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Elevations of cathepsin B and cathepsin L activities in forelimb and hind limb muscles of genetically dystrophic mice
EXPERIMENTAL
NEUROLOGY
93,642-646
(1986)
RESEARCH
NOTE
Elevations of Cathepsin B and Cathepsin L Activities in Forelimb and Hind Limb Muscles of Genetically Dystrophic Mice KEIJI KOMATSU, KAZUTOMO TSUKUDA, JUN HOSOYA, AND SUSUMU SATOH’ Department of Pharmacology. Pharmaceutical Institute, Tohoku University, Aobayama, Sendai 980, Japan Received November S, 1985; revision received May IS, 1986 The combined activities of cathepsin B and cathepsin L were studied in the forelimb and hind limb muscles of dystrophic mice. The activities of these proteases in the forelimb and hind limb muscles of young and adult dystrophic mice were significantly higher than those in normal mice. However, clinical involvement of dystrophy appeared in the hind limbs but not in the forelimbs. We therefore suggest that the increase in protease activity begins at a very early age and that the clinical involvement is not linked with the increase in cathepsins B and L. o 1986 Academic Pm, Inc.
Muscular dystrophy is a degenerative myopathy ascribable to some defect in the muscle fiber. The mechanisms involved remain to be elucidated. However, studies have shown that the activities of several proteases are markedly elevated in dystrophic muscles (3, 10, 11, 13, 17). Apparent clinical involvement of dystrophy in mice appears in the hind limbs, whereas in the forelimbs, no notable dystrophic symptoms are observed (9). The activity of cathepsin B, which may lead to muscle degeneration (12), is increased in dystrophic muscle of mice and chickens (11, 17). It Abbreviations: BANA-benzoyl-L-arginyl-p-naphthylamide, BAPA-benzoyl-L-arginyl-p nitroanilide. ’ We thank Dr. M. Ohzeki, Taisho Pharmaceutical Company, for providing succinyl-l-tyrosine-L-methionine-2-naphthylamide. 642 0014-4886/86 $3.00 Copyrig% Q 1986 by Academic Pres Inc. AU rights of reproduction in any form mend.
CATHEPSMS
B AND L IN DYSTROPHIC
MICE
643
would therefore be of interest to see whether or not protease activity is increased in the forelimb muscles of dystrophic mice. However, because cathepsin B activity is very low in skeletal muscles, it is not easy to determine the protease activity in limb muscles of dystrophic mice when benzoylL-arginyl+naphthylamide (BANA) or benzoyl-L-arginyl-pnitroanilide (BAPA) is used as a substrate. Katunuma et al. reported the use of new synthetic substrates for assays of cathepsin B which have a sensitivity to cathepsin B much greater than that of BANA (4). We therefore studied the relationship between protease activity and clinical involvement of dystrophy in mice using one of these new synthetic sub strates (succinyl-L-tyrosine-L-methionine-2-naphthylamide). Because the clinical manifestations of dystrophy in male and female mice are similar, in this study we used female homozygous dystrophic (dy/dy) and female phenotypically normal mice of the C57BL/6J-dy strain bred and raised in our department. Young mice were 3 weeks old, and adult mice between 11 and 14 weeks old. As young dystrophic mice did not show any clear clinical manifestation of the disease, they were distinguished from their normal litter-mates by the manifestations resulting from the application of pressure to the thighs. When the hind limbs were extended and released quickly and this procedure was repeated a few times, the hind limbs of dystrophic mice developed tonic spasms. The dystrophic mice thus selected showed the clinical manifestations of the disease reported by Michelson et al. (9) as they grew older. The animals were killed through ether anesthesia. All muscles of the foreand hind limbs were isolated, weighed, and minced finely with scissors. The hind limb muscles isolated from one mouse were homogenized in ice-cold phosphate-buffered saline (PH 7.4) consisting of 137.4 mM NaCl, 2.7 nGI4 KCl, 8.1 m&f NaHP04. 12H20, 1.5 m&I KHzP04, and Triton X-100 (1 ml/l), using an Ultra-Turrax homogenizer. Because the forelimb muscle mass isolated from one mouse was not enough for an assay of enzyme activity, the forelimb muscles isolated from two mice were combined, and then homogenized in the same manner as that described above. Each homogenate was centrifuged 10 min at 1600 g, and each supematant fluid was retained. Enzyme assays were carried out in tubes shaken at 37°C. The hydrolysis of succinyl-L-tyrosine-L-methionine-2-naphthylamide was measured in an incubation buffer solution (total volume, including enzyme, 2.0 ml) containing the substrate (2.0 n&I). The buffer consisted of 76.4 mA4 CH3COONa, 1 mMNa,EDTA, 2.0 rmirlcysteine, pure CH&OOH (3.8 ml/l), and dimethylsulfoxide ( 100 ml/l), pH 5.0. After a 20-min incubation, a coupling reagent (2.0 ml) was introduced to stop the reaction, n-butanol(4.0 ml) was added, and the tubes were thoroughly shaken and centrifuged 5 min at 1600 g. The absorbancy of the butanol layer was measured at 520 nm. An enzyme sample
644
KOMATSU
ET AL.
