/ . Biochem. 83, 1355-1360 (1978)
Muscle Serine Protease1 Nobumasa YASOGAWA,* Yukihiro SANADA,** and Nobuhiko KATUNUMA 1 ' •* *2nd Department of Internal Medicine, School of Medicine, Tokushima University, and **Department of Enzyme Chemistry, Institute for Enzyme Research, School of Medicine, Tokushima University, 3 Kuramotc-cho, Tokushima, Tokushima 770 Received for publication, December 17, 1977
The ability of serine protease of skeletal muscle to degrade native myofibrillar proteins, such as myosin, actin, troponin, tropomyosin, a-actinin, and M-protein from rabbit skeletal muscle was studied. The amino acids or peptides liberated from these proteins by the protease were determined fluorometrically using o-phthalaldehyde. The order of their susceptibilities at a molar ratio of the serine protease to substrate of 1:100 was: myosin^>troponin>tropomyosin > actin. a-Actinin and M-protein were not degraded. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the myosin heavy chain was degraded into two fragments, having molecular weights of 100,000 and 88,000, whereas the light chains were scarcely degraded. The serine protease degraded troponin-T rapidly and troponin-I slowly, but did not degrade troponin-C. Tropomyosin was degraded rapidly into two components with molecular weights of 21,500 and 19,000. Actin was degraded slowly, but no liberated fragment could be detected.
There are several reports on the turnover rates of individual myofibrillar proteins (1-4) and also on intracellular proteases (5-16) in skeletal muscle, but no information is available about the susceptibilities of individual myofibrillar proteins to intracellular protease, except that Ca l+ -dependent neutral protease can degrade intact myofibrils and purified individual myofibrillar proteins (17-20). Recently, we reported that the activity of serine 1
This work was supported in part by a grant for research on muscular dystrophy from the Ministry of Health and Welfare, Japan (1977). 1 To whom requests for reprints should be sent. Vol. 83, No. 5, 1978
1355
protease in skeletal muscle was much higher in dystrophic mice than in normal mice (21) and that the activity was also increased in the muscle in Duchenne or Becker type muscular dystrophy (22). Moreover, based on the patterns of myofibrillar proteins after incubation with a highly purified serine protease, we suggested that the serine protease was responsible for the marked decrease of myofibrillar proteins in muscle in muscular dysstrophy (21). This paper reports the results of studies on the susceptibilities of various purified myofibrillar proteins to the serine protease and discusses the role of serine protease in the degradation of myofibrillar proteins.
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Susceptibilities of Various Myofibrillar Proteins to
1356
N. YASOGAWA, Y. SANADA, and N. KATUNUMA
MATERIALS AND METHODS
Serine protease was prepared by the method of Katunuma et al. (23) with some modifications. Recently, we succeeded in crystallizing the enzyme, as will be reported elsewhere (Sanada Y. Yasogawa, N., & Katunuma, N., manuscript in preparation). Crystalline enzyme with a specific activity of 7,600 unit/min/mg protein was used in all experiments. Apo-ornithine aminotransferase [EC 2.6.1.13] used for determination of the serine protease activity was prepared by the method of Sanada et al. (24). Serine protease was assayed as reported previously (21). Protein concentration was determined by the biuret method (25) or by the method of Lowry et al. (26) using bovine serum albumin as a standard. Myofibrillar proteins were prepared from rabbit hind leg. Myosin, actin, troponin, tropomyosin, a-actinin, and M-protein were prepared by the methods of Perry (27), Ebashi and Ebashi (28), Ebashi et al. (29), Mueller (50), Masaki and Takaiti (31), and Masaki and Takaiti (32), respectively. The ability of serine protease to hydrolyze native myofibrillar proteins was studied as follows. The reaction mixture contained 0.25 M sodium phosphate buffer, pH 8.0, and a 10~J M concentration of the test myofibrillar protein with or without 2 X10"' to 10"7 M serine protease in a final volume of 1.0 ml. The mixture was incubated at 37°C for 0 to 60min. Samples of 0.2 ml were removed at intervals, mixed with 1.3 ml of distilled water and boiled for 3 min to stop the reaction. Then 1.5 ml of 0.01 % Fluescin was added and the mixtures were centrifuged at 3,000 rpm for 10 min. Material with fluorescence in the supernatant, with
RESULTS Table I shows the ability of serine protease to hydrolyze various purified myofibrillar proteins, determined by measuring the amounts of amino acids or peptides released during the incubation. The release from myosin was rapid and linear for 15 min on incubating a molar ratio of serine protease to myosin of 1 : 500, but then the release appeared to level off gradually, judging from the data obtained at a molar ratio of 1 :100. Troponin and tropomyosin showed similar patterns of degradation, all suggesting limited proteolysis. Small amounts of amino groups were released linearly from actin during the incubation, but no liberation of amino acids or peptides from aactinin or M-protein was observed. No release of amino acids or peptides from the myofibrillar proteins was observed in control experiments without the serine protease. These findings suggest that myosin, troponin, tropomyosin, and actin are susceptible to the serine protease but that a-actinin and M-protein are not. Next, the modes of degradation of the myofibrillar proteins were investigated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. /. Biochem.
