J. Biochem., 81, 243-247 (1977)
I-Protein, a New Regulatory Protein from Vertebrate
III. Function1 Koscak MARUYAMA, Sachio KUNITOMO, Sumiko KIMURA, and Kazuyo OHASHI Department of Biophysics, Faculty of Science, University of Kyoto, Sakyo-ku, Kyoto, Kyoto 606 Received for publication, June 3, 1976
I-protein inhibited the Mg-activated ATPase [EC 3.6.1.3] activity of actomyosin by approximately 50% at low ionic strength. Concomitantly, the onset of superprecipitation was retarded. I-protein was found to bind to myosin, but not to F-acttn. The inhibitory action of I-protein occurred only in the absence of Ca ions in the troponintropomyosin-actin-myosin system. Addition of Ca ions abolished the effect. Thus, it is very likely that I-protein prevents unnecessary hydrolysis of ATP in the relaxed state of muscle.
It is well established that the regulation of muscle •contraction by Ca ions is mediated by the troponintropomyosi.i system in vertebrate skeletal muscle
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Fig 4. Time course of superpreciprtation and ATPase activity of actomyosin under the influence of I-protein. Myosin, 0.2mg/ml; actin, 0 2mg/ml, pH 7.2. Other conditions were as in Fig. i. O, control; • , +1protein, 10% myosin. , turbidity; , Pi libration. •considerably retarded by I-protein, accompanied by
decreased ATPase activity. It should be mentioned that all the ATP added was completely hydrolyzed, though with some time lag in the presence of I-protein, and the supsrprecipitation "was also completed. Effect of the Troponin System—It was found that the action of I-protein on the actin-myosin interaction became Ca-dependent provided that tropomn and tropomyosin were present. The inhibitory effect of I-protein was only observed in the absence of Ca ions, and not at all in their presence. Figure 5 shows that the inhibition decreased on adding the troponin-tropomyosin system in the presence of Ca ions. The inhibition by I-protein "was abolished on adding 20% native tropomyosin by weight with respect to F-actin. Addition of troponin or tropomyosin alone did not affect the action of I-protein at all. The mixture of the two proteins worked well. The effects of various Ca concentrations are summarized in Fig. 6. The inhibitory action of I-protein was abolished around 2 x 10~6 M Ca. The time course clearly showed the inhibition by 1-protein of the actomyosin ATPase activity under relaxing conditions (Fig. 7). The •specific ATPase activity of actomyosin in the presence of I-protein and the troponin-tropomyosin complex was approximately 0.06-0.09 /Jmoles/mg/ min under the present relaxing conditions; 40-50% inhibition was produced by I-protein. It should be
Vol. 81, No. 1, 1977
0
10 20 30 40 50 Native tropomyosin /F-actin (%) Fig. 5. Effect of the troponin-tropomyosin complex on the action of I-protein under the influence of Ca ions. Myosin, 0.2 mg/ml, F-actin, 0.066 mg/ml, 0 1 mM CaCI,. Other conditions were as in Fig 1. O, control; • , + I-protein, 8% myosin
id*
(Ca2*) (M)
10s
10*
Fig. 6. Effect of concentration of Ca ions on the action of I-protein. Myosin, 0.2 mg/ml; F-actin, 0.066 mg/ml; native tropomyosin, 0.02 mg/ml; pH 7.0. Other conditions were as in Fig. 1. O, control; • , +l-protein, 8% myosin. noted that the ATPase activity was still higher than that of pure myosin under the same conditions (0.02 //moles/mg/min). The ATPase activity of myosin B or natural actomyosin was not affected at all by I-protein in the presence or absence of Ca ions. Presumably, this was due to the presence of I-protein in myosin B preparations. Binding of /-Protein to Myosin—To test the binding of I-protein to myosin, a suspension of myosin, 3 mg/ml, with or without 2 % I-protein in
Downloaded from https://academic.oup.com/jb/article-abstract/81/1/243/835963 by Leiden University / LUMC user on 16 January 2019
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246
