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Calpain proteolysis of free and bound forms of calponin, a troponin T-like protein in smooth muscle
Volume 250, number 2, 493-496
FEB 07336
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Calpain proteolysis of free and bound forms of calponin, a troponin T-like protein in smooth muscle S h o j i T s u n e k a w a *÷, K a t s u h i t o T a k a h a s h i °, M a s a h i r o A b e °, K u n i o H i w a d a °, K a z u e O z a w a ÷ a n d Takashi Murachi*
*Department of Clinical Science and Laboratory Medicine and +Second Department of Surgery, Faculty of Medicine, Kyoto University, Sakyo-ku, Kyoto 606, Japan and °Second Department of Internal Medicine, Ehime University School of Medicine, Onsen-gun, Ehime 791-02, Japan Received 6 May 1989 Calponin, a novel homologue of troponin T, purified from chicken gizzard was found to be one of the most susceptible proteins among smooth muscle contraction-associated proteins to hydrolysis by calpain I purified from human red blood cells. The high susceptibility of calponin was comparable to that reported for troponin T. The rate of degradation of calponin, unlike caldesmon and myosin light chain kinasc, was accelerated when bound to calmodulin. When calponin existed as a bound form in both reconstituted actin filament and native thin filament, the rate of proteolysis was markedly retarded, indicating close association of calponin with actin filament. These observations are compatible with the view that calponin is an integral part of the actin-linked contractile machinery in smooth muscle. Calpain; Proteolysis; Smooth muscle; Calmodulin-binding protein; Troponin T; Thin filament
1. I N T R O D U C T I O N R e c e n t d i s c o v e r y o f a t r o p o n i n T-like p r o t e i n in s m o o t h muscle, r e f e r r e d to as c a l p o n i n [1,2], elicited novel a p p r o a c h e s to the s t u d y o f s m o o t h muscle contraction. Calponin isolated from c h i c k e n g i z z a r d s m o o t h m u s c l e was f o u n d to b i n d c a l m o d u l i n , F - a c t i n , a n d t r o p o m y o s i n , suggesting its a c t u a l i n v o l v e m e n t in s m o o t h muscle c o n t r a c t i o n in a w a y e q u i v a l e n t to t h a t for t r o p o n i n T in skeletal muscle [3,4]. C a l p a i n ( E C 3.4.22.17) is a C a 2 + - d e p e n d e n t cysteine e n d o p e p t i d a s e , which is k n o w n to be very w i d e l y d i s t r i b u t e d in a n i m a l tissues (for review see [5]), i n c l u d i n g s m o o t h muscles o f b o v i n e b l o o d vessels [6] a n d c h i c k e n g i z z a r d [7]. I n c r e a s e d d e g r a d a t i o n o f m y o f i b r i l l a r p r o t e i n s at elevated int r a c e l l u l a r C a z+ c o n c e n t r a t i o n s have been freq u e n t l y r e p o r t e d [8,9]. Thus, the p u r p o s e o f the
Correspondence address: T. Murachi, Department of Clinical Science and Laboratory Medicine, Faculty of Medicine, Kyoto University, Sakyo-ku, Kyoto 606, Japan
p r e s e n t s t u d y was to d e t e r m i n e i f c a l p o n i n c o u l d be p r o t e o l y z e d b y c a l p a i n a n d , if so, h o w such p r o teolysis w o u l d be a f f e c t e d b y c a l p o n i n b i n d i n g to other contraction-associated proteins.
2. M A T E R I A L S A N D M E T H O D S
2.1. Smooth muscle proteins Actin [10], myosin [IlL myosin light chain kinase [12], calponin [3], calmodulin (Takahashi, K. et al., unpublished), tropomyosin and caldesmon [13] were purified from chicken gizzard. They were practically devoid of contamination (fig. 1). The thin filaments were prepared in the 'native' state from chicken gizzard as described [14]. F-actin-bound calponin was prepared at a molar ratio of actin monomer to calponin of 7 [15]. 2.2. Proteolysis by calpain Proteolysis was carried out at 25°C in 1.0 ml incubation mixture containing 0.4 mg of the substrate protein, 0.49 unit (or 3.3/zg) of calpain I purified from human red blood cells [16], 20 mM Tris-HCl buffer (pH 7.5), 100 mM NaCI, 0.5 mM DTT, 5 mM cysteine and 0.5 mM CaClz. Calmodulin was added to the reaction mixture to saturate the ealmodulin-binding sites of the substrate protein: molar ratios to calmodulin were 0.25 for calponin, and 0.5 for caldesmon monomer and myosin
Published by Elsevier Science Publishers B. V. (Bioinedical Division) 00145793/89/$3.50 © 1989 Federation of European Biochemical Societies
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Fig.1. SDS-PAGE of crude actomyosin and purified proteins from chicken gizzard smooth muscle. Lane 1, crude actomyosin (40/zg protein); lane 2, myosin (7/zg); lane 3, caldesmon (7/~g); lane 4, myosin light chain kinase (6/zg); lane 5, actin (10/eg); lane 6, tropomyosin (10/~g); lane 7, calponin (8/zg); lane 8, calmodulin (9/zg). The acrylamide concentration used for lanes 1-4 was 10070and for lanes 5-8, 12.5070. MHC, myosin heavy chain; CD, caldesmon; A, actin; TM, /5' subunit of tropomyosin; CN, calponin; MLC, myosin light chain; k, kDa.
