0020-711X/92S5.M)+ 0.00 Pergamon Press Ltd
Int. J. Biochem.Vol. 24, No. 12, Pp. 19674978, 1992 Printed in Great Britain
MODE OF ACTION OF RABBIT SKELETAL MUSCLE CATHEPSIN B TOWARDS MYOFIBRILLAR PROTEINS AND THE MYOFIBRILLAR STRUCTURE MASANORI MATSUISHI,~* TIBUYO MATSUMOT~,~* AKIHIRO OKITANI’ and HIROMICHI KATO~
‘Department of Food Science and Technology, Nippon Veterinary and Animal !kience University, Musashino-shi, Tokyo 180, Japan, *Department of Nutrition, Faculty of Home Economics, Tokyo Kasei University, Itabashi-ku, Tokyo 173, Japan and 3Department of Agricultural Chemistry, University of Tokyo, Bunkyo-ku, Tokyo 113, Japan (Received 22 April
1992)
Abstract-1. The mode of degradation of myofib~~r proteins and the structural changes in myofibrils due to the action of cathepsin B highly purified from rabbit skeletal muscle were studied. 2. Cathepsin B degraded myosin heavy chain, actin and troponin T, but not a-actinin, tropomyosin, troponin I or troponin C among myofibrillar proteins. 3. Cathepsin B optimally degraded myosin heavy chain,, actin and troponin T at around pH 5. Degradation of myosin heavy chain produced 6 fragments, 180,080, 150,080,87,000,81,000, 75,000 and 69,000 Da, respectively. Actin was hydrolyzed into fragments of 41,000, 38,008 and 30,080 Da. Troponin T was degraded into fra8rnents of 21,000, 12,008 and 10,000 Da. 4. Cathepsin B caused the fragmentation of myofibrils and disturbance of the lateral arrangement of myotibrils. 5. Cathepsin B partly disintegrated the Z-line and the M-line, and induced disordering of the arrangement of filaments in the I-band.
INTRODUCITON
Cathepsin B, which is considered to be one of the major proteinases involved in the lysosomal pathway in the proteolytic system of animal cells, has been purified from various tissues and its properties have been investigated. Skeletal muscle cathepsin B was highly purified from monkey (Hirao et al., 1984) and rabbit (Okitani et uf., 1988). In addition, there have been a few studies on the mode of degradation of myofibrillar proteins by cathepsin B in order to clarify its role in the degradation of the proteins in skeletal muscle cells. Hirao et al. (1984) investigated the action of cathepsin B highly purified from monkey skeietal muscle on skeletal muscle myosin and cardiac muscle myofibrils, and reported that myosin and actin were degraded. However, they did not determine whether or not regulatory proteins such as troponin and tro~myos~ were degraded. Schwartz and Bird (1977) reported that rat skeletal muscle cathepsin B degraded myosin. However, it was not certain that such degradation was due to cathepsin B alone, since there was no evidence that their cathepsin B preparation was really free from cathepsin L, which was later reported to be similar to cathepsin B in several properties (Okitani t-2 al., *To whom correspondence and reprint requests should be addressed. *Deceased.