TABLE 1 Combined Activities of Cathepsins B and L in the Forelimb and Hind Limb Muscles of Dystrophic Mice Protease activity (X lo-’ nmol/min/mg protein)”
Age Young Adult
Mice Normal Dystrophic Normal Dystrophic
Hind limb muscles 10.3 + 45.7 -t 6.1 + 38.4 +
0.8 (6) 5.8 (6)* 0.6 (9) 5.8 (6)*
Forelimb muscles 12.1 + 1.7(7) 48.9 -t 3.9 (5)* 8.4 k 2.1 (7) 39.5 + 9.5 (8)*
’ The protease activity was defined as the amount of 2naphthylamine released from the substrate in 1 min/l mg protein at 37°C. Each value represents the mean + SE. Numbers in parentheses represent the numbers of samples tested. * P < 0.0 1 compared with normal (Student’s t test).
withheld from incubation was used as the blank; recrystallized 2-naphthylamine was the standard. The substrate was dissolved in dimethylsulfoxide and used within 20 min. The coupling reagent was prepared as described by Barrett (1). The first garnet used in preparing the coupling reagent was from Sigma, the substrate used was provided by Taisho Pharmaceutical Company, Japan, and the other chemicals were from Wako Pure Chemical Industries, Ltd., Japan. The amount of substrate hydrolyzed was linear for the time period used in the assay. Protein was determined by the method of Lowry et al., using bovine serum albumin as a standard (7). Cathepsin B and cathepsin L have been separated from cathepsin B1 (5, 16). Because the substrate used in this study is sensitive to both forms of cathepsin (4), it was not possible to distinguish the respective activities of the two proteases. Their combined activities were therefore determined, and are shown in Table 1. The protease activities in the fore- and hind limb muscles of adult dystrophic mice were significantly higher than those in the normal mice. Similarly, the protease activities in the fore- and hind limb muscles of young dystrophic mice were also significantly higher than those in the normal mice. Morphologic abnormalities have been demonstrated in both forelimb (15) and hind limb muscles (14) of dystrophic mice at an early age. Although it is possible that these proteases participate in the degeneration of muscle protein, as the presence of cathepsin B was shown to lead to the degeneration of myofibrillar proteins in vitro (12), it was also suggested that lysosomal proteases are not involved in the initial, rate-limiting steps of myo-
CATHEPSINS
B AND L IN DYSTROPHIC
MICE
645
fibrillar protein degeneration (2). Therefore, it remains to be elucidated whether or not the increased protease activity triggers the muscle degeneration characteristic of dystrophy. Cathepsin B and L activity in the forelimb muscles of dystrophic mice was similar to that in the hind limb muscles, irrespective of age (Table 1). Nevertheless, no clinical involvement of dystrophy developed in the forelimbs during the lifespan of any mouse. As described in our previous report (6), clinically apparent involvement of dystrophy appeared in the hind limbs between 6 and 7 weeks of age and worsened gradually until the hind limbs were permanently extended with joint contractures. However, no such clinical involvement appeared in the forelimbs. The reason why the forelimbs of dystrophic mice can maintain an apparently normal function is not clear at present. Mastaglia et al. suggested that abortive regeneration of muscle cells is responsible for the aggravation of Duchenne muscular dystrophy (8). It seems distinctly possible that an imbalance of muscle regeneration and muscle necrosis may contribute to such aggravation in dystrophic mice. It was also observed that the hind limbs of dystrophic mice were able to retain apparently normal function at an early age. From these results, we hypothesize that muscle regeneration is more active in the forelimbs than in the hind limbs, so that muscle necrosis in the forelimbs, but not in the hind limbs, can be compensated for by muscle regeneration, producing manifestations of clinical involvement only in the hind limbs. As there are only scanty reports of studies comparing muscle regeneration in the forelimbs with that in the hind limbs of dystrophic mice, our hypothesis has still to be evaluated. However, our finding that the forelimbs can maintain apparently normal function despite the increased protease activity is of interest in helping to clarify the mechanism of aggravation of muscular dystrophy. REFERENCES 1. BARRETT, A. J. 1972.A new assayfor cathepsin B, and other thiol proteases. Anal. Biochem.