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Male white rabbits, weighing 3 to 4 kg, were used for the preparation of myofibrillar proteins. Male Wistar-strain rats, weighing 250 to 300 g, were used for the preparation of serine protease. Bovine serum albumin, used as a protein standard, was obtained from Miles Laboratory Inc. o-Phthalaldehyde (Fluescin) and its dilution buffer (Tritrisol) were obtained from Merck Japan Co. The marker proteins used in determination of molecular weights by sodium dodecyl sulfate-polyacrylamide gel electrophoresis were obtained from BDH Chemicals Ltd. All other chemicals used were commercial products of the highest grade available.
an appropriate level of L-leucine as a standard, was determined by the method of Benson et al. (33). Fluorescence was determined using a Hitachi MPF-2A fluorescence spectrophotometer. Degradation of myofibrillar protein was analyzed as follows. A reaction mixture similar to that for fluorometric analysis, but with different molar ratios of serine protease to substrate was used. At various times, samples of 0.2 ml were removed for sodium dodecyl sulfate-polyacrylamide gel electrophoresis. An equal volume of 2% w/v sodium dodecyl sulfate containing 50% glycerol and 20 fil of 2-mercaptoethanol were added and the mixture was boiled for 3 min. The samples were then mixed with 10 pi of 1 % w/v bromphenol blue and a sample of 20 pg of the protein was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Electrophoresis was carried out by the method of Weber and Osborn (34) except that a slab gel containing 8.0% acrylamide was used. The molar ratios of enzyme to protein in the incubation mixtures are given in the legends to the figures.
ACTION OF SERINE PROTEASE ON MYOFIBRILLAR PROTEINS
1357
TABLE I. Activities of the serine protease on various myofibrillar proteins. Substrate
Troponin Tropomyosin Actin or-Actinin M-Protein 1
0
5
10
15
1 500
0
16.0
27.0
1 500
0
1 100
0
38.5
1
100
0
7.0
52.6 15.5
1
41.4 34.0 57.8 24.0 24.5 16.0
30
60
51.5
73.5
28.0 30.0
35.0 38.0 13.0
100
0
1 100
0
1
100
0
5.5
9.$
1
100
0
0
0
0
1
100
0
0
0
0
Molar ratio of serine protease to substrate in the reaction mixture.
a
b c d e f g h
a
bc
e f
10DD00
ID-T
IS
—
T i l •* Tn C ~ • • M M |i
13000
Fig. 1. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic patterns of myosin after incubation with and without serine protease. Myosin was incubated in 0.25 M sodium phosphate buffer, pH 8.0, for 0 (a), 15 (b), 30 (c), and 60 (d) min without the serine protease, and for 0 (e), 15 (f), 30 (g), and 60 (h) min with the protease. The molar ratio of serine protease to myosin was 1 : 300. The experimental conditions are described in " MATERIALS AND METHODS." H, Heavy chain of myosin; L-l, L-2, L-3, light chains of myosin.
Fig. 2. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic patterns of troponin after incubation with and without serine protease. Experimental conditions were similar to those for Fig. 1. Troponin was incubated for 0 (a) and 60 (b) min without the protease, and for 0 (c), 15 (d), 30 (e), and 60 (f) min with the protease. The molar ratio of the serine protease to troponin was I : 300. Tn-T, Troponin-T; Tn-I, troponin-1; Tn-C, troponin-C.
Figure 1 shows results on the initial degradation of purified myosin by the serine protease. Two kinds of fragments, with molecular weights of approximately 100,000 and 88,000, seemed to be produced by the serine protease degradation of myosin heavy chain. Light chain 2 was degraded very slowly, but light chain 1 showed no detectable change. Although the myosin preparation was
slightly contaminated, the contaminants were not digested by the serine protease. These results suggest that myosin is subject to limited proteolysis by the serine protease. Figure 2 shows the results of a similar experiment with purified troponin. Troponin-T was completely digested within 15 min, and troponin-I was also digested, although more slowly. However, troponin-C was not susceptible
Vol. 83, No. 5, 1978
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Myosin
Time of incubation (min)
Molar1 ratio
1358
N. YASOGAWA, Y. SANADA, and N. KATUNUMA
a fc c i i f
a
b c d e f §
Fig. 3. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic patterns of tropomyosin after incubation with and without the serine protease. Experimental conditions were as for Fig. 1. Tropomyosin was incubated for 0 (a) and 60 (b) min without the protease, and for 0 (c), 15 (d), 30 (e), and 60 (f) min with the protease. The molar ratio of the serine protease to tropomyosin was 1 : 200.
to the serine protease. The troponin preparation contained a contaminant with a molecular weight of 13,000, which appeared to be the proteolytic product formed from troponin by the serine protease. Figure 3 shows the patterns obtained on incubation of purified tropomyosin with and without the protease. Tropomyosin was degraded completely within 30 min by the protease. At an early stage of digestion (15 min), an intermediate product with a molecular weight of 32,000 appeared, but the serine protease acted on tropomyosin to break it ultimately into two components with molecular weights of 21,500 and 19,000 as well as further smaller fragments which were not stained. Figure 4 shows the effect of serine protease on purified actin. The band of purified actin disappeared very slowly, but no new fragments were detected. In Fig. 5 the effect of serine protease on purified a-actinin is shown. tr-Actinin showed no detectable change during incubation with or without the serine protease. These results indicate that the serine protease hydrolyzed several native myofibrillar proteins and that its mode of hydrolysis was limited.
