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Caldesmon weakens the bonding between myosin heads and actin in ghost fibers
Biochimica et Biophysica Acta, 999 (1989) 289-292
289
Elsevier BBAPRO33506
Caldesmon weakens the bonding between myosin heads and actin in ghost fibers E w a N o w a k l, Y u r i i S. B o r o v i k o v 2 a n d R e n a t a D ] b r o w s k a 1 I Department of Muscle Biochemistry. Nencki Institute of Experimental Biology, Warsaw (Poland) and ~ Group of Cell Motility, Institute of Cytology of the Academy of Sciences of U. S.S.K, Leningrad (U. S. S.K )
(Received15 May 1989) (Revisedmanuscriptreceived14 July 1989)
Key words: Caldesmon;Actin-myosininteraction;Ghost fiber; Polanzeofluorescence Earlier studies using polarized mierophotometry have shown that caldesmon inhibits the alterations in structure and flexibility of actin in ghost fibers that take place upon the binding of myosin heads (Gal~kiewicz et al. (1987) Bioehim. Biophys. Acta 916, 368-375). The present investigations, pedormcd with an LAEDANS label attached to myosin subfragment I (S-l), revealed that this inhibition results from the weakening of the binding between myosin heads and aetin as indicated by the ealdesmon-induced increase in the random movement of S-I. Parallel experiments with aedn labeled at Cys-374 demonstrated that this effect of caldesmon is transmitted to the C-terminus of the actin molecule resulting in a eonformational adjustment in this region of the molecule.
Introduction Caldesmon is an actin- and calmodulin-binding protein which participates in a Ca2+-dependent actin-linked regulatory system of smooth muscle. In vitro its binding to actin causes inhibition of the actin-activated Mg2+-ATPase activity of myosin which is potentiated by tropomyosin [1-3]. With a view to gaining more insight into the molecular basis of this inhibition, we have studied the effect of caldesmon on the structure and flexibility of actin filaments in skeletal muscle fibers devoid of myosin and regulatory proteins (ghost fibers) in the presence and absence of both S-1 and tropomyosin. The regular arrangement of actin filaments in ghost fibers supported the use of polarized microphotometry in this study. Earlier investigations utilizing this technique have de~onstrate,d that binding of the myosin head to F-actin complexed with fluorescent phalloidin in ghost fibers ' turns on' actin filaments, i.e., induces change in orientation of actin protomers in polymer and increases its flexibility [4-6]. A similar effect on actin structure as found with S-1 was evoked by sm,~oth muscle tropomyosin [4,7]. On the other hand, our previous
Correspondence: R. Dgbrowska, Nenck/ Institute of Experimental Biology, Department Muscle Biochemistry, 3 Pasteur Str., 02-093 Warsaw, Poland.
studies showed that the alterations in actin filament structure occurring upon binding of S-1 in the presence of tropomyosin were significantly reduced by caldesmon [4]. To clarify the molecular nature of this phenomenon we have attached a fluorescent label (1,5IAEDANS) to the SH 1 thiol of S-1 and formed rigor links with actin in skeletal muscle ghost fibers and monitored caldesmon-induced changes in structure and flexibility of myosin heads. Caldesmon was prepared from fresh chicken gizzards according to the method of Bretscher [8]. Chicken gizzard tropomyosin was purified as described earlier [9]. Rabbit skeletal muscle S-1 devoid of the regulatory light chains was obtained according to Weeds and Pol~ [10]. To determine the concentration of these proteins, the following absorption coefficients and Mr values ~er¢ used: chicken gizzard caldesmon, Ez~6 --0.30, 140000 [8]; chicken giT:,ard tropomyosin, £27s ffi 0.22, 72000 [11l; rabbit skeletal muscle S-1, E~0 •0.75, 115000 [12]. Ghost fibers were obtained from single glycgrinatod fibers of rabbit psoas muscle by extraction of myosin and regulatory proteins with the solution containing 800 mM KCI, 1 mM MgCl 2, 10 mM ATP and 67 mM phosphate buffer (pH 7.0) as described previously [13l. Labeling of actin in fibers and S-1 in solution with 1,5-1AEDANS was performed essentially according to Borejdo and Putnam [14]. Incorporation of chicken ~Tzard caldesmon and chicken g i r a r d tropomyosin to
0167-4838/89/$03.50 © 1989 ElsevierSciencePublishersB.V.(BiomedicalDivision)
290 ghost fibers was performed by immersion of fibers into the solution containing 100 mM KCI, 1 mM MgCI:, 67 mM phosphate (pH 7.0) and 2-5 mg/ml of the respextire protein. The incorporation of skeletal muscle S-1 was performed in the solution containing 10 mM KCI, 1 mM MgCI 2, 30 mM phosphate buffer (pH 7.0) and 3 mg/ml of S-1. After removal of the unbound protein by washing the fibers in the respective buffer, the. composition of the fibers was controlled by SDS-polyacrylamide gel electrophoresis [4]. The molar ratios of caldesmon, tropomyosin and S-1 bound to actin in ghost fibers determined by densitometric scans of the gels, were in. the range 1 : 30, 1 : 6.5 and 1 : 5, respectively. The last value was independent of the presence or absence of caldesmon. The polarized fluorescence from 1,5-1AEDANS bound either to S-1 or actin in ghost fibers was