47280-293. 2. GERAD, K. W., AND D. L. SCHNEIDER. 1979. Evidence for degeneration of myofibrillar proteins in lysosomes. J. Biol. Chern. 254: 11798-l 1805. 3. KAR, N. C., AND C. M. PEARSON. 1976. A calcium-activated neutral protease in normal and dystrophic human muscle. Clin. Acta 73: 293-297. 4. KATUNUMA, N., T. TOWATARI, M. TAMAI, AND K. HANADA. 198 1. Use of new synthetic substrated for assaysof cathepsin L and cathepsin B. J. Biochem. 93: 1129- 1135. 5. KIRSCHKE, H., J. LANGNER, B. WIEDERANDERS, S. ANSORGE, AND P. BOHLEY. 1977. A new protease from rat-liver lysosomes. Eur. J. Biochem. 74: 293-301. 6. KOMATW,K.,K.INAZUKI,J.HOSOYA, AND SSATOH. 1986.Beneficialefktofnewthiol protease inhibitors, epoxide derivatives, on dystrophic mice. Exp. Neural. 91:23-29. 7. LOWRY, 0. H., N. J. ROSEBROUGH,A. L. FARR, AND R. J. RANDALL. 195 1. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193: 265-275.
646
KOMATSU
ET AL.
8. MASTAGLIA, F. L., J. M. PAPADIMURIOU, AND B. A. KAKULAS. 1970. Regeneration of muscle in Duchenne muscular dystrophy: an electron microscopic study. J. Neural. Sci. 11: 425-444. 9. MICHELSON, A. M., E. S. RUSSELL, AND P. J. HARMAN. 1955. Dystrophia muscularis: a hereditary primary myopathy in the house mouse. Proc.Natl. Acad.Sci. U.S.A.41: 1079-1084. 10. NEERUNJUN, T. S., AND V. Duwowrrz. 1979. Increased calcium-activated neutral protease activity in muscles of dystrophic hamsters and mice. J. Neural. Sci. 40: 105- 111. 11. NODA, T., K. ISOGAI, N. KATUNUMA, Y. TARUMOTO, AND M. OHZEKI. 1981. Effect of cathepsin B, H, and D in pectoral muscle of dystrophic chickens (Line 4 13) of in viva administration of E-64-c. J. Biochem. 90:893-896. 12. NODA, T., K. Iscxx, H. HAYASHI, AND N. KATUNUMA. 198 1. Susceptibilities of various myofibrillar proteins to cathepsin B and morphological alteration of isolated myofibrils by thisenzyme. J. B&hem. 90:37l-379. 13. F%NNIGTON, R. J., AND J. E. ROBINSON. 1968. Cathepsin activity in normal and dystrophic human muscle. Enzymol.Biol. Clin.9: 175- 182. 14. FUZI-ER, A. C., AND J. A. POWELL. 1975. Fine structure of prenatal and early postnatal dystrophic mouse muscle. J. Neural. Sci.24: 109-126. 15. TOTSUKA, T., AND K. WATANABE. 198 1. Some evidence for concurrent involvement of the fore- and hindleg muscles in murine muscular dystrophy. Exp.Anim.30:465-470. 16. TOWATAFU, T.,K.TANAKA,D. YOSHIKAWA,ANDN.KATUNUMA. 1976.Separationofa new protease from cathepsin B, of rat liver lysosomes. FEBSLett. 67:284-288. 17. SANADA,Y.,YAMGAl,ANDN.~TUNUhSA. 1978. Serine protease in mice with hereditary muscular dystrophy. J. Biochem. 8%27-33.