K. MARUYAMA, S. KUNITOMO, S. KIMURA, and K. OHASHJ
DISCUSSION I-protein is located in the A-band except for the center part and also in the area of the A-I junction (5). The present work showed that the I-protein 10 20 30 binds to myosin but not to F-actin in vitro. JudgTime (mln) ing from the behavior of I-protein during itsFig. 7. Time course of actomyosin ATPase activity purification procedure (3) and also from observaunder the influence of I-protein and relaxing conditions Myosin, 0.2mg/ml; F-actin, 0.06mg/ml; native tropo- tion with fluorescent anti-I-protein antibody (4), it myosin, 0 02mg/ml; pH 7.0; 1 ITIM EGTA. Other appears that I-protein can bind to F-actin under the conditions were as in Fig. 1. O, control; • , + I-pro- influence of unknown factor(s). Troponin and tein, 8% myosin. tropomyosin were found not to be involved in thisbinding phenomenon. Therefore, in the present study, I-protein presumably exerted its action through its binding to myosin. Here, a problem, arises: 1-protein fully inhibited the ATPase activity of actomyosin at far less than an equimolar ratio. Taking the molecular weights of I-protein and myosin to be 50,000 and 500,000, I-protein amounting to 10% by weight of myosin corresponds to an equimolar ratio. As indicated in Fig. 1, 2-3% of I-protein exerted an almost saturated effect. This means that one molecule of I-protein affects three tofivemolecules of myosin, which would imply that I-protein acts on a myosin aggregate (A-filament). Supporting this interpretation, I-protein was much, Fig. 8. Binding of I-protein to myosin aggregate, a, less effective with acto-heavy meromyosin than I-Protein (supernatant); b, myosin+I-protein (supernaactomyosin. As shown in Fig. 9, I-protein appretant); c, myosin (supernatant); d, myosin+I-protein ciably inhibited the ATPase activity of acto-heavy (precipitate). The arrow shows the band of I-protein meromyosin, but the inhibition was still progressive For further details, see the text. up to the same amount of I-protem as of heavy meromyosin, at which point I-protein was present 0.02 M KC1, 1 mM MgCI,, and 0.01 M Tris buffer, in afive-foldexcess over heavy meromyosin (molar pH 8.0, was centnfuged for 20 min at 13,000 rpm. ratio). Here, less than 25% inhibition occurred. Portions of the supernatant and the precipitate were Evidently the effect of I-protein on heavy merosubjected to SDS gel electrophoresis. As shown in myosin, the functional part of the myosin molecule, Fig. 8, the supernatants of myosin and of myosin is much weaker than that on myosin aggregate. plus I-protein did not contain I-protein, but the precipitate of myosin plus I-protein contained a The mechanism of action of I-protein on the band corresponding to I-protein. No such band actomyosin ATPase remains to be elucidated. was detected in the precipitate of myosin alone. However, judging from its ineffectiveness against I-protein was not sedimented under these experi- myosin ATPase (Ca-activated), it probably intermental conditions. Therefore, it seems very likely feres with the activating effect of actin on myosin that I-protein is bound to myosin. ATPase activity. This view was supported by
1
/. Biochem.
Downloaded from https://academic.oup.com/jb/article-abstract/81/1/243/835963 by Leiden University / LUMC user on 16 January 2019
On the other hand, I-protein was not observed to bind to F-actin. This was also the case when I-protein was first added to G-actin, followed by the addition of KC1 to polymerize the actin. After ultracentrifugation, all the I-protein remained in the; supernantant.
1-2
247
FUNCTION OF I-PROTEIN
50 100 150 I-protein added (*/•)
Fig. 9. Comparison of the effects of I-protein on the ATPase activities of acto-heavy meromyosin and actomyosin. Heavy meromyosin, 0.2 mg/ml. Other conditions were as in Fig. 1. O, actomyosin; • , actoheavy meromyosin. retardation of the onset of superprecipitation by I-protein.