1980). On the other hand, Noda et al. (1981) demonstrated that myosin, actin, tropomyosin and troponin were degraded when various myofibrillar proteins of rat skeletal muscle were treated with cathepsin B higbly purified from rat liver. However, the results obtained for the liver enzyme do not imply that the same results will be- obtained for the skeletal muscle enzyme, because it has not been examined whether this enxyme exhibits tissue specificity or not. As mentioned above, information on the mode of degradation of myofibrillar proteins by skeletal muscle cathepsin B is not sufficient yet. Therefore, we purified cathepsin B from rabbit skeletal muscle, definitely eliminating cathepsin L, according to the method reported previously (Okitani et al., 1988), and investigated its action towards various myofibrillar proteins. Moreover, we revealed structural changes in myofib~ls treated with cathepsin B. MATRRUIS AND METHOBS Materials
Rabbit muscles (longissimus dorsi and psoas) were obtained from carcasses immediately after death. The longissirnus dorsi muscle was minced and used to prepare cathepsin B, and myofibrils and their constituent proteins. The psoas muscle was used to prepare glycerinated muscle fibers. Pepstatin A was purchased from the Peptide Institute, Osaka, Japan. Bovine serum albumin, ovalbumin, chymotrypsinogcn and cytochrome c were purchased from
1967
Boehringer Mannheim GmbH. DEAE-Sephadex A-SO, Sephadex G-75, Sephadex G-100 and CNBr-activated Sepharose 4B were products of Pharmacia Pine Chemicals, Sweden. Phosph~~~o~ was obtained from Seikagaku Kogyo. DEAE-Toyopearl was obtained from Toyosoda Kogyo. Prewration of cathepsinB Cathepsin
B was prepared
according
to the method
Enzyme treatmentof myofibrifsfor observationof morphofogicaf changes Myofibrils (1.5 mgjml) were incubated with cathepsin B (1.45 mu/ml) at 4°C for 9 days in the presence of 40 mM T&-acetate buiTer (pH 5.5)lO.l M KCl/3 mM dithiothreitol/l mM EDTA/O. 1 mM pepstatin/lO mM NaN, . The morphological changes of myofibrils were observed under a phase-contrast microscope.
reported previously (Okitani et al., 1988). The crude enzyme
Enzyme tre&nent of gfycerinatedmusclefibers
extracted from the muscle homo~nate at pH 3.7 for 2hr was fractionated with @H&SO,. The precipitate between 25 and 45% saturation was applied to a Sephadex G-75 column and then to a phosphocellulose column. The active fractions obtained were purified on columns of APSepharose (an affinity adsorbent containing, as ligands, peptides carrying arginine at their C termini), DEAEToyopearl and Sephadex G-100. The purified enzyme was shown to be homogeneous on SD~poly~~la~de gel electrophoresis. The amount, in units, of cathepsin B activity was determined by incubation with 0.5mM N-abenzoyl-DL-arginine-@-naphthylamide at 37°C in 50 mM potassium phosphate buffer (pH 6.2)/0.4mM EDTA/ 0.3 mM dithiothreitol. One unit of activity represents the hydrolysis of 1 pmol substrate/~n,
Glycerinated muscle fibers were washed with 6.7mM potassium phosphate buffer (pH 7.01fO.lM KCl/l mM MgCl, and then cut into small pieces (ca 1cm in length) with a razor. Ten pieces were incubated with cathepsin B (54mU/ml) in 1 ml of 40mM Tris-acetate buffer (pH 55)/O. 1 M KC1/3 mM dithiothreitol/l mM EDTA/O.l mM pcpstatin at 37°C for 4.5 hr.
Preparationof myojbrffs Myofibrils were prepared as described by Yang et al. (1970). Preparationof gfy~erfnotedmuscfefibers Glycerinated muscle fibers were prepared from psoas muscle according to the procedure of Huxley (1963). Preparationof myojibriffarproteins Myosin was prepared as described by Perry (1955) and purified further by DEAE-Sephadex A-50 column chromatography as described by Richards et al. (1967). Actin was prepared according to the method of Mommaerts (1951). a-Actinin was prepared by the method of Masaki and Takaiti (1969). Preparation of troponin and its subunits was carried out according to the method of Ebashi et al. (1971). Tropomyosin was prepared from the residue obtained on lithium chlorideextraction of troponin by the method of Ebasi et 41. (1971), according to the methods of Mueller
(1966), Hartshome and Mueller (1969) and Woods (1969). Enzyme treatmentof myojibrifsandtheirconstituentproteins for efectrophoreticonafysis The substrate proteins (2.Omg/ml of myofibrils or 77-f256&nl of each of the constituent proteins) were incubated with cathepsin B (1.2-2.84 mU/ml) at 37°C in 5OmM sodium acetate-HCl buffer or Tris-acetate buffer/3 mM dithiothreitol/0.2-0.3 mM pepstatin/l mM NaN,, in a total volmne of 0.3 ml. The enzyme concentrations employed for the treatment of myofibrils and the constituent proteins were comparable to the values expected for the muscle homogenates, on the basis of the purification data previously reported (Okitani et al., 1988). Pep&tin was added to block cathepsin D type activity, if any, bound to the substrate proteins. At a given time, 0. IS ml of 60 mM sodium phosphate butl’er (pH 7.2)/5.24% SDS/32.4% 2-mercaptoethanol/20.2% glycerol/O.O29% bromophenol blue was added to the incubated mixtures, followed by boiling for 5 min to stop the enzymatic reaction.