light chain kinase. For F-actin-bound calponin, the incubation mixture contained 4.3 mg of substrate protein, corresponding to 0.4 mg of calponin as estimated by densitometric analysis on SDS-PAGE. For the proteolysis of native thin filament, the final protein concentration was 3.5 mg/ml, corresponding to approximately 0.15 mg of calponin and 0.15 mg of caldesmon. The reaction was stopped at time intervals by transferring an aliquot of the reaction mixture into SDS-PAGE sample buffer. 2.3. Other materials and methods The pattern of proteolysis was analyzed by SDS-PAGE in the buffer system of Laemmli [17]. Densitometric scanning was performed with a Toyo digital densitometer, Densitol, model DMU-33C (Tokyo, Japan) at 620 nm. Protein concentration was determined by the method of Bradford [18] using bovine immunoglobulin as the standard. Immunoblot analysis was carried out with anti-calponin and anti-caldesmon antibody as described [19]. 3. R E S U L T S
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Fig.2. Susceptibility of individual proteins purified from smooth muscle to calpaln I. The logarithmic ordinate shows the percentage of densitometric intensity of a remaining protein band having the same mobility on SDS-PAGE as the starting material after various times of incubation. (Or) Actin; (×) myosin; (<>) tropomyosin; (+) calmodulin; (o,e) calponin; ( n , a) myosin light chain kinase; (A, A) caldesmon. For the last three substrate proteins: open symbols indicate an incubation in the absence of calmodulin; closed symbols indicate an incubation in the presence of calmodulin.
ting m a t e r i a l , was p l o t t e d as a f u n c t i o n o f the inc u b a t i o n time. S e m i - l o g a r i t h m i c plots gave an a l m o s t linear decrease in the early p h a s e o f the r e a c t i o n , i n d i c a t i n g t h a t the p r o t e o l y t i c r e a c t i o n f o l l o w e d a p p a r e n t f i r s t - o r d e r kinetics. This led us t o calculate, in c o m m o n l o g a r i t h m s , a p p a r e n t f i r s t - o r d e r rate c o n s t a n t , kapp, for each reaction. T h e kavp values for c a l p o n i n , c a l d e s m o n a n d m y o s i n light c h a i n kinase were 8.1 x 10 -3 s -1, 9.6 x 10 -3 s -1 a n d 3.1 x 10 -3 s -1, respectively. C a l p o n i n (34 k D a ) was d e g r a d e d into 30, 27, a n d 19.5 k D a f r a g m e n t s b e f o r e f u r t h e r extensive d i g e s t i o n (fig.3A). A l t h o u g h t r o p o m y o s i n r e m a i n ed i n t a c t within 90 s, a smaller p o r t i o n o f its 7 s u b u n i t with a n a p p a r e n t m o l e c u l a r m a s s o f 50 k D a was g r a d u a l l y d e g r a d e d t h e r e a f t e r , p r o d u c i n g a m a j o r f r a g m e n t at 43 k D a a n d several m i n o r f r a g m e n t s at 3 0 - 2 0 k D a . F - a c t i n , m y o s i n , a n d c a l m o d u l i n were resistant to c a l p a i n u n d e r the s a m e c o n d i t i o n s (fig.2).