recorded at 480-600 nm after excitation at 365 + 5 nm. The intensity of four components of polarized fluorescence was measured in parallel (ul~, ulII) and perpendicular (~I,1, ~I±) orientations of the fiber axis to the polarization plane of the exciting light. From these four components we calculated the degree of fluorescence polarization (Pn and Pj.), the angles of absorption (O^) and emission (O~) dipoles of the fluorophore relative to the long axis of F-actin and the number of randomly oriented fluorophores (N). The theoretical basis for these calculations is given in [5,15-1~1. Table I illustrates the effect of ealdesmon on the degree of fluorescence polarization of 1,5-1AEDANSbound either directly to F-actin or to S-1 attached to actin in ghost fibers containing chicken ~i,.ard tropomyosin. In agreement with the data obtained previoasly with phalloidin-rhodamine~labeled F-actin [4], the results with 1,5-1AEDANS-Iabeled actin show thet: (a) S-1 evokes the changes in the light polarized perpendicular (P.0 (52~) and parallel (Ptl) (28~) to fiber axis; (b) tropomyosin potentiates these changes (to 129 and 34~, respectively); and (c) caldesmon reduces the changes induced by S-1 and tropomyosin (to 76 and 32~, respectively). It should be noted, however, that the effect of caldesmon on tropomyosin- and S-l-induced changes in the IAEDANS-labeled C-terminal region of actin is weaker than that observed earlier [4] in the region of phalloidin-rhodamine attachment site (i.e., in the region of the cleft between the two domains of actin). This difference can be related to the higher flexibility of the C-terminal part of actin molecule with respect to the entire filament [18], which was shown to be strongly inhibited upon binding of myosin heads [19,20]. The degree of fluorescence polarization (PjL and PU) of IAEDANS-iabeled S-1 bound to actin was altered by tropomyosin. On the other hand, both these fluorescence parameters of the labeled S-1 bound to actintropomyosin complex were practically unaffected by
caldesmon (Table I). Computer analysis of the data presented in Table I showed that the angles of absorption and emission dipoles of the fluorophore attached to S-l, which was in rigor link with actin, were 48.0 ° and 45.1 °, respectively. The label located on the S-1 flagment of myosin reflects, however, not only the motions of myosin head relative to the actin monomer but also the motions of entire actin fdament itself. The participation of the latter one, was determined (by estimatimi of 01/2 value - the mean angle between F-actin and fiber long axes) in parallel experiments performed with phalloidin-rhodamine-labeled actin. Taking account of this it was possible to calculate the number of randomly oriented fluorophores attached to S-1 reflectln~ t h e actual changes in the mobility of myosin heads relative to actin filament. The N value for acto-S-1 complex was equal to 0.192. The incorporation of chicken ~-,ard tropomyosin to the ghost fibers containing S-1 caused an increase in the value of the • A and Oe angles by 9.0 and 1.5~, respectively, whereas the number of randomly oriented fluorophores on S-1 was decreased by 60~. The incorporation of caldesmon diminished the changes in • A and O e angles as well as in the N value evoked by S-1 and tropomyosin by 53, 86 and 29~, respectively (Fig. 1A). These data indicate that caldesmon-inde_~eed inhibition of the alterations of actin structure and flexibility which occur in the presence of smooth muscle tropomyosin, affects actin-myosin interaction. As could be judged from the increase in the number of rando~y oriented fluorophores attached to S-1 (Fig. 1A) and the lack of the changes in the degree of fluorescence polar. ization of the light polarized parallel to the fiber axis (Table I), caldesmon does not affect the anglar distribu. tion of myosin heads relative to the fiber axis but increases their random movement. This indicates that caldesmon weakens the bonding between mycein heads and actin. Recent data of Borovikov et al. [21] suggest that the interaction of 20 kDa C-terminal domain of myosin (which contains 1,5-1AEDANS-binding SH! group) with actin is responsible for inducing the conformational changes in F-actin structure. On the other hand, several lines of experimental evidence have implicated three regions of actin as providing sites of contact with skeletal muscle myosin: two of them interact with the heavy chain of myosin and one with the light chain LCl [22-25]. The site of the interaction with the heavy chain C-terminal 20 kDa fragment of myosin was shown to be located in N-terminal part of actin molecule [22-24], which is also a docking region for caldesmon [26I. The occupation of the closely sequential sites by these two proteins on aztin may result in the weakening or breaking of one of the bonds between S-1 and actin by caldesmon. As could be judged from the similar changes in the
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The authors thank Dr. lan P. Trayer for critical reading of the manuscript. This work was supported by the Pofish Academy of Science within the project CPBP 04.01 and by the Academy of Sciences of the U.S.S.R.