Fig. 4. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic patterns of actin after incubation with and without serine protease. Experimental conditions were as for Fig. 1. Actin was incubated for 0 (a), 30 (b), and 60 (c) min without the protease, and for 0 (d), 15 (e), 30 (f), and 60 (g) min with the protease. The molar ratio of the serine protease to actin was 1 :200.
a I e i i f i
|
Fig. 5. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic patterns of a-actinin after incubation with and without serine protease. Experimental conditions were as for Fig. 1. cr-Actinin was incubated for 0 (a), 15 (b), 30 (c), and 60 (d) min without the protease, and for 0 (e), 15 (f), 30 (g), and 60 (h) min with the protease. The molar ratio of the serine protease to ir-actinin was 1 : 200. DISCUSSION
Dayton et al. (J8) showed that Cat+-dependent neutral protease degraded troponin-T, troponin-I, tropomyosin, and C-protein, but did not hydroJ. Biochem.
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3200D 21500 19000
ACTION OF SERINE PROTEASE ON MYOFIBRILLAR PROTEINS
Previously we reported that the activity of serine protease was greatly increased in the muscles of mice with hereditary muscular dystrophy (21) and also in the muscles of patients with progressive muscular dystrophy (22), and we detected several abnormal protein fragments formed by limited proteolysis of the muscles in cases of muscular dystrophy (21). Furthermore, when myofibrillar proteins of normal and dystrophic muscles were incubated with highly purified serine protease, their myosin, a-actinin and tropomyosin disappeared completely (21). The present results are consistent with these previous data, except with respect to a-actinin; the reason for the discrepancy in the case of a-actinin is not known. In spite of this discrepancy, the results show that serine protease, like CaI+-dependent neutral protease, is responsible for the initial degradation of several myofibrillar proteins and is involved in the turnover of myofibrillar proteins. Recently, Uchida et al. (35) reported another protease which readily degrades myosin of rat cardiac muscle. There also appear to be other Vol. 83, No. 5, 1978
proteases, including the lysosomal cathepsin group. Thus, the degradation of myofibrillar proteins and the turnover rate of each myofibrillar protein must depend on the cooperative actions of all these proteases under physiological conditions. A change in this balanced cooperation could result in increase in the action of some protease, such as serine protease (21, 22) or Ca1+-dependent neutral protease (20, 36) causing marked loss of certain myofibrillar proteins (21) and development of the characteristic pattern of decrease of myofibrillar proteins seen in muscular dystrophy, as reported by Sugita et al. (37-39). Intracellular protease activity seems to be regulated by the concentration of its specific inhibitor, which coexists with the protease (22). Further studies are required on the inhibitor of this serine protease. REFERENCES 1. Funabiki, R. & Cassens, R.G. (1973) / . Nutr. Sci. Vitaminol. 19, 361-368 2. Koizumi, T. (1974) / . Biochem. 76, 431^439 3. Zak, R., Martin, A.F., Prior, G., & Rabinowitz, M. (1977) /. Biol. Chem. 7S1, 3430-3435 4. Martin, A.F., Rabinowitz, M., Blough, R., Prior, G., & Zak, R. (1977) / . Biol. Chem. 252, 3422-3429 5. Tappel, A.L., Zalkin, H., Caldwell, K.A., Desai, I.D., & Shibko, S. (1962) Arch. Biochem. Biophys. 96,340-346 6. Iodice, A.A., Chin, J., Perker, S., & Weinstock, I.M. (1972) Arch. Biochem. Biophys. 152, 166-174 7. Iodice, A.A. (1976) Life Sci. 19, 1351-1358 8. Pearson, CM. & Kar, N.C. (1973) in Clinical Studies in Myology (Kakulas, B.A., cd.) pp. 89-97, Exccrpta Medica, Amsterdam 9. Koszalka, T.R. & Miller, L.L. (1960) / . Biol. Chem. 235,665-668 10. Koszalka, T.R. & Miller, L.L. (1960) / . Biol. Chem. 235, 669-672 11. Goldspink, D.F., Holmes, D., & Pennington, R J . (1971) Biochem. J. 125, 865-868 12. Mayer, M., Amin, R., & Shafrir, E. (1974) Arch. Biochem. Biophys. 161, 20-25 13. Brush, J.S. (1971) Diabetes 20, 140-145 14. Noguchi, T. & Kandatsu, M. (1971) Agric. Biol. Chem. 35, 1092-1100 15. Noguchi, T., Miyazawa, E., & Kametaka, M. (1974) Agric. Biol. Chem. 38, 253-257 16. Noguchi, T. & Kandatsu, M. (1976) Agric. Biol. Chem. 40, 927-933 17. Busch, W.A., Stromer, M.H., Goll, D.E., & Suzuki, A. (1972) / . Cell Biol. 52, 367-331