3.1. Susceptibifity to calpain
3.2. Effect o f calmodulin
A m o n g the p r o t e i n s s h o w n in f i g . l , c a l p o n i n a n d c a l d e s m o n were the m o s t susceptible to h y d r o l y s i s b y c a l p a i n I, f o l l o w e d b y m y o s i n light c h a i n k i n a s e (fig.2). T h e p e r c e n t a g e o f densitometric intensity on SDS-PAGE of a remaining b a n d , which r e t a i n e d the s a m e m o b i l i t y as the star-
C a l p o n i n was d e g r a d e d m o r e r a p i d l y w h e n inc u b a t e d with c a l p a i n in the presence o f 4 times m o l a r excess o f c a l m o d u l i n . T h e kavv value c a l c u l a t e d was 1.8 x 10 -2 s -1, which was 2.2 times h i g h e r t h a n t h a t f o u n d in t h e absence o f c a l m o d u l i n (fig.2). H o w e v e r , the d e g r a d a t i o n p a t -
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Fig.3. Degradation pattern of calponin by calpain I in the absence (A) and in the presence (B) of calmodulin (12.5% SDS-PAGE gels). The incubation periods were: lane 1, 0 min; lane 2, 30 s; lane 3, 60 s; lane 4, 90 s; lane 5, 2 min; lane 6, 3 min; lane 7, 5 min; lane 8, 15 min; lane 9, 30 min, lane 10, 60 min. Lane M, molecular mass markers (k, kDa). The weakly stained proteins of 31, 30 and 22 kDa found even at 0 min represent the contamination of degradation products of calponin (34 kDa) during the purification procedures. terns o f calponin o n S D S - P A G E were similar in the presence and absence o f calmodulin (fig.3). C a l m o d u l i n did not markedly affect the d e g r a d a t i o n o f caldesmon (kapv = 8.3 × 10 -3 s-X), while it decreased t h e rate o f degradation o f m y o s i n light chain kinase (k~pp = 1.1 × 10 -3 s-X). These observations were in agreement with the previous report o f Kosaki et al. [20]. 3.3. Degradation of F-actin-bound calponin T h e degradation rate o f F - a c t i n - b o u n d calponin was r e m a r k a b l y retarded. The degradation o f c a l p o n i n became visible on S D S - P A G E only after 10 min o f incubation (graphic d a t a not shown), a n d calculated kavp value was 5.8 x 10 -5 s -1, which was 1/140th o f that f o u n d without F-actin, i.e. free f o r m o f calponin.
n o t be identified, because n o n e o f anti-actin ( A m e r s h a m ) , anti-caldesmon, or anti-calponin antibodies corss-reacted with 31 k D a band. It could have originated f r o m intermediate filaments possibly contaminating the native thin filament p r e p a r a t i o n used, since intermediate filaments are also k n o w n to be g o o d substrates o f calpain [21]. 4. D I S C U S S I O N The present study d e m o n s t r a t e d that calponin was a g o o d substrate for calpain I a m o n g the
3.4. Degradation of calponin located on native
thin filament Since F - a c t i n - b o u n d calponin was highly resistant to calpain digestion, 9.8 times m o r e calpain I was added to the incubation mixture. C a l p o n i n was degraded completely after 3 h-incubation (kapp = 1.6 × 10 -4 s-l), while caldesmon was d e g r a d e d almost completely within 3 min (kapp = 7.3 X 10 -3 s-l). Actin and t r o p o m y o s i n were well preserved t h r o u g h o u t the incubation period (fig.4). By i m m u n o b l o t analysis, several fragments o f 1 7 - 1 4 k D a seen after 20-s incubation were identified as the degradation products o f actin. The origin o f 31 k D a f r a g m e n t (lanes 2 - 5 , fig.4) could
Fig.4. Degradation of native thin filament by calpain I (12.5% SDS-PAGE gel). The incubation periods were: lane 1, 0 min; lane 2, 20 s; lane 3, 50 s; lane 4, 80 s; lane 5, 3 min; lane 6, 20 min; lane 7, 40 min; lane 8, 60 min; lane 9, 2 h; lane 10, 3 h; lane 11, 4 h. Lane M, molecular mass markers (k, kDa). CD, caldesmon; CP, heavy subunit of calpain I; A, actin; TM, B subunit of tropomyosin; CN, calponin. 495
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smooth muscle contraction-associated proteins. Note that calponin is similar in its characteristics to troponin T of skeletal and cardiac muscle [1], and troponin T is also reported to be quite susceptible to calpain [8,22]. In the presence of calmodulin, calponin was degraded by calpain about twice more rapidly than in the absence of calmodulin when kapp values were compared. Calmodulin may have caused a conformational change of the calponin molecule resulting in higher susceptibility to calpain. This is compatible with the loss of the tropomyosin-binding ability of calponin in the presence of Ca 2+ and calmodulin [41. When calponin was bound to F-actin, it became markedly resistant to calpain digestion. It was degraded about 140 times more slowly in terms of kapp values. The kapp value for calponin in the native thin filaments was 1.6 x 10 -4 s -1, which may correspond to 1.6 x 10-3 s -1 under the assay conditions used for free calponin, since 9.8 times more enzyme was added in the experiment with native thin filament (fig.4) than in the experiment with free calponin (figs 2 and 3). We concluded that the thin filament-bound form of calponin was degraded 500 times more slowly than the free form of calponin. Such a marked decrease in susceptibility may have been caused not only by actin filament but also by the other components such as tropomyosin and caldesmon. Caldesmon bound to the thin filaments was degraded 13 times more slowly than the free form of caldesmon. Calponin and caldesmon are Factin-binding proteins [3,13,23] and susceptible to calpain to almost the same extent when tested in each free form. However, calponin was degraded about 50 times less rapidly than caldesmon when tested as the thin filament-bound forms. This may be related to the structural difference between caldesmon and calponin in constituting thin filament. For example, caldesmon is an elongated and highly flexible protein [24-26] which would extend over the length of 70-150 nm along the axis of the actin filaments, while calponin may be a globular protein [1,3] which is more deeply buried in actin filaments than caldesmon. Such close association of calponin with actin filament could further support the view that calponin is an integral part of smooth muscle thin filament, which is vital to the actin-linked mechanism of contraction [3,15]. 496
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Acknowledgements: We thank Dr T.R. Shearer for his critical reading of the manuscript, and Nobuko Ohmori for her clerical assistance. This work was supported by Grants-in-Aid for Scientific Research and for Special Project Research (Metabolic Studies of Blood Vessels, no.6212005) from the Ministry of Education, Science and Culture, Japan, and by a Japan Heart Foundation Research Grant (K.T.).