It is most remarkable that the inhibitory action of I-protein is only operative in the troponintropomyosin-actomyosin system under relaxing conditions. This suggests that the action of Iprotein is under regulation by Ca ions. Physiologically, this is important, because unnecessary splitting of ATP is inhibited in the relaxed state of muscle. C-protein, which strongly binds to myosin {11), has an appreciable inhibitory effect on the interaction of myosin filaments with F-actin (12). However, C-protein differs from I-protein in that the effect is independent of whether Ca ions are present or not. Finally, it should be mentioned that the ATPase activity of reconstituted actomyosin in the
Vol. 81V No. 1, 1977
The authors thank Prof. S. Ebashi of the University of Tokyo for his helpful advice REFERENCES 1. Ebashi, S. (1975) in Essays in Biochemistry (Campbell, D.N. & Dickens, F., eds.) Vol. 10, pp. 1-36,. Academic Press, New York 2. Weber, A. (1959) /. Bwl. Chem. 234, 2764-2769 3. Ohashi, K., Kimura, S., Deguchi, K., & Maruyama, K. (1977) / . Biochem. 81, 233-236 4. Ohashi, K., Masaki, T., & Maruyama, K. (1977) J. Biochem. 81, 237-242 5. Fujn, T. & Maruyama, K. (1971) 5c/. Pap. Coll. Gen. Educ. Univ. Tokyo 21, 45-61 6. Ebashi, S. (1961) / . Biochem. 50, 236-244 7. Taussky, H.H. & Shorr, E. (1953) /. Bwl. Chem. 203, 675-681 8. Ogawa, Y. (1968) /. Biochem. 64, 255-257 9. Weber, K. & Osborn, M. (1969) J. Biol. Chem. 244, 4406-4412 10. Maruyama, K & Ishikawa, Y. (1964) Annot. Zool. Jap. 37, 20-25 11. Offer, G., Moos, C , & Starr, R. (1973) / . Mol. Biol. 74, 653-676 12. Moos, C , Offer, G , Starr, R., & Benett, P. (1975) / . Mol. Biol. 97, 1-9 13. Kawamura, M. & Maruyama, K. (1969) J Biochem. 66, 619-626
Downloaded from https://academic.oup.com/jb/article-abstract/81/1/243/835963 by Leiden University / LUMC user on 16 January 2019
presence of the troponin-tropomyosin system and I-protein is still higher than that of myosin alone under the relaxation conditions. The latter lowlevel of ATPase activity is easily observed with. myosin B or myofibnls. It is thought that long F-actin filaments in the reconstituted actomyosin (13) may be entangled with each other and are thus not easily dissociable from myosin aggregatesShort F-actin in myosin B (13) is expected to dissociate completely under the relaxation conditions.
100
I-Protein, a New Regulatory Protein from Vertebrate
III. Function1 Koscak MARUYAMA, Sachio KUNITOMO, Sumiko KIMURA, and Kazuyo OHASHI Department of Biophysics, Faculty of Science, University of Kyoto, Sakyo-ku, Kyoto, Kyoto 606 Received for publication, June 3, 1976
I-protein inhibited the Mg-activated ATPase [EC 3.6.1.3] activity of actomyosin by approximately 50% at low ionic strength. Concomitantly, the onset of superprecipitation was retarded. I-protein was found to bind to myosin, but not to F-acttn. The inhibitory action of I-protein occurred only in the absence of Ca ions in the troponintropomyosin-actin-myosin system. Addition of Ca ions abolished the effect. Thus, it is very likely that I-protein prevents unnecessary hydrolysis of ATP in the relaxed state of muscle.
It is well established that the regulation of muscle •contraction by Ca ions is mediated by the troponintropomyosi.i system in vertebrate skeletal muscle
c
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2-0 5
K.