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lyze myosin, actin or a-actinin. The effects of our serine protease on troponin, tropomyosin, and a-actinin were very similar to those of the Ca1+dependent neutral protease, except that the products of limited proteolysis by the two enzymes were slightly different. Serine protease hydrolyzed tropomyosin mainly into two fragments with molecular weights of 21,500 and 19,000, whereas CaI+-dependent neutral protease hydrolyzed it to fragments with molecular weights in the range of 13,000 to 18,000. Moreover serine protease produced only one fragment with a molecular weight of approximately 13,000 from troponin-T and troponin-I, whereas CaI+-dependent neutral protease produced two fragments with molecular weights of 10,000 and 14,000 from troponin-T and troponin-I. The actions of serine protease and Ca1+-dependent neutral protease on myosin and actin were also different: serine protease hydrolyzed myosin very rapidly and actin slightly, but Ca1+dependent neutral protease did not hydrolyze either protein. Thus both enzymes exhibited marked specificity in their actions on myofibrillar proteins, suggesting that the two enzymes have different roles in the degradation of individual myofibrillar proteins under physiological and pathological conditions.
1359
1360
28. Ebashi, S. & Ebashi, F. (1955) / . Biochem. 55, 604-613 29. Ebashi, S., Kodama, A., & Ebashi, F. (1968) J. Biochem. 64, 465-477 30. Mueller, H. (1966) Biochem. Z. 345, 300-321 31. Masaki, T. & Takaiti, O. (1969) / . Biochem. 66, 637-643 32. Masaki, T. & Takaiti, O. (1974) / . Biochem. IS, 367-380 33. Benson, J.R. & Hare, P.E. (1975) Proc. Natl. Acad. Sci. U.S. 72, 619-622 34. Weber, K. & Osborn, M. (1969) /. Biol. Chem. 244, 4406-4412 35. Uchida, K., Murakami, U., & Hiratuka, T. (1977) / . Biochem. 82, 469^176 36. Kar, N.C. & Pearson, CM. (1976) Clin. Chim. Ada 73, 293-297 37. Sugita, H. & Toyokura, Y. (1976) Proc. Jap. Acad. 52, 256-259 38. Sugita, H. & Toyokura, Y. (1976) Proc. Jap. Acad. 52, 260-263 39. Sugita, H., Katagiri, T , Simizu, T , & Toyokura, Y. (1974) in Basic Research in Myology (Kakulas, B.A., ed.) pp. 291-297, Excerpta Medica, Amsterdam
/ . Biochem.
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18. Dayton, W.R., GoU, D.E., Stromer, M.H., Reville, W.J., Zeece, M.G., & Robson, R.M. (1975) Cold Spring Harbor Conferences on Cell Proliferation, Vol. II. Proteases and Biological Control pp. 551577, Cold Spring Harbor, New York 19. Dayton, W.R., Goll, D.E., Zeece, M.G., Robson, R.M., & Reville, WJ. (1976) Biochemistry 15, 2150-2158 20. Dayton, W.R., Reville, WJ., Goll, D.E., & Stromer, M.H. (1976) Biochemistry 15, 2159-2167 21. Sanada, Y., Yasogawa, N., & Katunuma, N. (1978) / . Biochem. 83, 27-33 22. Katunuma, N., Yasogawa, N., Kito, K., Sanada, Y., Kawai, H., & Miyoshi, K. (1978) /. Biochem. 83, 625-628 23. Katunuma, N., Kominami, E., Kobayashi, K., Banno, Y., Suzuki, K., Chichibu, K., Hamaguchi, Y., & Katsunuma, T. (1975) Eur. J. Biochem. 52, 37-50 24. Sanada, Y., Shiotani, T., Okuno, E., & Katunuma, N. (1976) Eur. J. Biochem. 69, 507-515 25. Gornall, A.G., Bardawill, C.S., & David, M.M. (1949) / . Biol. Chem. 177, 751-766 26. Lowry, O.H., Rosebrough, N.J., Farr, A.L., & Randall, R.J. (1951) / . Biol. Chem. 193, 265-275 27. Perry, S.V. (1955) in Methods in Enzymology (Colowick, S.P. & Kaplan, N.O., eds.) Vol. 2, pp. 583-586, Academic Press, New York
N. YASOGAWA, Y. SANADA, and N. KATUNUMA
Muscle Serine Protease1 Nobumasa YASOGAWA,* Yukihiro SANADA,** and Nobuhiko KATUNUMA 1 ' •* *2nd Department of Internal Medicine, School of Medicine, Tokushima University, and **Department of Enzyme Chemistry, Institute for Enzyme Research, School of Medicine, Tokushima University, 3 Kuramotc-cho, Tokushima, Tokushima 770 Received for publication, December 17, 1977
The ability of serine protease of skeletal muscle to degrade native myofibrillar proteins, such as myosin, actin, troponin, tropomyosin, a-actinin, and M-protein from rabbit skeletal muscle was studied. The amino acids or peptides liberated from these proteins by the protease were determined fluorometrically using o-phthalaldehyde. The order of their susceptibilities at a molar ratio of the serine protease to substrate of 1:100 was: myosin^>troponin>tropomyosin > actin. a-Actinin and M-protein were not degraded. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the myosin heavy chain was degraded into two fragments, having molecular weights of 100,000 and 88,000, whereas the light chains were scarcely degraded. The serine protease degraded troponin-T rapidly and troponin-I slowly, but did not degrade troponin-C. Tropomyosin was degraded rapidly into two components with molecular weights of 21,500 and 19,000. Actin was degraded slowly, but no liberated fragment could be detected.
