usually lessthan 10% of the control maximum tension by changingCa2+-concentrations.The densitometricpattern of the SDS-gel of the fiber treated with cardiac troponin T showedthat the amount of troponins C, I and T decreasedand a new band at the positionof cardiac troponin T appeared(Fig. 2-B). This indicated that troponin C.1.T complex asa whole wasreplacedwith addedtroponin T to someextent in the skeletalfiber, in accord with 1024
Vol.
181, No. 3, 1991
A
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
LCl
Fiber
treated with traponin T
i’ (II Fiber treated with troponin T.1.C
Fig. 2. Densitometric profiles of the SDS-gel electrophoretic pattern of single glycerinated rabbit skeletal muscle fibers treated with either rabbit skeletal (A) or bovine cardiac (B) troponin components.Upper, control; middle, after treatmentwith troponinT, lowest,after treatmentwith troponinT, I andC. Abbreviation; HC, myosinheavy chain;A, actin ; LC-I, -2 and-3, myosinlight chains-l,-2 and-3respectively;TM, tropomyosin; T, I and C, troponin-T, -I and -C, respectively.Positionof skeletal(A) and cardiac ( V ) troponincomponents areindicatedin thefigure.
recent findings on the rabbit skeletal myofibrils treated with chicken skeletal troponin T or its 26K fragment (9). The tension of the fiber treated with cardiac troponin T was inhibited regardlessof Caz+-concentmtions after treatment with troponin I and resensitized to Ca2+ by the further addition of troponin C. The SDS-gel electrophoretic pattern of the fully reconstituted fiber revealed that cardiac troponins C, I and T were incorporated into the fiber (Fig. 2-B4owest ). It was noted that the pCa at half maximum tension (pK) for the skeletal fiber reconstituted with cardiac troponin C.1.T was slightly but definitely larger than that reconstituted with skeletal troponin C.1.T (Fig. 3, 1025
Vol.
181, No. 3, 1991
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
1
0.75 s a f 0.5 s '.Z 2 t
0.25
0 9
6
7
6
5
4
PCs
Fig. 3. Tension-pCa relation of single glycerinated skeletal muscle fibers, of which three troponin components were replaced with either skeletal or cardiac troponin components. 0, control; 0, replaced with skeletal troponin C.I.T, A, replaced with cardiac troponin C.1.T. Parameters of the tension-pCa curves are summarized in Table I.
Table I). This would meanthat, in the skeletal fiber, the affinity of cardiac troponin complex to Ca2+is higher than that of skeletaltroponin complex. The Hill coefficient value of the Ca2+-activated tension averaged reconstituted with cardiac troponin
C.1.T was 1.78 and much lower
from
the three fibers
than the value of the fiber
reconstituted with skeletal troponin components,3.13 (Table I). Kitazawa first showedthat the Table I. Change in the parameters of the Ca2+-activated tension of single glycerinated rabbit skeletal muscle fibers reconstituted with either skeletal or cardiac troponin C.I.T.1)
Condition of the muscle fibers
number of experiments
Hill coefficient2)
6
4.51kO.78
5.78fo.02
Fibers reconstituted with
skeletaltroponinC1.T
3
3.13f0.17
5.97f0.02
Fibers reconstituted with cardiactroponinCe1.T
3
I .78+0.22
6.17S.02
Controlfibers
pK2)
(4
1)Data of Fig. 3 were expressed relative to the maximum tension of the each experimental stage and fitted to the equation described below by using least-square method (Simplex algorithm).
Relativetension= [Ca2+ln/(Kn+fCaz+]n) n isHill coefficientandK isconstant.pK(-IogK)isthe pCa value at half maximum tension. 2)Values are expressed as means f standard errors.
1026
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181,
No. 3, 1991
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
tensionof the glycerinated rabbit cardiac muscle wasactivated much lesscooperatively by Ca2+ than the single glycerinated rabbit skeletal fiber (10). Recent studieshave also reported that the Hill coefficient values of the Ca2+-activatedtension of the cardiac muscle bundlesand skeletal musclefibers of the rabbit were 1.8 and 4.3, respectively (11, 12). The Hill coefficient values of Ca2+-activation of the myofibrillar ATPase were also reported to be 2.0 for porcine cardiac myofibrils and 3.6 for rabbit skeletal myofibrils, respectively (13). These findings strongly suggest that one of the characteristicpropertiesin the contraction of cardiac muscle, i.e., the lower
cooperativity of the Ca2+-activation comparedto the fast skeletal muscle,is mainly due to the property of troponin C1.T complex. The resultsof the presentstudy demonstratedthat each of the troponin musclefiber can be replacedwith troponin componentsof various muscles.
componentsin a single Thus, the troponin T-
treatment procedureseems useful for investigating the mechanismof troponin componentsunder physiological conditions,in situ. Acknowledgment : This work was supportedin part by Grant-in-Aid for Cooperative Research from the Ministry of Education, Scienceand Culture of Japan.
REFERENCES 1. Ohtsuki, I., Maruyama, K., and Ebashi, S. (1986) Adv. Prot. Chem. 38, l-67. 2. Nakayama, Y., Aoki, H., Watanabe, K., and Yamaguchi, M. (1990) Jpn. J. Physiol. 40, 403-410. 3. Hatakenaka,M., and Ohtsuki, I. (1990) Abstracts in 28th Annual Meeting of the Biophysical Society of JapanS281 (in Japanese) 4. Ebashi, S. (1974) In “Lipmann Symposium:Energy, Biosynthesisand Regulation in Molecular Biology” (Richter, D., ed.), pp. 165-178,Walter du Gruyter, Berlin. 5. Tsukui, R., and Ebashi, S. (1973) J. Biochem. (Tokyo), 73, 1119-l 121. 6. Tobacman, L. S., and Lee, R. (1987) J. Biol. Chem. 262, 4059-4064. 7. Morimoto, S., Fujiwara, T., and Ohtsuki, I. (1988) J. Biochem. 104, 873-874. 8. Laemmli, U. K. (1970) Nature. 227, 680-685. 9. Shiraishi, F., Kambara, M., and Ohtsuki, I. (1992) J. Biochem. 111 (l), in press. 10. Kitazawa, T. (1976) J. Biochem. 80, 1129-1147. 11. Putkey, J. A., Liu, W., and Sweeney, H. L. (1991) .f. Biol. Chem.266, 14481-14484. 12. Solaro, R. J., Lee, J. A., Kentish, J. C., and Allen, D. J. (1988) Circ. Res.63, 779-787. 13. Morimoto, S., and Ohtsuki, I. (1987) J. Biochem. 104, 149-154.
1027