REFERENCES [1] Takahashi, K., Hiwada, K. and Kokubu, T. (1988) Hypertension 11,620-626. [2] Takahashi, K., Abe, M., Hiwada, K. and Kokubu, T. (1988) J. Hypertension 6, $40-$43. [3] Takahashi, K., Hiwada, K. and Kokubu, T. (1986) Biochem. Biophys. Res. Commun. 141, 20-26. [4] Takahashi, K., Abe, M., Hiwada, K. and Kokubu, T. (1988) Cell Structure Function 13, 691. [5] Murachi, T. (1989) Biochem. Int. 18, 263-294. [6] Coolican, S.A. and Hathaway, D.R. (1984) J. Biol. Chem. 259, 11627-11630. [7] Hathaway, D.R., Werth, D.K. and Haeberle, J.R. (1982) J. Biol. Chem. 257, 9072-9077. [8] Toyo-oka, T. and Masaki, T. (1979) J. Mol. Cell. Cardiol. ll, 769-786. [9] Leonard, J.P. and Salpeter, M.M. (1979) J. Cell Biol. 82, 811-819. [10] Spudich, J.A. and Watt, S. (1971) J. Biol. Chem. 246, 4866-4871. Ill] Persechini, A., Mrwa, U. and Hartshorne, D.J. (1981) Biochem. Biophys. Res. Commun. 98, 800-805. [12] Sobue, K., Morimoto, K., Kanda, K., Fukunaga, K., Miyamoto, E. and Kakiuchi, S. (1982) Biochem. Int. 5, 503-510. [13] Bretscher, A. (1984) J. Biol. Chem. 259, 12873-12880. [14] Marston, S.B. and Lehman, W. 0985) Biochem. J. 231, 517-522. [15] Takahashi, K., Abe, M., Hiwada, K. and Kokubu, T. (1988) Abstr., Sixth Int. Symp. on Calcium-binding Proteins in Health and Disease, July, Nagoya, p,69. [16] Hatanaka, M., Kikuchi, T. and Murachi, T. (1983) Biomed. Res. 4, 381-388. [17] Laemmli, U.K. (1970) Nature 227, 680-685. [18] Bradford, M.M. (1976) Anal. Biochem. 72, 248-254. [19] Takahashi, K., Hiwada, K. and Kokuhu, T. (1987) Life Sci. 41,291-296. [20] Kosaki, G., Tsujinaka, T., Kambayashi, J., Morimoto, K., Yamamoto, K., Yamagami, K., Sobue, K. and Kakiuchi, S. (1983) Biochem. Int. 6, 767-775. [21] Nelson, W.J. and Traub, P. (1982) J. Biol. Chem. 257, 5544-5553. [22] Ishiura, S., Sugita, H., Suzuki, K. and Imahori, K. (1979) J. Biochem. (Tokyo) 86, 579-581. [23] Sobue, K., Muramoto, Y., Fujita, M. and Kakiuchi, S. (1981) Proc. Natl. Acad. Sci. USA 78, 5652-5655. [24] Fiirst, D.O., Cross, R.A., De Mey, J. and Small, J.V. (1986) EMBO J. 5, 251-257. [25] Lynch, W.P., Riseman, V.M. and Bretscher, A. (1987) J. Biol. Chem. 262, 7429-7437. [26] Graceffa, P., Wang, C.-L.A. and Stafford, W.F. (1988) J. Biol. Chem. 263, 14196-14202.