There are several reports on the turnover rates of individual myofibrillar proteins (1-4) and also on intracellular proteases (5-16) in skeletal muscle, but no information is available about the susceptibilities of individual myofibrillar proteins to intracellular protease, except that Ca l+ -dependent neutral protease can degrade intact myofibrils and purified individual myofibrillar proteins (17-20). Recently, we reported that the activity of serine 1
This work was supported in part by a grant for research on muscular dystrophy from the Ministry of Health and Welfare, Japan (1977). 1 To whom requests for reprints should be sent. Vol. 83, No. 5, 1978
1355
protease in skeletal muscle was much higher in dystrophic mice than in normal mice (21) and that the activity was also increased in the muscle in Duchenne or Becker type muscular dystrophy (22). Moreover, based on the patterns of myofibrillar proteins after incubation with a highly purified serine protease, we suggested that the serine protease was responsible for the marked decrease of myofibrillar proteins in muscle in muscular dysstrophy (21). This paper reports the results of studies on the susceptibilities of various purified myofibrillar proteins to the serine protease and discusses the role of serine protease in the degradation of myofibrillar proteins.
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Susceptibilities of Various Myofibrillar Proteins to
1356
N. YASOGAWA, Y. SANADA, and N. KATUNUMA
MATERIALS AND METHODS
Serine protease was prepared by the method of Katunuma et al. (23) with some modifications. Recently, we succeeded in crystallizing the enzyme, as will be reported elsewhere (Sanada Y. Yasogawa, N., & Katunuma, N., manuscript in preparation). Crystalline enzyme with a specific activity of 7,600 unit/min/mg protein was used in all experiments. Apo-ornithine aminotransferase [EC 2.6.1.13] used for determination of the serine protease activity was prepared by the method of Sanada et al. (24). Serine protease was assayed as reported previously (21). Protein concentration was determined by the biuret method (25) or by the method of Lowry et al. (26) using bovine serum albumin as a standard. Myofibrillar proteins were prepared from rabbit hind leg. Myosin, actin, troponin, tropomyosin, a-actinin, and M-protein were prepared by the methods of Perry (27), Ebashi and Ebashi (28), Ebashi et al. (29), Mueller (50), Masaki and Takaiti (31), and Masaki and Takaiti (32), respectively. The ability of serine protease to hydrolyze native myofibrillar proteins was studied as follows. The reaction mixture contained 0.25 M sodium phosphate buffer, pH 8.0, and a 10~J M concentration of the test myofibrillar protein with or without 2 X10"' to 10"7 M serine protease in a final volume of 1.0 ml. The mixture was incubated at 37°C for 0 to 60min. Samples of 0.2 ml were removed at intervals, mixed with 1.3 ml of distilled water and boiled for 3 min to stop the reaction. Then 1.5 ml of 0.01 % Fluescin was added and the mixtures were centrifuged at 3,000 rpm for 10 min. Material with fluorescence in the supernatant, with
RESULTS Table I shows the ability of serine protease to hydrolyze various purified myofibrillar proteins, determined by measuring the amounts of amino acids or peptides released during the incubation. The release from myosin was rapid and linear for 15 min on incubating a molar ratio of serine protease to myosin of 1 : 500, but then the release appeared to level off gradually, judging from the data obtained at a molar ratio of 1 :100. Troponin and tropomyosin showed similar patterns of degradation, all suggesting limited proteolysis. Small amounts of amino groups were released linearly from actin during the incubation, but no liberation of amino acids or peptides from aactinin or M-protein was observed. No release of amino acids or peptides from the myofibrillar proteins was observed in control experiments without the serine protease. These findings suggest that myosin, troponin, tropomyosin, and actin are susceptible to the serine protease but that a-actinin and M-protein are not. Next, the modes of degradation of the myofibrillar proteins were investigated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. /. Biochem.