NEUROLOGY
93,642-646
(1986)
RESEARCH
NOTE
Elevations of Cathepsin B and Cathepsin L Activities in Forelimb and Hind Limb Muscles of Genetically Dystrophic Mice KEIJI KOMATSU, KAZUTOMO TSUKUDA, JUN HOSOYA, AND SUSUMU SATOH’ Department of Pharmacology. Pharmaceutical Institute, Tohoku University, Aobayama, Sendai 980, Japan Received November S, 1985; revision received May IS, 1986 The combined activities of cathepsin B and cathepsin L were studied in the forelimb and hind limb muscles of dystrophic mice. The activities of these proteases in the forelimb and hind limb muscles of young and adult dystrophic mice were significantly higher than those in normal mice. However, clinical involvement of dystrophy appeared in the hind limbs but not in the forelimbs. We therefore suggest that the increase in protease activity begins at a very early age and that the clinical involvement is not linked with the increase in cathepsins B and L. o 1986 Academic Pm, Inc.
Muscular dystrophy is a degenerative myopathy ascribable to some defect in the muscle fiber. The mechanisms involved remain to be elucidated. However, studies have shown that the activities of several proteases are markedly elevated in dystrophic muscles (3, 10, 11, 13, 17). Apparent clinical involvement of dystrophy in mice appears in the hind limbs, whereas in the forelimbs, no notable dystrophic symptoms are observed (9). The activity of cathepsin B, which may lead to muscle degeneration (12), is increased in dystrophic muscle of mice and chickens (11, 17). It Abbreviations: BANA-benzoyl-L-arginyl-p-naphthylamide, BAPA-benzoyl-L-arginyl-p nitroanilide. ’ We thank Dr. M. Ohzeki, Taisho Pharmaceutical Company, for providing succinyl-l-tyrosine-L-methionine-2-naphthylamide. 642 0014-4886/86 $3.00 Copyrig% Q 1986 by Academic Pres Inc. AU rights of reproduction in any form mend.
CATHEPSMS
B AND L IN DYSTROPHIC
MICE
643
would therefore be of interest to see whether or not protease activity is increased in the forelimb muscles of dystrophic mice. However, because cathepsin B activity is very low in skeletal muscles, it is not easy to determine the protease activity in limb muscles of dystrophic mice when benzoylL-arginyl+naphthylamide (BANA) or benzoyl-L-arginyl-pnitroanilide (BAPA) is used as a substrate. Katunuma et al. reported the use of new synthetic substrates for assays of cathepsin B which have a sensitivity to cathepsin B much greater than that of BANA (4). We therefore studied the relationship between protease activity and clinical involvement of dystrophy in mice using one of these new synthetic sub strates (succinyl-L-tyrosine-L-methionine-2-naphthylamide). Because the clinical manifestations of dystrophy in male and female mice are similar, in this study we used female homozygous dystrophic (dy/dy) and female phenotypically normal mice of the C57BL/6J-dy strain bred and raised in our department. Young mice were 3 weeks old, and adult mice between 11 and 14 weeks old. As young dystrophic mice did not show any clear clinical manifestation of the disease, they were distinguished from their normal litter-mates by the manifestations resulting from the application of pressure to the thighs. When the hind limbs were extended and released quickly and this procedure was repeated a few times, the hind limbs of dystrophic mice developed tonic spasms. The dystrophic mice thus selected showed the clinical manifestations of the disease reported by Michelson et al. (9) as they grew older. The animals were killed through ether anesthesia. All muscles of the foreand hind limbs were isolated, weighed, and minced finely with scissors. The hind limb muscles isolated from one mouse were homogenized in ice-cold phosphate-buffered saline (PH 7.4) consisting of 137.4 mM NaCl, 2.7 nGI4 KCl, 8.1 m&f NaHP04. 12H20, 1.5 m&I KHzP04, and Triton X-100 (1 ml/l), using an Ultra-Turrax homogenizer. Because the forelimb muscle mass isolated from one mouse was not enough for an assay of enzyme activity, the forelimb muscles isolated from two mice were combined, and then homogenized in the same manner as that described above. Each homogenate was centrifuged 10 min at 1600 g, and each supematant fluid was retained. Enzyme assays were carried out in tubes shaken at 37°C. The hydrolysis of succinyl-L-tyrosine-L-methionine-2-naphthylamide was measured in an incubation buffer solution (total volume, including enzyme, 2.0 ml) containing the substrate (2.0 n&I). The buffer consisted of 76.4 mA4 CH3COONa, 1 mMNa,EDTA, 2.0 rmirlcysteine, pure CH&OOH (3.8 ml/l), and dimethylsulfoxide ( 100 ml/l), pH 5.0. After a 20-min incubation, a coupling reagent (2.0 ml) was introduced to stop the reaction, n-butanol(4.0 ml) was added, and the tubes were thoroughly shaken and centrifuged 5 min at 1600 g. The absorbancy of the butanol layer was measured at 520 nm. An enzyme sample
644
KOMATSU
ET AL.
