COMMUNICATION
J. Biochem. 83, 625-628 (1978)
Abnormal Expression of a Serine Protease in Human Dystrophic Muscle1 Nobuhiko KATUNUMA,* Nobumasa YASOGAWA," Kiyoaki KITO,* Yukihiro SANADA,* Hisaomi KAWAI,*** and Kazuo MIYOSHI*** •Department of Enzyme Chemistry, Institute for Enzyme Research, School of Medicine, Tokushima University, "2nd Department of Internal Medicine, School of Medicine, Tokushima University, and ***lst Department of Internal Medicine, School of Medicine, Tokushima University, Kuramoto-cho 3, Tokushima, Tokushima 770 Received for publication, October 12, 1977
The activities of serine protease in muscles from normal persons and from patients with progressive muscular and neuromuscular diseases have been determined. A significant increase in the level of serine protease was found in muscle of patients with Duchenne-type muscular dystrophy and with Becker-type muscular dystrophy, but the activity was not increased in muscle of a patient with amyotrophic lateral sclerosis.
The intracellular concentrations of structural proteins in myofibrils are regulated by their rates of synthesis and degradation. Thus the abnormal decrease of these proteins in muscular dystrophy may be due to increased catabolism or decreased anabolism of myofibrillar proteins, or both. Proteases are responsible for degradation of myofibrillar proteins, and Simon et al. (1) reported that in mice with muscular dystrophy the turnovers of liver proteins were normal but the turnovers of muscle proteins and the catabolism of myofibrillar proteins were increased. These results show that certain intracellular proteases are at least partly responsible for the decrease of myofibrillar proteins in dystrophic muscle. Since the activities of lyso-
1
This work was supported in part by a grant for research on muscular dystrophy from the Ministry of Health and Welfare, Japan (1976). Vol. 83, No. 2, 1978
soma! catheptic enzymes are slightly increased in dystrophic muscle {2-4), these enzymes have been thought to be responsible for the degradation of myofibrillar proteins. However, it seems unlikely that the characteristic decrease of myofibrillar proteins in muscular dystrophy (5, 6) is caused entirely by lysosomal catheptic enzymes (7, 8). Several proteases besides catheptic enzymes have been found in muscle (9-11) but only the properties of the Ca1+-dependent neutral protease have been reported (12, 13). Recently, we discovered new intracellular serine proteases in rats in skeletal muscle (14, 15), liver mitochondria (15), and the muscle layer of the intestine (15), and we suggested that these proteases are involved in the initial degradation of intracellular proteins in the various organs (16-18). This paper reports an abnormal increase in the activity of the new serine protease in the muscles of patients with muscular dystrophy.
625
626
COMMUNICATION
Muscle biopsy specimens were obtained from 5 patients with muscular dystrophy, diagnosed on the basis of clinical and biochemical tests. Biopsy specimens from 2 patients with no evidence of muscle disease were used as controls. The samples were frozen as soon as possible and their enzyme activities were assayed within a week. The activity of the serine protease was assayed as reported previously {15). One unit of serine protease was defined as the amount inactivating 50% of apoornithine aminotransferase [EC 2.6.1.13] in 60 min at 37°C. Ornithine aminotransferase, creatine phosphokinase [EC 2.7.3.2] and lactate dehydrogenase [EC 1.1.1.27] were assayed by the methods of Matsuzawa et al. {19), Kuby et al. (20), and Wroblewsky and La Due (21), respectively. Protein concentrations were determined by the Biuret method (22) or by the method of Lowry et al. (23) using bovine serum albumin as the standard. For assay of serine protease, acetone powder of 300 mg wet weight of muscle obtained by biopsy was extracted with 4 ml of 10 mM potassium phosphate buffer, pH 8.0. The extract was centrifuged and the precipitate was resuspended in a final volume of 1.5 ml of the same buffer. The serine protease activity of this preparation was assayed. For assay of lactate dehydrogenase, 100 mg wet weight of muscle was homogenized with 0.25 M sucrose in 10 mM Tris-HCl buffer, pH 7.5, the