Downloaded from https://academic.oup.com/jb/article-abstract/83/5/1355/775308 by University of Western Sydney user on 12 January 2019
Male white rabbits, weighing 3 to 4 kg, were used for the preparation of myofibrillar proteins. Male Wistar-strain rats, weighing 250 to 300 g, were used for the preparation of serine protease. Bovine serum albumin, used as a protein standard, was obtained from Miles Laboratory Inc. o-Phthalaldehyde (Fluescin) and its dilution buffer (Tritrisol) were obtained from Merck Japan Co. The marker proteins used in determination of molecular weights by sodium dodecyl sulfate-polyacrylamide gel electrophoresis were obtained from BDH Chemicals Ltd. All other chemicals used were commercial products of the highest grade available.
an appropriate level of L-leucine as a standard, was determined by the method of Benson et al. (33). Fluorescence was determined using a Hitachi MPF-2A fluorescence spectrophotometer. Degradation of myofibrillar protein was analyzed as follows. A reaction mixture similar to that for fluorometric analysis, but with different molar ratios of serine protease to substrate was used. At various times, samples of 0.2 ml were removed for sodium dodecyl sulfate-polyacrylamide gel electrophoresis. An equal volume of 2% w/v sodium dodecyl sulfate containing 50% glycerol and 20 fil of 2-mercaptoethanol were added and the mixture was boiled for 3 min. The samples were then mixed with 10 pi of 1 % w/v bromphenol blue and a sample of 20 pg of the protein was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Electrophoresis was carried out by the method of Weber and Osborn (34) except that a slab gel containing 8.0% acrylamide was used. The molar ratios of enzyme to protein in the incubation mixtures are given in the legends to the figures.
ACTION OF SERINE PROTEASE ON MYOFIBRILLAR PROTEINS
1357
TABLE I. Activities of the serine protease on various myofibrillar proteins. Substrate
Troponin Tropomyosin Actin or-Actinin M-Protein 1
0
5
10
15
1 500
0
16.0
27.0
1 500
0
1 100
0
38.5
1
100
0
7.0
52.6 15.5
1
41.4 34.0 57.8 24.0 24.5 16.0
30
60
51.5
73.5
28.0 30.0
35.0 38.0 13.0
100
0
1 100
0
1
100
0
5.5
9.$
1
100
0
0
0
0
1
100
0
0
0
0
Molar ratio of serine protease to substrate in the reaction mixture.
a
b c d e f g h
a
bc
e f
10DD00
ID-T
IS
—
T i l •* Tn C ~ • • M M |i
13000
Fig. 1. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic patterns of myosin after incubation with and without serine protease. Myosin was incubated in 0.25 M sodium phosphate buffer, pH 8.0, for 0 (a), 15 (b), 30 (c), and 60 (d) min without the serine protease, and for 0 (e), 15 (f), 30 (g), and 60 (h) min with the protease. The molar ratio of serine protease to myosin was 1 : 300. The experimental conditions are described in " MATERIALS AND METHODS." H, Heavy chain of myosin; L-l, L-2, L-3, light chains of myosin.
Fig. 2. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic patterns of troponin after incubation with and without serine protease. Experimental conditions were similar to those for Fig. 1. Troponin was incubated for 0 (a) and 60 (b) min without the protease, and for 0 (c), 15 (d), 30 (e), and 60 (f) min with the protease. The molar ratio of the serine protease to troponin was I : 300. Tn-T, Troponin-T; Tn-I, troponin-1; Tn-C, troponin-C.
Figure 1 shows results on the initial degradation of purified myosin by the serine protease. Two kinds of fragments, with molecular weights of approximately 100,000 and 88,000, seemed to be produced by the serine protease degradation of myosin heavy chain. Light chain 2 was degraded very slowly, but light chain 1 showed no detectable change. Although the myosin preparation was
slightly contaminated, the contaminants were not digested by the serine protease. These results suggest that myosin is subject to limited proteolysis by the serine protease. Figure 2 shows the results of a similar experiment with purified troponin. Troponin-T was completely digested within 15 min, and troponin-I was also digested, although more slowly. However, troponin-C was not susceptible
Vol. 83, No. 5, 1978
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Myosin
Time of incubation (min)
Molar1 ratio
1358
N. YASOGAWA, Y. SANADA, and N. KATUNUMA
a fc c i i f
a
b c d e f §
Fig. 3. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic patterns of tropomyosin after incubation with and without the serine protease. Experimental conditions were as for Fig. 1. Tropomyosin was incubated for 0 (a) and 60 (b) min without the protease, and for 0 (c), 15 (d), 30 (e), and 60 (f) min with the protease. The molar ratio of the serine protease to tropomyosin was 1 : 200.
to the serine protease. The troponin preparation contained a contaminant with a molecular weight of 13,000, which appeared to be the proteolytic product formed from troponin by the serine protease. Figure 3 shows the patterns obtained on incubation of purified tropomyosin with and without the protease. Tropomyosin was degraded completely within 30 min by the protease. At an early stage of digestion (15 min), an intermediate product with a molecular weight of 32,000 appeared, but the serine protease acted on tropomyosin to break it ultimately into two components with molecular weights of 21,500 and 19,000 as well as further smaller fragments which were not stained. Figure 4 shows the effect of serine protease on purified actin. The band of purified actin disappeared very slowly, but no new fragments were detected. In Fig. 5 the effect of serine protease on purified a-actinin is shown. tr-Actinin showed no detectable change during incubation with or without the serine protease. These results indicate that the serine protease hydrolyzed several native myofibrillar proteins and that its mode of hydrolysis was limited.