E
a>
^~
1.0 I
c c
13
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-0-5
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15
Fig 4. Time course of superpreciprtation and ATPase activity of actomyosin under the influence of I-protein. Myosin, 0.2mg/ml; actin, 0 2mg/ml, pH 7.2. Other conditions were as in Fig. i. O, control; • , +1protein, 10% myosin. , turbidity; , Pi libration. •considerably retarded by I-protein, accompanied by
decreased ATPase activity. It should be mentioned that all the ATP added was completely hydrolyzed, though with some time lag in the presence of I-protein, and the supsrprecipitation "was also completed. Effect of the Troponin System—It was found that the action of I-protein on the actin-myosin interaction became Ca-dependent provided that tropomn and tropomyosin were present. The inhibitory effect of I-protein was only observed in the absence of Ca ions, and not at all in their presence. Figure 5 shows that the inhibition decreased on adding the troponin-tropomyosin system in the presence of Ca ions. The inhibition by I-protein "was abolished on adding 20% native tropomyosin by weight with respect to F-actin. Addition of troponin or tropomyosin alone did not affect the action of I-protein at all. The mixture of the two proteins worked well. The effects of various Ca concentrations are summarized in Fig. 6. The inhibitory action of I-protein was abolished around 2 x 10~6 M Ca. The time course clearly showed the inhibition by 1-protein of the actomyosin ATPase activity under relaxing conditions (Fig. 7). The •specific ATPase activity of actomyosin in the presence of I-protein and the troponin-tropomyosin complex was approximately 0.06-0.09 /Jmoles/mg/ min under the present relaxing conditions; 40-50% inhibition was produced by I-protein. It should be
Vol. 81, No. 1, 1977
0
10 20 30 40 50 Native tropomyosin /F-actin (%) Fig. 5. Effect of the troponin-tropomyosin complex on the action of I-protein under the influence of Ca ions. Myosin, 0.2 mg/ml, F-actin, 0.066 mg/ml, 0 1 mM CaCI,. Other conditions were as in Fig 1. O, control; • , + I-protein, 8% myosin
id*
(Ca2*) (M)
10s
10*
Fig. 6. Effect of concentration of Ca ions on the action of I-protein. Myosin, 0.2 mg/ml; F-actin, 0.066 mg/ml; native tropomyosin, 0.02 mg/ml; pH 7.0. Other conditions were as in Fig. 1. O, control; • , +l-protein, 8% myosin. noted that the ATPase activity was still higher than that of pure myosin under the same conditions (0.02 //moles/mg/min). The ATPase activity of myosin B or natural actomyosin was not affected at all by I-protein in the presence or absence of Ca ions. Presumably, this was due to the presence of I-protein in myosin B preparations. Binding of /-Protein to Myosin—To test the binding of I-protein to myosin, a suspension of myosin, 3 mg/ml, with or without 2 % I-protein in
Downloaded from https://academic.oup.com/jb/article-abstract/81/1/243/835963 by Leiden University / LUMC user on 16 January 2019
•o
2-5 £ o
246
K. MARUYAMA, S. KUNITOMO, S. KIMURA, and K. OHASHJ
DISCUSSION I-protein is located in the A-band except for the center part and also in the area of the A-I junction (5). The present work showed that the I-protein 10 20 30 binds to myosin but not to F-actin in vitro. JudgTime (mln) ing from the behavior of I-protein during itsFig. 7. Time course of actomyosin ATPase activity purification procedure (3) and also from observaunder the influence of I-protein and relaxing conditions Myosin, 0.2mg/ml; F-actin, 0.06mg/ml; native tropo- tion with fluorescent anti-I-protein antibody (4), it myosin, 0 02mg/ml; pH 7.0; 1 ITIM EGTA. Other appears that I-protein can bind to F-actin under the conditions were as in Fig. 1. O, control; • , + I-pro- influence of unknown factor(s). Troponin and tein, 8% myosin. tropomyosin were found not to be involved in thisbinding phenomenon. Therefore, in the present study, I-protein presumably exerted its action through its binding to myosin. Here, a problem, arises: 1-protein fully inhibited the ATPase activity of actomyosin at far less than an equimolar ratio. Taking the molecular weights of I-protein and myosin to be 50,000 and 500,000, I-protein amounting to 10% by weight of myosin corresponds to an equimolar ratio. As indicated in Fig. 1, 2-3% of I-protein exerted an almost saturated effect. This means that one molecule of I-protein affects three tofivemolecules of myosin, which would imply that I-protein acts on a myosin aggregate (A-filament). Supporting this interpretation, I-protein was much, Fig. 8. Binding of I-protein to myosin aggregate, a, less effective with acto-heavy meromyosin than I-Protein (supernatant); b, myosin+I-protein (supernaactomyosin. As shown in Fig. 9, I-protein appretant); c, myosin (supernatant); d, myosin+I-protein ciably inhibited the ATPase activity of acto-heavy (precipitate). The arrow shows the band of I-protein meromyosin, but the inhibition was still progressive For further details, see the text. up to the same amount of I-protem as of heavy meromyosin, at which point I-protein was present 0.02 M KC1, 1 mM MgCI,, and 0.01 M Tris buffer, in afive-foldexcess over heavy meromyosin (molar pH 8.0, was centnfuged for 20 min at 13,000 rpm. ratio). Here, less than 25% inhibition occurred. Portions of the supernatant and the precipitate were Evidently the effect of I-protein on heavy merosubjected to SDS gel electrophoresis. As shown in myosin, the functional part of the myosin molecule, Fig. 8, the supernatants of myosin and of myosin is much weaker than that on myosin aggregate. plus I-protein did not contain I-protein, but the precipitate of myosin plus I-protein contained a The mechanism of action of I-protein on the band corresponding to I-protein. No such band actomyosin ATPase remains to be elucidated. was detected in the precipitate of myosin alone. However, judging from its ineffectiveness against I-protein was not sedimented under these experi- myosin ATPase (Ca-activated), it probably intermental conditions. Therefore, it seems very likely feres with the activating effect of actin on myosin that I-protein is bound to myosin. ATPase activity. This view was supported by
1
/. Biochem.