SD~-~fy~ry~~
gel efectrophores~
SDS-polyacrylamide gel electrophoresis was carried out as described by Weber and Osbom (1969) using 5 or 10% gels containing 0.1% SDS. The gels were stained with Coomassie brilliant blue R-250. The molecular masses of myofibrillar proteins and their degradation products were determined by comparing their mobilities with those of myosin heavy chain (2OOkDa), a-actinin (95 kDa), bovine serum albumin (68 kDa), ovalbumin (45 kDa), chymotrypsinogen (25 kDa) and cytochrome c (12.5 kDa). Electronmicroscopy Double fixation in 3.0% glutaraldehyde and 1.0% osmium tetraoxide was followed by dehydration through a graded ethanol and embedding in an Epon mixture. Thin sections were stained with saturated many1 acetate in water and poststained with lead citrate (Suzuki et al., 1978). Specimens were examined under a Hitachi H 300 electron microscope operated at 75 kV. Protein ~te~~~tion Protein concentrations of myofibrils and their constituent proteins were determined by the biuret method (Gomall et al., 1949) or the method of Lowry et al. (1951), using bovine serum albumin as a standard. RESULTS
Myofibrils were incubated with cathepsin 3 at 37°C and pH 3.8-6.7 for 17 hr, and then their changes were investigated by SDS-polyacrylamide gel electrophoresis. As shown in Fig. 1, myosin heavy chain was degraded at pH 3.8-5.1, two bands of 180,000 and 15O,~Da, which seemed to represent the degradation products of myosin heavy chain, being produced. These changes were most remarkable at pH 5.1. The intensity of the band corresponding to actin decreased, and a 30,000 Da band appeared at pH 4.2 and 5.1. Figure 2 shows the time-course of changes in myofibrils treated with cathepsin B at pH 5.0 and 37°C for up to 24 hr. The intensity of the bands of myosin heavy chain and actin decreased with the elapse of the time up to 24 hr. The 180,000 and 150,000 Da bands appeared after 2 hr incubation, and their intensity increased up to 8 ht, but decreased after 24 hr incubation. 87,000, 81,000,75,000,69,000
Myofibrils treated with cathepsin B and 30,000 Da bands appeared after 4 hr incubation, but disappeared after 24 hr incubation. These degradation products were thought to be further degraded into smaller polypeptides before the elapse of 24 hr. Action towards donated myo~bri~~arprotein In order to determine the origins of various degradation products observed on the treatment of myofibrils with cathepsin B and to clarify the susceptibility of troponin T and/or tropomyosin, both of which comigrated on the electrophoresis of myofibrils, the changes in various isolated myofibrillar proteins on treatment with cathepsin B were examined by SDS-polyacrylamide gel electrophoresis. Action towards myosin. Figure 3 shows the changes in myosin on Trident with cathepsin B at 37°C and pH 3.9-6.8 for 17 hr. The degradation of myosin heavy chain and the generation of a 150,000 Da fragment were observed at pH 4.3-6.8. These changes were most notable at pH 5.1-6.2. In this pH range, 87,000, 81,000, 75,000 and 69,000 Da fragments also appeared. Figure 4 shows the time-course of changes in myosin on treatment with cathepsin B at pH 5.5 for up to 24 hr. The reason why pH 5.5 was used for the treatment is that it is close to the ultimate pH of postmortem muscles, i.e. pH 5.6 and the optimum pH for the cathepsin B action towards myosin. Thus, treatment at this pH also provided information on the autolysis of postmortem muscles. In the following experiments, pH 5.5 was used for the same reason. After 3 hr incubation, 180,000 and 150,000 Da fragments emerged, the amounts of which were almost constant until 24 hr. Fragments of 87,000, 81,000, 75,000 and 69,000 Da also appeared after 3 hr incubation. After 24hr incubation, myosin heavy chain decreased considerably, These results demonstrated that myosin heavy chain was easily hydroly~d almost completely into the 150,OOf1Da fragment, which was relatively resistant to further degradation. Action towarak actin. The changes in actin on treatment with cathepsin B at 37°C and pH 4.4-6.8 for 17 hr are shown in Fig. 5. Fragments of 41,000, 38,000 and 30,000 Da appeared, and these changes were most remarkable at pH 5.1. Figure 6 shows the time+ourse of changes