Fig. 4. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic patterns of actin after incubation with and without serine protease. Experimental conditions were as for Fig. 1. Actin was incubated for 0 (a), 30 (b), and 60 (c) min without the protease, and for 0 (d), 15 (e), 30 (f), and 60 (g) min with the protease. The molar ratio of the serine protease to actin was 1 :200.
a I e i i f i
|
Fig. 5. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic patterns of a-actinin after incubation with and without serine protease. Experimental conditions were as for Fig. 1. cr-Actinin was incubated for 0 (a), 15 (b), 30 (c), and 60 (d) min without the protease, and for 0 (e), 15 (f), 30 (g), and 60 (h) min with the protease. The molar ratio of the serine protease to ir-actinin was 1 : 200. DISCUSSION
Dayton et al. (J8) showed that Cat+-dependent neutral protease degraded troponin-T, troponin-I, tropomyosin, and C-protein, but did not hydroJ. Biochem.
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3200D 21500 19000
ACTION OF SERINE PROTEASE ON MYOFIBRILLAR PROTEINS
Previously we reported that the activity of serine protease was greatly increased in the muscles of mice with hereditary muscular dystrophy (21) and also in the muscles of patients with progressive muscular dystrophy (22), and we detected several abnormal protein fragments formed by limited proteolysis of the muscles in cases of muscular dystrophy (21). Furthermore, when myofibrillar proteins of normal and dystrophic muscles were incubated with highly purified serine protease, their myosin, a-actinin and tropomyosin disappeared completely (21). The present results are consistent with these previous data, except with respect to a-actinin; the reason for the discrepancy in the case of a-actinin is not known. In spite of this discrepancy, the results show that serine protease, like CaI+-dependent neutral protease, is responsible for the initial degradation of several myofibrillar proteins and is involved in the turnover of myofibrillar proteins. Recently, Uchida et al. (35) reported another protease which readily degrades myosin of rat cardiac muscle. There also appear to be other Vol. 83, No. 5, 1978
proteases, including the lysosomal cathepsin group. Thus, the degradation of myofibrillar proteins and the turnover rate of each myofibrillar protein must depend on the cooperative actions of all these proteases under physiological conditions. A change in this balanced cooperation could result in increase in the action of some protease, such as serine protease (21, 22) or Ca1+-dependent neutral protease (20, 36) causing marked loss of certain myofibrillar proteins (21) and development of the characteristic pattern of decrease of myofibrillar proteins seen in muscular dystrophy, as reported by Sugita et al. (37-39). Intracellular protease activity seems to be regulated by the concentration of its specific inhibitor, which coexists with the protease (22). Further studies are required on the inhibitor of this serine protease. REFERENCES 1. Funabiki, R. & Cassens, R.G. (1973) / . Nutr. Sci. Vitaminol. 19, 361-368 2. Koizumi, T. (1974) / . Biochem. 76, 431^439 3. Zak, R., Martin, A.F., Prior, G., & Rabinowitz, M. (1977) /. Biol. Chem. 7S1, 3430-3435 4. Martin, A.F., Rabinowitz, M., Blough, R., Prior, G., & Zak, R. (1977) / . Biol. Chem. 252, 3422-3429 5. Tappel, A.L., Zalkin, H., Caldwell, K.A., Desai, I.D., & Shibko, S. (1962) Arch. Biochem. Biophys. 96,340-346 6. Iodice, A.A., Chin, J., Perker, S., & Weinstock, I.M. (1972) Arch. Biochem. Biophys. 152, 166-174 7. Iodice, A.A. (1976) Life Sci. 19, 1351-1358 8. Pearson, CM. & Kar, N.C. (1973) in Clinical Studies in Myology (Kakulas, B.A., cd.) pp. 89-97, Exccrpta Medica, Amsterdam 9. Koszalka, T.R. & Miller, L.L. (1960) / . Biol. Chem. 235,665-668 10. Koszalka, T.R. & Miller, L.L. (1960) / . Biol. Chem. 235, 669-672 11. Goldspink, D.F., Holmes, D., & Pennington, R J . (1971) Biochem. J. 125, 865-868 12. Mayer, M., Amin, R., & Shafrir, E. (1974) Arch. Biochem. Biophys. 161, 20-25 13. Brush, J.S. (1971) Diabetes 20, 140-145 14. Noguchi, T. & Kandatsu, M. (1971) Agric. Biol. Chem. 35, 1092-1100 15. Noguchi, T., Miyazawa, E., & Kametaka, M. (1974) Agric. Biol. Chem. 38, 253-257 16. Noguchi, T. & Kandatsu, M. (1976) Agric. Biol. Chem. 40, 927-933 17. Busch, W.A., Stromer, M.H., Goll, D.E., & Suzuki, A. (1972) / . Cell Biol. 52, 367-331