FEB 07336
July 1989
Calpain proteolysis of free and bound forms of calponin, a troponin T-like protein in smooth muscle S h o j i T s u n e k a w a *÷, K a t s u h i t o T a k a h a s h i °, M a s a h i r o A b e °, K u n i o H i w a d a °, K a z u e O z a w a ÷ a n d Takashi Murachi*
*Department of Clinical Science and Laboratory Medicine and +Second Department of Surgery, Faculty of Medicine, Kyoto University, Sakyo-ku, Kyoto 606, Japan and °Second Department of Internal Medicine, Ehime University School of Medicine, Onsen-gun, Ehime 791-02, Japan Received 6 May 1989 Calponin, a novel homologue of troponin T, purified from chicken gizzard was found to be one of the most susceptible proteins among smooth muscle contraction-associated proteins to hydrolysis by calpain I purified from human red blood cells. The high susceptibility of calponin was comparable to that reported for troponin T. The rate of degradation of calponin, unlike caldesmon and myosin light chain kinasc, was accelerated when bound to calmodulin. When calponin existed as a bound form in both reconstituted actin filament and native thin filament, the rate of proteolysis was markedly retarded, indicating close association of calponin with actin filament. These observations are compatible with the view that calponin is an integral part of the actin-linked contractile machinery in smooth muscle. Calpain; Proteolysis; Smooth muscle; Calmodulin-binding protein; Troponin T; Thin filament
1. I N T R O D U C T I O N R e c e n t d i s c o v e r y o f a t r o p o n i n T-like p r o t e i n in s m o o t h muscle, r e f e r r e d to as c a l p o n i n [1,2], elicited novel a p p r o a c h e s to the s t u d y o f s m o o t h muscle contraction. Calponin isolated from c h i c k e n g i z z a r d s m o o t h m u s c l e was f o u n d to b i n d c a l m o d u l i n , F - a c t i n , a n d t r o p o m y o s i n , suggesting its a c t u a l i n v o l v e m e n t in s m o o t h muscle c o n t r a c t i o n in a w a y e q u i v a l e n t to t h a t for t r o p o n i n T in skeletal muscle [3,4]. C a l p a i n ( E C 3.4.22.17) is a C a 2 + - d e p e n d e n t cysteine e n d o p e p t i d a s e , which is k n o w n to be very w i d e l y d i s t r i b u t e d in a n i m a l tissues (for review see [5]), i n c l u d i n g s m o o t h muscles o f b o v i n e b l o o d vessels [6] a n d c h i c k e n g i z z a r d [7]. I n c r e a s e d d e g r a d a t i o n o f m y o f i b r i l l a r p r o t e i n s at elevated int r a c e l l u l a r C a z+ c o n c e n t r a t i o n s have been freq u e n t l y r e p o r t e d [8,9]. Thus, the p u r p o s e o f the
Correspondence address: T. Murachi, Department of Clinical Science and Laboratory Medicine, Faculty of Medicine, Kyoto University, Sakyo-ku, Kyoto 606, Japan
p r e s e n t s t u d y was to d e t e r m i n e i f c a l p o n i n c o u l d be p r o t e o l y z e d b y c a l p a i n a n d , if so, h o w such p r o teolysis w o u l d be a f f e c t e d b y c a l p o n i n b i n d i n g to other contraction-associated proteins.
2. M A T E R I A L S A N D M E T H O D S
2.1. Smooth muscle proteins Actin [10], myosin [IlL myosin light chain kinase [12], calponin [3], calmodulin (Takahashi, K. et al., unpublished), tropomyosin and caldesmon [13] were purified from chicken gizzard. They were practically devoid of contamination (fig. 1). The thin filaments were prepared in the 'native' state from chicken gizzard as described [14]. F-actin-bound calponin was prepared at a molar ratio of actin monomer to calponin of 7 [15]. 2.2. Proteolysis by calpain Proteolysis was carried out at 25°C in 1.0 ml incubation mixture containing 0.4 mg of the substrate protein, 0.49 unit (or 3.3/zg) of calpain I purified from human red blood cells [16], 20 mM Tris-HCl buffer (pH 7.5), 100 mM NaCI, 0.5 mM DTT, 5 mM cysteine and 0.5 mM CaClz. Calmodulin was added to the reaction mixture to saturate the ealmodulin-binding sites of the substrate protein: molar ratios to calmodulin were 0.25 for calponin, and 0.5 for caldesmon monomer and myosin
Published by Elsevier Science Publishers B. V. (Bioinedical Division) 00145793/89/$3.50 © 1989 Federation of European Biochemical Societies
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Fig.1. SDS-PAGE of crude actomyosin and purified proteins from chicken gizzard smooth muscle. Lane 1, crude actomyosin (40/zg protein); lane 2, myosin (7/zg); lane 3, caldesmon (7/~g); lane 4, myosin light chain kinase (6/zg); lane 5, actin (10/eg); lane 6, tropomyosin (10/~g); lane 7, calponin (8/zg); lane 8, calmodulin (9/zg). The acrylamide concentration used for lanes 1-4 was 10070and for lanes 5-8, 12.5070. MHC, myosin heavy chain; CD, caldesmon; A, actin; TM, /5' subunit of tropomyosin; CN, calponin; MLC, myosin light chain; k, kDa.