homogenate was centrifuged for 20 min at 15,000 rpm and the activity of the supernatant was assayed. Table I shows the serine protease activities of specimens of muscle from patients with various types of muscular dystrophy. The total protein concentration was normal in the muscles of patients with amyotrophic lateral sclerosis and Becker muscular dystrophy, but was greatly decreased in the muscles of patients with Duchenne muscular dystrophy. Serum creatine phosphokinase activity, measured by the method of Ebashi et al. (24), was higher than normal in the muscles of all the cases of Duchenne muscular dystrophy and Becker muscular dystrophy. The serine protease activity was significantly increased in the cases of myogenic muscular dystrophy, such as Duchenne or Becker muscular dystrophy. It is interesting that in the cases of Becker muscular dystrophy the activity of serine protease increased before there was any marked decrease of muscular proteins. However, the one patient with amyotrophic lateral sclerosis, who had neurogenic muscular atrophy, had only slightly increased activity. We have reported (25) that the serine protease activity in skeletal muscle during atrophy caused by denervation increased to three times that in control muscle, and we observed a similar tendency in the muscle of the case with amyotrophic lateral sclerosis. Thus, the activities of serine protease were different in myogenic and neurogenic muscular atrophies. Use of two dif-
TABLE I. Enzyme activities in dystrophic muscle. Serum Case No. 1 2
3
Position
Type
Age
DMD DMD DMD
12
M. vast us lateral is
7.5
4
M. vastus lateral is
6.3 ,
20
M. gastrocnemius
—
Creatine phosphokinase (nng/lOOmg wet wt.) (units/ml/min)
4
BMD
20
M. vastus lateral is
5
BMD
25
M. vastus lateralis
6
ALS
52
M. deltoideus
7
Normal 56 M. deltoideus Normal 78 M. iliopsoas
8
Total protein
18.9 19.0 21.6 22.3 19.6
570
1,400
Muscle Lactate dehydrogenase (/imol/mg/min)
Serine protease (units/mg/h)
1.02 2.14
24.0 11.0 25.3 15.4 16.0
167
—
200
3.50 2.15 1.66 1.26 0.72
140 12
J. Biochem. 83, 625-628 (1978)
Abnormal Expression of a Serine Protease in Human Dystrophic Muscle1 Nobuhiko KATUNUMA,* Nobumasa YASOGAWA," Kiyoaki KITO,* Yukihiro SANADA,* Hisaomi KAWAI,*** and Kazuo MIYOSHI*** •Department of Enzyme Chemistry, Institute for Enzyme Research, School of Medicine, Tokushima University, "2nd Department of Internal Medicine, School of Medicine, Tokushima University, and ***lst Department of Internal Medicine, School of Medicine, Tokushima University, Kuramoto-cho 3, Tokushima, Tokushima 770 Received for publication, October 12, 1977
The activities of serine protease in muscles from normal persons and from patients with progressive muscular and neuromuscular diseases have been determined. A significant increase in the level of serine protease was found in muscle of patients with Duchenne-type muscular dystrophy and with Becker-type muscular dystrophy, but the activity was not increased in muscle of a patient with amyotrophic lateral sclerosis.
The intracellular concentrations of structural proteins in myofibrils are regulated by their rates of synthesis and degradation. Thus the abnormal decrease of these proteins in muscular dystrophy may be due to increased catabolism or decreased anabolism of myofibrillar proteins, or both. Proteases are responsible for degradation of myofibrillar proteins, and Simon et al. (1) reported that in mice with muscular dystrophy the turnovers of liver proteins were normal but the turnovers of muscle proteins and the catabolism of myofibrillar proteins were increased. These results show that certain intracellular proteases are at least partly responsible for the decrease of myofibrillar proteins in dystrophic muscle. Since the activities of lyso-
1
This work was supported in part by a grant for research on muscular dystrophy from the Ministry of Health and Welfare, Japan (1976). Vol. 83, No. 2, 1978
soma! catheptic enzymes are slightly increased in dystrophic muscle {2-4), these enzymes have been thought to be responsible for the degradation of myofibrillar proteins. However, it seems unlikely that the characteristic decrease of myofibrillar proteins in muscular dystrophy (5, 6) is caused entirely by lysosomal catheptic enzymes (7, 8). Several proteases besides catheptic enzymes have been found in muscle (9-11) but only the properties of the Ca1+-dependent neutral protease have been reported (12, 13). Recently, we discovered new intracellular serine proteases in rats in skeletal muscle (14, 15), liver mitochondria (15), and the muscle layer of the intestine (15), and we suggested that these proteases are involved in the initial degradation of intracellular proteins in the various organs (16-18). This paper reports an abnormal increase in the activity of the new serine protease in the muscles of patients with muscular dystrophy.