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Fig. i. The effect of caldesmon on the structure and mobility of IAEDANS-labeled S-I (A) and IAEDANS-labeled actin (B) bound to each other in ghost fibers conutining chicken ~ r d tropomyosin. The chanses in the q'A, 4be angles and the number of randomly oriented fluomphores (N) were calculated from the data presented in Table I as a d i f f ~ between the values estimated for acto-S-I complex prior and after the addition of tropomyosin in the absence (13)and presence (g) of caldesmon.
orientation of IAEDANS bound either to actin or the myosin head and in the mobility of both S-1 and the C-terminal region of actin evoked by caldesmon (compare Fig. 1A and 1B), its effect on the interaction of S-1 with N-terminal part of actin molecule is transmitted to C-terminus. This supports earlier predictions made by others [27-29] that N-terminal region of actin is close to its C-terminus. Our results indicating that caldesmon causes a weakeni~ of the bondin$ between actin and myosin in skeletal muscle fibers confLrm earlier observations of Chalovich ¢t al. [30] who found in sedimentation assays that the binding of l~l-labeled caldesmon to actin under rigor conditions weakened the acto-S-1 interaction. Thus, it can be concluded that the basic physiological role of caldesmon may be to modulate the transition of the actomyosin complex between weakbinding and strong-bindin 8 states.
1 Sobue, K, Morimoto, K., lnui, M., Kanda, K. and Kakiuchi, S. (1982) Biomed. Res. 3, 188-196. 2 Dcbrowska, I~, Goch, A., GAt~_,kiewicT.,B. and Osifiska, H. (1985) Biochim. Biophys. Acta 842, 70-75. 3 Smith, C.W.J., Pritchard, IL and Marston. S.B. (1987) £ Biol. Chem. 262, 116-122. 4 GJ~Jdewicz, B., Borovikov, Y.S. and D#browska, R. (1987) Biochim. Biophys. Acta 916, 368-375. 5 Ymmglda, T. and Oosawa, F. (1978) J. Mol. Biol. 126, 507-524. 6 Prb~hniewicg-Nakayama,E, Yanasida, 1". and Oosawa, F. (1983) J. Cell Biol. 97, 1663-1667. 7 Dobrowolsld, Z., Borovikov, Y.S., Nowak, E., Gal~klewicT., B. and D~tbrowska, R. (1988) Biochim. Biophys. Acta 956, 140-150. 8 Bretscher, A. (1984) J. Biol. Chem. 259, 12873-12880. 9 Dabrowska, R., Nowak, E. and Drabikowsld, W. (1980) Comp. Biochem. Physiol. 65B, 75-83. 10 Weeds, A.G. and Pope, B. (1977) J. MoL Biol. 111, 129-157. 11 Woods, E.F. (1969) Biochemistry 8, 4336-4344. 12 Matgossian, S.S. and Lowey, S. (1978) Biochemistry 17, 5431-5439. 13 Borovikov, Y.S. and ~ , N.B. (1983) Eur. J. Biofhem. 136, 363-369. 14 Borejdo, J. and Putnam, S. (1977) Biochim. Biophys. Acta 459, 578-595. 15 Wilson, M.G. and Mendelson, R.A. (1983) .I. Muscle Res. Cell MotH. 4, 671-693. 16 Yanagida, T. (1985) J. Muscle Res. Cell MotH. 6, 43-52. 17 ILlkol, I., Borovikov, Y.S., S,~,~na, D., KinSna, V.P. and Levitsky, D.I- (1987) Biochim. Biophys. Acta 913-919. 18 Ikkai, 1"., Wahl, P. and Author, J.-C. (1979) Ear. J. Biochem. 93, 397-408. 19 Thomas, D.D., Seidd, J.C. and Gergely, J. (1979) J. Mol. Biol. 132, 257-273. 20 Hegyi, G., Szilagyi, L. and lklagyi, J. (1988) Eur. J. Biochem. 175, 271-274. 21 Borovikov, Y.S., Kulova, N.I, Khoros~cv, M.L and Vdovina, I.B. (1989) Ear. J. Biochem., in press. 22 Sutoh, K. (1983) Biochemistry 22, 1579-1586. 23 Moil A3.G., Levine, B.A., Goodearl, A. and Trayer, I.P. (1987) J. Muscle Res. Cell MotH. 8, 68-69. 