light chain kinase. For F-actin-bound calponin, the incubation mixture contained 4.3 mg of substrate protein, corresponding to 0.4 mg of calponin as estimated by densitometric analysis on SDS-PAGE. For the proteolysis of native thin filament, the final protein concentration was 3.5 mg/ml, corresponding to approximately 0.15 mg of calponin and 0.15 mg of caldesmon. The reaction was stopped at time intervals by transferring an aliquot of the reaction mixture into SDS-PAGE sample buffer. 2.3. Other materials and methods The pattern of proteolysis was analyzed by SDS-PAGE in the buffer system of Laemmli [17]. Densitometric scanning was performed with a Toyo digital densitometer, Densitol, model DMU-33C (Tokyo, Japan) at 620 nm. Protein concentration was determined by the method of Bradford [18] using bovine immunoglobulin as the standard. Immunoblot analysis was carried out with anti-calponin and anti-caldesmon antibody as described [19]. 3. R E S U L T S
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Fig.2. Susceptibility of individual proteins purified from smooth muscle to calpaln I. The logarithmic ordinate shows the percentage of densitometric intensity of a remaining protein band having the same mobility on SDS-PAGE as the starting material after various times of incubation. (Or) Actin; (×) myosin; (<>) tropomyosin; (+) calmodulin; (o,e) calponin; ( n , a) myosin light chain kinase; (A, A) caldesmon. For the last three substrate proteins: open symbols indicate an incubation in the absence of calmodulin; closed symbols indicate an incubation in the presence of calmodulin.
ting m a t e r i a l , was p l o t t e d as a f u n c t i o n o f the inc u b a t i o n time. S e m i - l o g a r i t h m i c plots gave an a l m o s t linear decrease in the early p h a s e o f the r e a c t i o n , i n d i c a t i n g t h a t the p r o t e o l y t i c r e a c t i o n f o l l o w e d a p p a r e n t f i r s t - o r d e r kinetics. This led us t o calculate, in c o m m o n l o g a r i t h m s , a p p a r e n t f i r s t - o r d e r rate c o n s t a n t , kapp, for each reaction. T h e kavp values for c a l p o n i n , c a l d e s m o n a n d m y o s i n light c h a i n kinase were 8.1 x 10 -3 s -1, 9.6 x 10 -3 s -1 a n d 3.1 x 10 -3 s -1, respectively. C a l p o n i n (34 k D a ) was d e g r a d e d into 30, 27, a n d 19.5 k D a f r a g m e n t s b e f o r e f u r t h e r extensive d i g e s t i o n (fig.3A). A l t h o u g h t r o p o m y o s i n r e m a i n ed i n t a c t within 90 s, a smaller p o r t i o n o f its 7 s u b u n i t with a n a p p a r e n t m o l e c u l a r m a s s o f 50 k D a was g r a d u a l l y d e g r a d e d t h e r e a f t e r , p r o d u c i n g a m a j o r f r a g m e n t at 43 k D a a n d several m i n o r f r a g m e n t s at 3 0 - 2 0 k D a . F - a c t i n , m y o s i n , a n d c a l m o d u l i n were resistant to c a l p a i n u n d e r the s a m e c o n d i t i o n s (fig.2).