625
626
COMMUNICATION
Muscle biopsy specimens were obtained from 5 patients with muscular dystrophy, diagnosed on the basis of clinical and biochemical tests. Biopsy specimens from 2 patients with no evidence of muscle disease were used as controls. The samples were frozen as soon as possible and their enzyme activities were assayed within a week. The activity of the serine protease was assayed as reported previously {15). One unit of serine protease was defined as the amount inactivating 50% of apoornithine aminotransferase [EC 2.6.1.13] in 60 min at 37°C. Ornithine aminotransferase, creatine phosphokinase [EC 2.7.3.2] and lactate dehydrogenase [EC 1.1.1.27] were assayed by the methods of Matsuzawa et al. {19), Kuby et al. (20), and Wroblewsky and La Due (21), respectively. Protein concentrations were determined by the Biuret method (22) or by the method of Lowry et al. (23) using bovine serum albumin as the standard. For assay of serine protease, acetone powder of 300 mg wet weight of muscle obtained by biopsy was extracted with 4 ml of 10 mM potassium phosphate buffer, pH 8.0. The extract was centrifuged and the precipitate was resuspended in a final volume of 1.5 ml of the same buffer. The serine protease activity of this preparation was assayed. For assay of lactate dehydrogenase, 100 mg wet weight of muscle was homogenized with 0.25 M sucrose in 10 mM Tris-HCl buffer, pH 7.5, the
homogenate was centrifuged for 20 min at 15,000 rpm and the activity of the supernatant was assayed. Table I shows the serine protease activities of specimens of muscle from patients with various types of muscular dystrophy. The total protein concentration was normal in the muscles of patients with amyotrophic lateral sclerosis and Becker muscular dystrophy, but was greatly decreased in the muscles of patients with Duchenne muscular dystrophy. Serum creatine phosphokinase activity, measured by the method of Ebashi et al. (24), was higher than normal in the muscles of all the cases of Duchenne muscular dystrophy and Becker muscular dystrophy. The serine protease activity was significantly increased in the cases of myogenic muscular dystrophy, such as Duchenne or Becker muscular dystrophy. It is interesting that in the cases of Becker muscular dystrophy the activity of serine protease increased before there was any marked decrease of muscular proteins. However, the one patient with amyotrophic lateral sclerosis, who had neurogenic muscular atrophy, had only slightly increased activity. We have reported (25) that the serine protease activity in skeletal muscle during atrophy caused by denervation increased to three times that in control muscle, and we observed a similar tendency in the muscle of the case with amyotrophic lateral sclerosis. Thus, the activities of serine protease were different in myogenic and neurogenic muscular atrophies. Use of two dif-
TABLE I. Enzyme activities in dystrophic muscle. Serum Case No. 1 2
3
Position
Type
Age
DMD DMD DMD
12
M. vast us lateral is
7.5
4
M. vastus lateral is
6.3 ,
20
M. gastrocnemius
—
Creatine phosphokinase (nng/lOOmg wet wt.) (units/ml/min)
4
BMD
20
M. vastus lateral is
5
BMD
25
M. vastus lateralis
6
ALS
52
M. deltoideus
7
Normal 56 M. deltoideus Normal 78 M. iliopsoas
8
Total protein
18.9 19.0 21.6 22.3 19.6
570
1,400
Muscle Lactate dehydrogenase (/imol/mg/min)
Serine protease (units/mg/h)
1.02 2.14
24.0 11.0 25.3 15.4 16.0
167
—
200
3.50 2.15 1.66 1.26 0.72
140 12