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lyze myosin, actin or a-actinin. The effects of our serine protease on troponin, tropomyosin, and a-actinin were very similar to those of the Ca1+dependent neutral protease, except that the products of limited proteolysis by the two enzymes were slightly different. Serine protease hydrolyzed tropomyosin mainly into two fragments with molecular weights of 21,500 and 19,000, whereas CaI+-dependent neutral protease hydrolyzed it to fragments with molecular weights in the range of 13,000 to 18,000. Moreover serine protease produced only one fragment with a molecular weight of approximately 13,000 from troponin-T and troponin-I, whereas CaI+-dependent neutral protease produced two fragments with molecular weights of 10,000 and 14,000 from troponin-T and troponin-I. The actions of serine protease and Ca1+-dependent neutral protease on myosin and actin were also different: serine protease hydrolyzed myosin very rapidly and actin slightly, but Ca1+dependent neutral protease did not hydrolyze either protein. Thus both enzymes exhibited marked specificity in their actions on myofibrillar proteins, suggesting that the two enzymes have different roles in the degradation of individual myofibrillar proteins under physiological and pathological conditions.
1359
1360
28. Ebashi, S. & Ebashi, F. (1955) / . Biochem. 55, 604-613 29. Ebashi, S., Kodama, A., & Ebashi, F. (1968) J. Biochem. 64, 465-477 30. Mueller, H. (1966) Biochem. Z. 345, 300-321 31. Masaki, T. & Takaiti, O. (1969) / . Biochem. 66, 637-643 32. Masaki, T. & Takaiti, O. (1974) / . Biochem. IS, 367-380 33. Benson, J.R. & Hare, P.E. (1975) Proc. Natl. Acad. Sci. U.S. 72, 619-622 34. Weber, K. & Osborn, M. (1969) /. Biol. Chem. 244, 4406-4412 35. Uchida, K., Murakami, U., & Hiratuka, T. (1977) / . Biochem. 82, 469^176 36. Kar, N.C. & Pearson, CM. (1976) Clin. Chim. Ada 73, 293-297 37. Sugita, H. & Toyokura, Y. (1976) Proc. Jap. Acad. 52, 256-259 38. Sugita, H. & Toyokura, Y. (1976) Proc. Jap. Acad. 52, 260-263 39. Sugita, H., Katagiri, T , Simizu, T , & Toyokura, Y. (1974) in Basic Research in Myology (Kakulas, B.A., ed.) pp. 291-297, Excerpta Medica, Amsterdam
/ . Biochem.
Downloaded from https://academic.oup.com/jb/article-abstract/83/5/1355/775308 by University of Western Sydney user on 12 January 2019
18. Dayton, W.R., GoU, D.E., Stromer, M.H., Reville, W.J., Zeece, M.G., & Robson, R.M. (1975) Cold Spring Harbor Conferences on Cell Proliferation, Vol. II. Proteases and Biological Control pp. 551577, Cold Spring Harbor, New York 19. Dayton, W.R., Goll, D.E., Zeece, M.G., Robson, R.M., & Reville, WJ. (1976) Biochemistry 15, 2150-2158 20. Dayton, W.R., Reville, WJ., Goll, D.E., & Stromer, M.H. (1976) Biochemistry 15, 2159-2167 21. Sanada, Y., Yasogawa, N., & Katunuma, N. (1978) / . Biochem. 83, 27-33 22. Katunuma, N., Yasogawa, N., Kito, K., Sanada, Y., Kawai, H., & Miyoshi, K. (1978) /. Biochem. 83, 625-628 23. Katunuma, N., Kominami, E., Kobayashi, K., Banno, Y., Suzuki, K., Chichibu, K., Hamaguchi, Y., & Katsunuma, T. (1975) Eur. J. Biochem. 52, 37-50 24. Sanada, Y., Shiotani, T., Okuno, E., & Katunuma, N. (1976) Eur. J. Biochem. 69, 507-515 25. Gornall, A.G., Bardawill, C.S., & David, M.M. (1949) / . Biol. Chem. 177, 751-766 26. Lowry, O.H., Rosebrough, N.J., Farr, A.L., & Randall, R.J. (1951) / . Biol. Chem. 193, 265-275 27. Perry, S.V. (1955) in Methods in Enzymology (Colowick, S.P. & Kaplan, N.O., eds.) Vol. 2, pp. 583-586, Academic Press, New York
N. YASOGAWA, Y. SANADA, and N. KATUNUMA