3.1. Susceptibifity to calpain
3.2. Effect o f calmodulin
A m o n g the p r o t e i n s s h o w n in f i g . l , c a l p o n i n a n d c a l d e s m o n were the m o s t susceptible to h y d r o l y s i s b y c a l p a i n I, f o l l o w e d b y m y o s i n light c h a i n k i n a s e (fig.2). T h e p e r c e n t a g e o f densitometric intensity on SDS-PAGE of a remaining b a n d , which r e t a i n e d the s a m e m o b i l i t y as the star-
C a l p o n i n was d e g r a d e d m o r e r a p i d l y w h e n inc u b a t e d with c a l p a i n in the presence o f 4 times m o l a r excess o f c a l m o d u l i n . T h e kavv value c a l c u l a t e d was 1.8 x 10 -2 s -1, which was 2.2 times h i g h e r t h a n t h a t f o u n d in t h e absence o f c a l m o d u l i n (fig.2). H o w e v e r , the d e g r a d a t i o n p a t -
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Fig.3. Degradation pattern of calponin by calpain I in the absence (A) and in the presence (B) of calmodulin (12.5% SDS-PAGE gels). The incubation periods were: lane 1, 0 min; lane 2, 30 s; lane 3, 60 s; lane 4, 90 s; lane 5, 2 min; lane 6, 3 min; lane 7, 5 min; lane 8, 15 min; lane 9, 30 min, lane 10, 60 min. Lane M, molecular mass markers (k, kDa). The weakly stained proteins of 31, 30 and 22 kDa found even at 0 min represent the contamination of degradation products of calponin (34 kDa) during the purification procedures. terns o f calponin o n S D S - P A G E were similar in the presence and absence o f calmodulin (fig.3). C a l m o d u l i n did not markedly affect the d e g r a d a t i o n o f caldesmon (kapv = 8.3 × 10 -3 s-X), while it decreased t h e rate o f degradation o f m y o s i n light chain kinase (k~pp = 1.1 × 10 -3 s-X). These observations were in agreement with the previous report o f Kosaki et al. [20]. 3.3. Degradation of F-actin-bound calponin T h e degradation rate o f F - a c t i n - b o u n d calponin was r e m a r k a b l y retarded. The degradation o f c a l p o n i n became visible on S D S - P A G E only after 10 min o f incubation (graphic d a t a not shown), a n d calculated kavp value was 5.8 x 10 -5 s -1, which was 1/140th o f that f o u n d without F-actin, i.e. free f o r m o f calponin.
n o t be identified, because n o n e o f anti-actin ( A m e r s h a m ) , anti-caldesmon, or anti-calponin antibodies corss-reacted with 31 k D a band. It could have originated f r o m intermediate filaments possibly contaminating the native thin filament p r e p a r a t i o n used, since intermediate filaments are also k n o w n to be g o o d substrates o f calpain [21]. 4. D I S C U S S I O N The present study d e m o n s t r a t e d that calponin was a g o o d substrate for calpain I a m o n g the
3.4. Degradation of calponin located on native
thin filament Since F - a c t i n - b o u n d calponin was highly resistant to calpain digestion, 9.8 times m o r e calpain I was added to the incubation mixture. C a l p o n i n was degraded completely after 3 h-incubation (kapp = 1.6 × 10 -4 s-l), while caldesmon was d e g r a d e d almost completely within 3 min (kapp = 7.3 X 10 -3 s-l). Actin and t r o p o m y o s i n were well preserved t h r o u g h o u t the incubation period (fig.4). By i m m u n o b l o t analysis, several fragments o f 1 7 - 1 4 k D a seen after 20-s incubation were identified as the degradation products o f actin. The origin o f 31 k D a f r a g m e n t (lanes 2 - 5 , fig.4) could
Fig.4. Degradation of native thin filament by calpain I (12.5% SDS-PAGE gel). The incubation periods were: lane 1, 0 min; lane 2, 20 s; lane 3, 50 s; lane 4, 80 s; lane 5, 3 min; lane 6, 20 min; lane 7, 40 min; lane 8, 60 min; lane 9, 2 h; lane 10, 3 h; lane 11, 4 h. Lane M, molecular mass markers (k, kDa). CD, caldesmon; CP, heavy subunit of calpain I; A, actin; TM, B subunit of tropomyosin; CN, calponin. 495
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smooth muscle contraction-associated proteins. Note that calponin is similar in its characteristics to troponin T of skeletal and cardiac muscle [1], and troponin T is also reported to be quite susceptible to calpain [8,22]. In the presence of calmodulin, calponin was degraded by calpain about twice more rapidly than in the absence of calmodulin when kapp values were compared. Calmodulin may have caused a conformational change of the calponin molecule resulting in higher susceptibility to calpain. This is compatible with the loss of the tropomyosin-binding ability of calponin in the presence of Ca 2+ and calmodulin [41. When calponin was bound to F-actin, it became markedly resistant to calpain digestion. It was degraded about 140 times more slowly in terms of kapp values. The kapp value for calponin in the native thin filaments was 1.6 x 10 -4 s -1, which may correspond to 1.6 x 10-3 s -1 under the assay conditions used for free calponin, since 9.8 times more enzyme was added in the experiment with native thin filament (fig.4) than in the experiment with free calponin (figs 2 and 3). We concluded that the thin filament-bound form of calponin was degraded 500 times more slowly than the free form of calponin. Such a marked decrease in susceptibility may have been caused not only by actin filament but also by the other components such as tropomyosin and caldesmon. Caldesmon bound to the thin filaments was degraded 13 times more slowly than the free form of caldesmon. Calponin and caldesmon are Factin-binding proteins [3,13,23] and susceptible to calpain to almost the same extent when tested in each free form. However, calponin was degraded about 50 times less rapidly than caldesmon when tested as the thin filament-bound forms. This may be related to the structural difference between caldesmon and calponin in constituting thin filament. For example, caldesmon is an elongated and highly flexible protein [24-26] which would extend over the length of 70-150 nm along the axis of the actin filaments, while calponin may be a globular protein [1,3] which is more deeply buried in actin filaments than caldesmon. Such close association of calponin with actin filament could further support the view that calponin is an integral part of smooth muscle thin filament, which is vital to the actin-linked mechanism of contraction [3,15]. 496
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Acknowledgements: We thank Dr T.R. Shearer for his critical reading of the manuscript, and Nobuko Ohmori for her clerical assistance. This work was supported by Grants-in-Aid for Scientific Research and for Special Project Research (Metabolic Studies of Blood Vessels, no.6212005) from the Ministry of Education, Science and Culture, Japan, and by a Japan Heart Foundation Research Grant (K.T.).