Downloaded from https://academic.oup.com/jb/article-abstract/81/1/243/835963 by Leiden University / LUMC user on 16 January 2019
On the other hand, I-protein was not observed to bind to F-actin. This was also the case when I-protein was first added to G-actin, followed by the addition of KC1 to polymerize the actin. After ultracentrifugation, all the I-protein remained in the; supernantant.
1-2
247
FUNCTION OF I-PROTEIN
50 100 150 I-protein added (*/•)
Fig. 9. Comparison of the effects of I-protein on the ATPase activities of acto-heavy meromyosin and actomyosin. Heavy meromyosin, 0.2 mg/ml. Other conditions were as in Fig. 1. O, actomyosin; • , actoheavy meromyosin. retardation of the onset of superprecipitation by I-protein.
It is most remarkable that the inhibitory action of I-protein is only operative in the troponintropomyosin-actomyosin system under relaxing conditions. This suggests that the action of Iprotein is under regulation by Ca ions. Physiologically, this is important, because unnecessary splitting of ATP is inhibited in the relaxed state of muscle. C-protein, which strongly binds to myosin {11), has an appreciable inhibitory effect on the interaction of myosin filaments with F-actin (12). However, C-protein differs from I-protein in that the effect is independent of whether Ca ions are present or not. Finally, it should be mentioned that the ATPase activity of reconstituted actomyosin in the
Vol. 81V No. 1, 1977
The authors thank Prof. S. Ebashi of the University of Tokyo for his helpful advice REFERENCES 1. Ebashi, S. (1975) in Essays in Biochemistry (Campbell, D.N. & Dickens, F., eds.) Vol. 10, pp. 1-36,. Academic Press, New York 2. Weber, A. (1959) /. Bwl. Chem. 234, 2764-2769 3. Ohashi, K., Kimura, S., Deguchi, K., & Maruyama, K. (1977) / . Biochem. 81, 233-236 4. Ohashi, K., Masaki, T., & Maruyama, K. (1977) J. Biochem. 81, 237-242 5. Fujn, T. & Maruyama, K. (1971) 5c/. Pap. Coll. Gen. Educ. Univ. Tokyo 21, 45-61 6. Ebashi, S. (1961) / . Biochem. 50, 236-244 7. Taussky, H.H. & Shorr, E. (1953) /. Bwl. Chem. 203, 675-681 8. Ogawa, Y. (1968) /. Biochem. 64, 255-257 9. Weber, K. & Osborn, M. (1969) J. Biol. Chem. 244, 4406-4412 10. Maruyama, K & Ishikawa, Y. (1964) Annot. Zool. Jap. 37, 20-25 11. Offer, G., Moos, C , & Starr, R. (1973) / . Mol. Biol. 74, 653-676 12. Moos, C , Offer, G , Starr, R., & Benett, P. (1975) / . Mol. Biol. 97, 1-9 13. Kawamura, M. & Maruyama, K. (1969) J Biochem. 66, 619-626
Downloaded from https://academic.oup.com/jb/article-abstract/81/1/243/835963 by Leiden University / LUMC user on 16 January 2019
presence of the troponin-tropomyosin system and I-protein is still higher than that of myosin alone under the relaxation conditions. The latter lowlevel of ATPase activity is easily observed with. myosin B or myofibnls. It is thought that long F-actin filaments in the reconstituted actomyosin (13) may be entangled with each other and are thus not easily dissociable from myosin aggregatesShort F-actin in myosin B (13) is expected to dissociate completely under the relaxation conditions.
100