in actin on treatment with cathepsin B at pH 5.5 for up to 24 hr. The 38,000 Da fragment emerged after 3 hr incubation and the 41,000 Da fragment after 6 hr incubation. The results in Figs 5 and 6 reveafed that the major degradation product of actin was the 38,000 Da fragment. Moreover, the combination of these results and the following results demonstrated that the 30,000 Da fragment was generated only from actin, suggesting that actin was the origin of the 30,000 Da fragment observed on treatment of myofibrils with cathepsin B. Action towards a-actinin and tropomyosin. As shown in Figs 7 and 8, a-actinin and tropomyosin did not undergo any change on treatment with cathepsin B at 37°C for 17 hr, at pH 3.9-6.8 or pH 3.8-6.7, respectively.
1969
Action towura!s troponin. Figure 9 shows the changes in troponin on treatment with cathepsin B at 37°C and pH 3.8-6.8 for 17 hr: 30,000 and 12,000 Da fragments emerged in the case of incubation at pH 5.1 without cathepsin B (lane 1). This phenomenon was also seen in the case of incubation at pH 5.5 without cathepsin B, as shown in Fig. 10 (lane 6). These changes were considered to be caused by some proteinase, other than cathepsin B, contaminating the troponin (see Discussion). Taking into account the phenomena observed in Fig. 9, the degradation product with cathepsin B alone is conceivably the 10,000 Da fragment which was observed in the case of incubation at pH 5.1-6.1 with cathepsin B (lanes 4, 5 and 6), but not in a control (lane 1). Since this fragment was generated most noticeably at pH 5.1, this pH was judged to be the optimum pH for the action of cathepsin B towards troponin. Figure 10 shows the time-course of the changes in troponin on treatment with cathepsin B at pH 5.5 for up to 24 hr. With the elapse of time, troponin T decreased, and the fragments of 12,000 and 10,000 Da increased. Since the generation of the 12,000 Da fragment after 24 hr incubation was more remarkable in the mixture with cathepsin B (lane 5) than in that without cathepsin B (lane 6), a part of the fragment was suggested to be generated by cathepsin B. It was clear that the 10,000 Da fragment was produced by cathepsin B. The density of the 30,000 Da band observed on all treatments with cathepsin B for more than 3 hr (lanes 2, 3, 4 and 5) was lower than without cathepsin B (lane 6). Moreover, its density after 24 hr incubation was lower than that after 10 hr incubation. Therefore, this fragment was thought to be produced by unknown proteinases, other than cathepsin B, contaminating the troponin and then further degraded by cathepsin B. The density of the band of troponin I on all treatments for more than 3 hr was much higher than at 0 hr, which might be due to the appearance of the degradation product of troponin T at the same place as troponin I. Therefore, whether or not troponin I was degraded cannot be determined from these results. No change in the band of troponin C was observed. Troponin T, and a mixture of troponins I and C, which were isolated from the troponin complex, were treated with cathepsin B at pH 5.5 in order to clarify the su~ptibility of troponin I, and the origin of the fragments observed in Figs 9 and 10. As shown in Fig. 11, troponin T was degraded into 21,000, 12,000 and 10,008 Da fragments. On the other hand, troponins I and C were not degraded at all, as shown in Fig. 12. Structural changes in myofibrils induced by cathepsin B treatment The changes in myofibrils treated with cathepsin B at pH 5.5 and 4°C for 9 days were examined under a phase-contrast microscope. As shown in Fig. 13,
1970
MASANORIMATSUIS?~ et al.
more fragmented myofibrils were observed in the presence of cathepsin B than in its absence. This indicated that cathepsin B possesses the ability to cause the fragmentation of myofibrils. Figure 14 shows the changes in the ultrastructure of the glycerinated muscle fibers incubated with cathepsin B at pH 5.5 and 37°C for 4.5 hr. The disturbance of the lateral arrangement of myofibrils occurred in the muscle fibers incubated with cathepsin B, as compared to without cathepsin B. Furthermore, the Z-line was destroyed in places, the H-zone partly disappeared and the arrangement of the filaments at the I-band was disturbed.