REFERENCES [1] Takahashi, K., Hiwada, K. and Kokubu, T. (1988) Hypertension 11,620-626. [2] Takahashi, K., Abe, M., Hiwada, K. and Kokubu, T. (1988) J. Hypertension 6, $40-$43. [3] Takahashi, K., Hiwada, K. and Kokubu, T. (1986) Biochem. Biophys. Res. Commun. 141, 20-26. [4] Takahashi, K., Abe, M., Hiwada, K. and Kokubu, T. (1988) Cell Structure Function 13, 691. [5] Murachi, T. (1989) Biochem. Int. 18, 263-294. [6] Coolican, S.A. and Hathaway, D.R. (1984) J. Biol. Chem. 259, 11627-11630. [7] Hathaway, D.R., Werth, D.K. and Haeberle, J.R. (1982) J. Biol. Chem. 257, 9072-9077. [8] Toyo-oka, T. and Masaki, T. (1979) J. Mol. Cell. Cardiol. ll, 769-786. [9] Leonard, J.P. and Salpeter, M.M. (1979) J. Cell Biol. 82, 811-819. [10] Spudich, J.A. and Watt, S. (1971) J. Biol. Chem. 246, 4866-4871. Ill] Persechini, A., Mrwa, U. and Hartshorne, D.J. (1981) Biochem. Biophys. Res. Commun. 98, 800-805. [12] Sobue, K., Morimoto, K., Kanda, K., Fukunaga, K., Miyamoto, E. and Kakiuchi, S. (1982) Biochem. Int. 5, 503-510. [13] Bretscher, A. (1984) J. Biol. Chem. 259, 12873-12880. [14] Marston, S.B. and Lehman, W. 0985) Biochem. J. 231, 517-522. [15] Takahashi, K., Abe, M., Hiwada, K. and Kokubu, T. (1988) Abstr., Sixth Int. Symp. on Calcium-binding Proteins in Health and Disease, July, Nagoya, p,69. [16] Hatanaka, M., Kikuchi, T. and Murachi, T. (1983) Biomed. Res. 4, 381-388. [17] Laemmli, U.K. (1970) Nature 227, 680-685. [18] Bradford, M.M. (1976) Anal. Biochem. 72, 248-254. [19] Takahashi, K., Hiwada, K. and Kokuhu, T. (1987) Life Sci. 41,291-296. [20] Kosaki, G., Tsujinaka, T., Kambayashi, J., Morimoto, K., Yamamoto, K., Yamagami, K., Sobue, K. and Kakiuchi, S. (1983) Biochem. Int. 6, 767-775. [21] Nelson, W.J. and Traub, P. (1982) J. Biol. Chem. 257, 5544-5553. [22] Ishiura, S., Sugita, H., Suzuki, K. and Imahori, K. (1979) J. Biochem. (Tokyo) 86, 579-581. [23] Sobue, K., Muramoto, Y., Fujita, M. and Kakiuchi, S. (1981) Proc. Natl. Acad. Sci. USA 78, 5652-5655. [24] Fiirst, D.O., Cross, R.A., De Mey, J. and Small, J.V. (1986) EMBO J. 5, 251-257. [25] Lynch, W.P., Riseman, V.M. and Bretscher, A. (1987) J. Biol. Chem. 262, 7429-7437. [26] Graceffa, P., Wang, C.-L.A. and Stafford, W.F. (1988) J. Biol. Chem. 263, 14196-14202.