TABLE 1 Combined Activities of Cathepsins B and L in the Forelimb and Hind Limb Muscles of Dystrophic Mice Protease activity (X lo-’ nmol/min/mg protein)”
Age Young Adult
Mice Normal Dystrophic Normal Dystrophic
Hind limb muscles 10.3 + 45.7 -t 6.1 + 38.4 +
0.8 (6) 5.8 (6)* 0.6 (9) 5.8 (6)*
Forelimb muscles 12.1 + 1.7(7) 48.9 -t 3.9 (5)* 8.4 k 2.1 (7) 39.5 + 9.5 (8)*
’ The protease activity was defined as the amount of 2naphthylamine released from the substrate in 1 min/l mg protein at 37°C. Each value represents the mean + SE. Numbers in parentheses represent the numbers of samples tested. * P < 0.0 1 compared with normal (Student’s t test).
withheld from incubation was used as the blank; recrystallized 2-naphthylamine was the standard. The substrate was dissolved in dimethylsulfoxide and used within 20 min. The coupling reagent was prepared as described by Barrett (1). The first garnet used in preparing the coupling reagent was from Sigma, the substrate used was provided by Taisho Pharmaceutical Company, Japan, and the other chemicals were from Wako Pure Chemical Industries, Ltd., Japan. The amount of substrate hydrolyzed was linear for the time period used in the assay. Protein was determined by the method of Lowry et al., using bovine serum albumin as a standard (7). Cathepsin B and cathepsin L have been separated from cathepsin B1 (5, 16). Because the substrate used in this study is sensitive to both forms of cathepsin (4), it was not possible to distinguish the respective activities of the two proteases. Their combined activities were therefore determined, and are shown in Table 1. The protease activities in the fore- and hind limb muscles of adult dystrophic mice were significantly higher than those in the normal mice. Similarly, the protease activities in the fore- and hind limb muscles of young dystrophic mice were also significantly higher than those in the normal mice. Morphologic abnormalities have been demonstrated in both forelimb (15) and hind limb muscles (14) of dystrophic mice at an early age. Although it is possible that these proteases participate in the degeneration of muscle protein, as the presence of cathepsin B was shown to lead to the degeneration of myofibrillar proteins in vitro (12), it was also suggested that lysosomal proteases are not involved in the initial, rate-limiting steps of myo-
CATHEPSINS
B AND L IN DYSTROPHIC
MICE
645
fibrillar protein degeneration (2). Therefore, it remains to be elucidated whether or not the increased protease activity triggers the muscle degeneration characteristic of dystrophy. Cathepsin B and L activity in the forelimb muscles of dystrophic mice was similar to that in the hind limb muscles, irrespective of age (Table 1). Nevertheless, no clinical involvement of dystrophy developed in the forelimbs during the lifespan of any mouse. As described in our previous report (6), clinically apparent involvement of dystrophy appeared in the hind limbs between 6 and 7 weeks of age and worsened gradually until the hind limbs were permanently extended with joint contractures. However, no such clinical involvement appeared in the forelimbs. The reason why the forelimbs of dystrophic mice can maintain an apparently normal function is not clear at present. Mastaglia et al. suggested that abortive regeneration of muscle cells is responsible for the aggravation of Duchenne muscular dystrophy (8). It seems distinctly possible that an imbalance of muscle regeneration and muscle necrosis may contribute to such aggravation in dystrophic mice. It was also observed that the hind limbs of dystrophic mice were able to retain apparently normal function at an early age. From these results, we hypothesize that muscle regeneration is more active in the forelimbs than in the hind limbs, so that muscle necrosis in the forelimbs, but not in the hind limbs, can be compensated for by muscle regeneration, producing manifestations of clinical involvement only in the hind limbs. As there are only scanty reports of studies comparing muscle regeneration in the forelimbs with that in the hind limbs of dystrophic mice, our hypothesis has still to be evaluated. However, our finding that the forelimbs can maintain apparently normal function despite the increased protease activity is of interest in helping to clarify the mechanism of aggravation of muscular dystrophy. REFERENCES 1. BARRETT, A. J. 1972.A new assayfor cathepsin B, and other thiol proteases. Anal. Biochem.