24 Mejean, C., Boyer, M., Labbe, J.P, Marlier, L, Benyamin, Y. and Roustan, C. (1987) Biochem. J. 244, 571-577. 25 Trayer, I.P., Trayet, H.I~Land Levine, B.A. (1987) Eur. J. Biochem. 164. 259-256. 26 Mok, AJ.G., Patchel, V.B., Perry, S.V. and Levine, B.A. (1988) S)~posium on Smooth Muscle, London 13-15 April, Abstr. Cornmum. A53. 27 Sutoh, K. and Mabuchi, 1. (1984) Biochemistry 23, 6757-6761. 28 Boyer, M., Roustan, C. and lk,nyamin, Y. (1985) Biosci. Rcp. 5, 39-46. 29 Kab~ch, W., Mannherz, H.G. and Suck, D. (1985) EMBO J. 4, 2113-2128. 30 Chalovich, J.M., Come5us, P. and Benson, C.E. (1987) J. Biol. Chem. 262, 5711-5716.
289
Elsevier BBAPRO33506
Caldesmon weakens the bonding between myosin heads and actin in ghost fibers E w a N o w a k l, Y u r i i S. B o r o v i k o v 2 a n d R e n a t a D ] b r o w s k a 1 I Department of Muscle Biochemistry. Nencki Institute of Experimental Biology, Warsaw (Poland) and ~ Group of Cell Motility, Institute of Cytology of the Academy of Sciences of U. S.S.K, Leningrad (U. S. S.K )
(Received15 May 1989) (Revisedmanuscriptreceived14 July 1989)
Key words: Caldesmon;Actin-myosininteraction;Ghost fiber; Polanzeofluorescence Earlier studies using polarized mierophotometry have shown that caldesmon inhibits the alterations in structure and flexibility of actin in ghost fibers that take place upon the binding of myosin heads (Gal~kiewicz et al. (1987) Bioehim. Biophys. Acta 916, 368-375). The present investigations, pedormcd with an LAEDANS label attached to myosin subfragment I (S-l), revealed that this inhibition results from the weakening of the binding between myosin heads and aetin as indicated by the ealdesmon-induced increase in the random movement of S-I. Parallel experiments with aedn labeled at Cys-374 demonstrated that this effect of caldesmon is transmitted to the C-terminus of the actin molecule resulting in a eonformational adjustment in this region of the molecule.
Introduction Caldesmon is an actin- and calmodulin-binding protein which participates in a Ca2+-dependent actin-linked regulatory system of smooth muscle. In vitro its binding to actin causes inhibition of the actin-activated Mg2+-ATPase activity of myosin which is potentiated by tropomyosin [1-3]. With a view to gaining more insight into the molecular basis of this inhibition, we have studied the effect of caldesmon on the structure and flexibility of actin filaments in skeletal muscle fibers devoid of myosin and regulatory proteins (ghost fibers) in the presence and absence of both S-1 and tropomyosin. The regular arrangement of actin filaments in ghost fibers supported the use of polarized microphotometry in this study. Earlier investigations utilizing this technique have de~onstrate,d that binding of the myosin head to F-actin complexed with fluorescent phalloidin in ghost fibers ' turns on' actin filaments, i.e., induces change in orientation of actin protomers in polymer and increases its flexibility [4-6]. A similar effect on actin structure as found with S-1 was evoked by sm,~oth muscle tropomyosin [4,7]. On the other hand, our previous
Correspondence: R. Dgbrowska, Nenck/ Institute of Experimental Biology, Department Muscle Biochemistry, 3 Pasteur Str., 02-093 Warsaw, Poland.
studies showed that the alterations in actin filament structure occurring upon binding of S-1 in the presence of tropomyosin were significantly reduced by caldesmon [4]. To clarify the molecular nature of this phenomenon we have attached a fluorescent label (1,5IAEDANS) to the SH 1 thiol of S-1 and formed rigor links with actin in skeletal muscle ghost fibers and monitored caldesmon-induced changes in structure and flexibility of myosin heads. Caldesmon was prepared from fresh chicken gizzards according to the method of Bretscher [8]. Chicken gizzard tropomyosin was purified as described earlier [9]. Rabbit skeletal muscle S-1 devoid of the regulatory light chains was obtained according to Weeds and Pol~ [10]. To determine the concentration of these proteins, the following absorption coefficients and Mr values ~er¢ used: chicken gizzard caldesmon, Ez~6 --0.30, 140000 [8]; chicken giT:,ard tropomyosin, £27s ffi 0.22, 72000 [11l; rabbit skeletal muscle S-1, E~0 •0.75, 115000 [12]. Ghost fibers were obtained from single glycgrinatod fibers of rabbit psoas muscle by extraction of myosin and regulatory proteins with the solution containing 800 mM KCI, 1 mM MgCl 2, 10 mM ATP and 67 mM phosphate buffer (pH 7.0) as described previously [13l. Labeling of actin in fibers and S-1 in solution with 1,5-1AEDANS was performed essentially according to Borejdo and Putnam [14]. Incorporation of chicken ~Tzard caldesmon and chicken g i r a r d tropomyosin to
0167-4838/89/$03.50 © 1989 ElsevierSciencePublishersB.V.(BiomedicalDivision)
290 ghost fibers was performed by immersion of fibers into the solution containing 100 mM KCI, 1 mM MgCI:, 67 mM phosphate (pH 7.0) and 2-5 mg/ml of the respextire protein. The incorporation of skeletal muscle S-1 was performed in the solution containing 10 mM KCI, 1 mM MgCI 2, 30 mM phosphate buffer (pH 7.0) and 3 mg/ml of S-1. After removal of the unbound protein by washing the fibers in the respective buffer, the. composition of the fibers was controlled by SDS-polyacrylamide gel electrophoresis [4]. The molar ratios of caldesmon, tropomyosin and S-1 bound to actin in ghost fibers determined by densitometric scans of the gels, were in. the range 1 : 30, 1 : 6.5 and 1 : 5, respectively. The last value was independent of the presence or absence of caldesmon. The polarized fluorescence from 1,5-1AEDANS bound either to S-1 or actin in ghost fibers was recorded at 480-600 nm after excitation at 365 + 5 nm. The intensity of four components of polarized fluorescence was measured in parallel (ul~, ulII) and perpendicular (~I,1, ~I±) orientations of the fiber axis to the polarization plane of the exciting light. From these four components we calculated the degree of fluorescence polarization (Pn and Pj.), the angles of absorption (O^) and emission (O~) dipoles of the fluorophore relative to the long axis of F-actin and the number of randomly oriented fluorophores (N). The theoretical basis for these calculations is given in [5,15-1~1. Table I illustrates the effect of ealdesmon on the degree of fluorescence polarization of 1,5-1AEDANSbound either directly to F-actin or to S-1 attached to actin in ghost fibers containing chicken ~i,.ard tropomyosin. In agreement with the data obtained previoasly with phalloidin-rhodamine~labeled F-actin [4], the results with 1,5-1AEDANS-Iabeled actin show thet: (a) S-1 evokes the changes in the light polarized perpendicular (P.0 (52~) and parallel (Ptl) (28~) to fiber axis; (b) tropomyosin potentiates these changes (to 129 and 34~, respectively); and (c) caldesmon reduces the changes induced by S-1 and tropomyosin (to 76 and 32~, respectively). It should be noted, however, that the effect of caldesmon on tropomyosin- and S-l-induced changes in the IAEDANS-labeled C-terminal region of actin is weaker than that observed earlier [4] in the region of phalloidin-rhodamine attachment site (i.e., in the region of the cleft between the two domains of actin). This difference can be related to the higher flexibility of the C-terminal part of actin molecule with respect to the entire filament [18], which was shown to be strongly inhibited upon binding of myosin heads [19,20]. The degree of fluorescence polarization (PjL and PU) of IAEDANS-iabeled S-1 bound to actin was altered by tropomyosin. On the other hand, both these fluorescence parameters of the labeled S-1 bound to actintropomyosin complex were practically unaffected by