47280-293. 2. GERAD, K. W., AND D. L. SCHNEIDER. 1979. Evidence for degeneration of myofibrillar proteins in lysosomes. J. Biol. Chern. 254: 11798-l 1805. 3. KAR, N. C., AND C. M. PEARSON. 1976. A calcium-activated neutral protease in normal and dystrophic human muscle. Clin. Acta 73: 293-297. 4. KATUNUMA, N., T. TOWATARI, M. TAMAI, AND K. HANADA. 198 1. Use of new synthetic substrated for assaysof cathepsin L and cathepsin B. J. Biochem. 93: 1129- 1135. 5. KIRSCHKE, H., J. LANGNER, B. WIEDERANDERS, S. ANSORGE, AND P. BOHLEY. 1977. A new protease from rat-liver lysosomes. Eur. J. Biochem. 74: 293-301. 6. KOMATW,K.,K.INAZUKI,J.HOSOYA, AND SSATOH. 1986.Beneficialefktofnewthiol protease inhibitors, epoxide derivatives, on dystrophic mice. Exp. Neural. 91:23-29. 7. LOWRY, 0. H., N. J. ROSEBROUGH,A. L. FARR, AND R. J. RANDALL. 195 1. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193: 265-275.
646
KOMATSU
ET AL.
8. MASTAGLIA, F. L., J. M. PAPADIMURIOU, AND B. A. KAKULAS. 1970. Regeneration of muscle in Duchenne muscular dystrophy: an electron microscopic study. J. Neural. Sci. 11: 425-444. 9. MICHELSON, A. M., E. S. RUSSELL, AND P. J. HARMAN. 1955. Dystrophia muscularis: a hereditary primary myopathy in the house mouse. Proc.Natl. Acad.Sci. U.S.A.41: 1079-1084. 10. NEERUNJUN, T. S., AND V. Duwowrrz. 1979. Increased calcium-activated neutral protease activity in muscles of dystrophic hamsters and mice. J. Neural. Sci. 40: 105- 111. 11. NODA, T., K. ISOGAI, N. KATUNUMA, Y. TARUMOTO, AND M. OHZEKI. 1981. Effect of cathepsin B, H, and D in pectoral muscle of dystrophic chickens (Line 4 13) of in viva administration of E-64-c. J. Biochem. 90:893-896. 12. NODA, T., K. Iscxx, H. HAYASHI, AND N. KATUNUMA. 198 1. Susceptibilities of various myofibrillar proteins to cathepsin B and morphological alteration of isolated myofibrils by thisenzyme. J. B&hem. 90:37l-379. 13. F%NNIGTON, R. J., AND J. E. ROBINSON. 1968. Cathepsin activity in normal and dystrophic human muscle. Enzymol.Biol. Clin.9: 175- 182. 14. FUZI-ER, A. C., AND J. A. POWELL. 1975. Fine structure of prenatal and early postnatal dystrophic mouse muscle. J. Neural. Sci.24: 109-126. 15. TOTSUKA, T., AND K. WATANABE. 198 1. Some evidence for concurrent involvement of the fore- and hindleg muscles in murine muscular dystrophy. Exp.Anim.30:465-470. 16. TOWATAFU, T.,K.TANAKA,D. YOSHIKAWA,ANDN.KATUNUMA. 1976.Separationofa new protease from cathepsin B, of rat liver lysosomes. FEBSLett. 67:284-288. 17. SANADA,Y.,YAMGAl,ANDN.~TUNUhSA. 1978. Serine protease in mice with hereditary muscular dystrophy. J. Biochem. 8%27-33.