caldesmon (Table I). Computer analysis of the data presented in Table I showed that the angles of absorption and emission dipoles of the fluorophore attached to S-l, which was in rigor link with actin, were 48.0 ° and 45.1 °, respectively. The label located on the S-1 flagment of myosin reflects, however, not only the motions of myosin head relative to the actin monomer but also the motions of entire actin fdament itself. The participation of the latter one, was determined (by estimatimi of 01/2 value - the mean angle between F-actin and fiber long axes) in parallel experiments performed with phalloidin-rhodamine-labeled actin. Taking account of this it was possible to calculate the number of randomly oriented fluorophores attached to S-1 reflectln~ t h e actual changes in the mobility of myosin heads relative to actin filament. The N value for acto-S-1 complex was equal to 0.192. The incorporation of chicken ~-,ard tropomyosin to the ghost fibers containing S-1 caused an increase in the value of the • A and Oe angles by 9.0 and 1.5~, respectively, whereas the number of randomly oriented fluorophores on S-1 was decreased by 60~. The incorporation of caldesmon diminished the changes in • A and O e angles as well as in the N value evoked by S-1 and tropomyosin by 53, 86 and 29~, respectively (Fig. 1A). These data indicate that caldesmon-inde_~eed inhibition of the alterations of actin structure and flexibility which occur in the presence of smooth muscle tropomyosin, affects actin-myosin interaction. As could be judged from the increase in the number of rando~y oriented fluorophores attached to S-1 (Fig. 1A) and the lack of the changes in the degree of fluorescence polar. ization of the light polarized parallel to the fiber axis (Table I), caldesmon does not affect the anglar distribu. tion of myosin heads relative to the fiber axis but increases their random movement. This indicates that caldesmon weakens the bonding between mycein heads and actin. Recent data of Borovikov et al. [21] suggest that the interaction of 20 kDa C-terminal domain of myosin (which contains 1,5-1AEDANS-binding SH! group) with actin is responsible for inducing the conformational changes in F-actin structure. On the other hand, several lines of experimental evidence have implicated three regions of actin as providing sites of contact with skeletal muscle myosin: two of them interact with the heavy chain of myosin and one with the light chain LCl [22-25]. The site of the interaction with the heavy chain C-terminal 20 kDa fragment of myosin was shown to be located in N-terminal part of actin molecule [22-24], which is also a docking region for caldesmon [26I. The occupation of the closely sequential sites by these two proteins on aztin may result in the weakening or breaking of one of the bonds between S-1 and actin by caldesmon. As could be judged from the similar changes in the
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The authors thank Dr. lan P. Trayer for critical reading of the manuscript. This work was supported by the Pofish Academy of Science within the project CPBP 04.01 and by the Academy of Sciences of the U.S.S.R.
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Fig. i. The effect of caldesmon on the structure and mobility of IAEDANS-labeled S-I (A) and IAEDANS-labeled actin (B) bound to each other in ghost fibers conutining chicken ~ r d tropomyosin. The chanses in the q'A, 4be angles and the number of randomly oriented fluomphores (N) were calculated from the data presented in Table I as a d i f f ~ between the values estimated for acto-S-I complex prior and after the addition of tropomyosin in the absence (13)and presence (g) of caldesmon.
orientation of IAEDANS bound either to actin or the myosin head and in the mobility of both S-1 and the C-terminal region of actin evoked by caldesmon (compare Fig. 1A and 1B), its effect on the interaction of S-1 with N-terminal part of actin molecule is transmitted to C-terminus. This supports earlier predictions made by others [27-29] that N-terminal region of actin is close to its C-terminus. Our results indicating that caldesmon causes a weakeni~ of the bondin$ between actin and myosin in skeletal muscle fibers confLrm earlier observations of Chalovich ¢t al. [30] who found in sedimentation assays that the binding of l~l-labeled caldesmon to actin under rigor conditions weakened the acto-S-1 interaction. Thus, it can be concluded that the basic physiological role of caldesmon may be to modulate the transition of the actomyosin complex between weakbinding and strong-bindin 8 states.
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