DISCUSSION
Rabbit skeletal muscle cathepsin B degraded myosin heavy chain, actin and troponin, but not a-actinin or tropomyosin. The susceptibility of troponin, a-actinin and tropomyosin to skeletal muscle cathepsin B was demonstrated first in this work. Rabbit skeletal muscle cathepsin B mainly produced a 150,000 Da fragment from myosin heavy chain, which is similar to the fact that monkey (Hirao et al., 1984) and rat (Schwartz et al., 1977) skeletal muscle cathepsins B hydrolyze myosin heavy chain mainly into 130,000-150,000 Da fragments.
(Figs I-3--+pposite)
Fig. 1. Eleetrophoretograms of myofibrils treated with cathepsin B at various pHs. Myofibrils (2 mg/ml) were incubated with cathepsin B (1.8 mu/ml) at 37°C and the pH indicated below each lane for 17 hr in 50 mM sodium acetate-HCl buffer (lanes 2, 3 and 4) or 50 mM Tris-acetate buffer (lanes 1, 5, 6 and 7) containing 3 mM dithiothreitol/l mM NaNJO. mM pepstatin. Lane 1 was without added cathepsin B. The incubated myofibrils (13 pg) were applied to a 5% polyacrylamide gel containing 0.1% SDS. Fig. 2. Electrophoretograms of myofibrils treated with cathepsin B. Myofibrils (2 mg/ml) were incubated with cathepsin B (1.8 mu/ml) at 37°C and pH 5.0 for the period indicated below each lane in 50 mM Tris-acetate buffer containing 3 mM dithiothreitol/l mM NaN,/0.25 mM pepstatin. Lanes 6 and 7 were without added cathepsin B. The incubated myofibrils (13 p(g) were applied to a 5% polyacrylamide gel containing 0.1% SDS. Fig. 3. Electrophoretograms of myosin treated with cathepsin B at various pHs. Myosin (400 yg/ml) was incubated with cathepsin B (1.2 mu/ml) at 37°C and the pH indicated below each lane for 17 hr in 50 mM sodium acetate-HCl buffer (lanes 2, 3 and 4) or 50 mM Tris-acetate buffer (lanes 1, 5,6 and 7) containing 3 mM dithiothreitol/l mM NaNJO. mM pepstatin. Lane 1 was without added cathepsin B. The incubated myosin (2.7 pg) was applied to a 5% polyacrylamide gel containing 0.1% SDS. (Figs 4-9-on
pp. 19724973)
Fig. 4. Electrophoretograms of myosin treated with cathepsin B. Myosin (400 pg/ml) was incubated with cathepsin B (1.2 mu/ml) at 37°C and pH 5.5 for the period indicated below each lane in 50mM Tris-acetate buffer containing 3 mM dithiothreitol/l mM NaNJO. mM pepstatin. Lane 6 was without added cathepsin B. The incubated myosin (3pg) was applied to a 5% polyacrylamide gel containing 0.1% SDS. Fig. 5. Electrophoretograms of actin treated with cathepsin B at various pHs. Actin (780pg/ml) was incubated with cathepsin B (1.2 mu/ml) at 37°C and the pH indicated below each lane for 17 hr in 50 mM sodium acetate-HCl buffer (lanes 2 and 3) or 50 mM Tris-acetate buffer (lanes 1,4, 5 and 6) containing 3 mM dithiothreitol/l mM NaNJO. mM pepstatin. Lane 1 was without added cathepsin B. The incubated actin (5.2pg) was applied to a 10% polyacrylamide gel containing 0.1% SDS. Fig. 6. Electrophoretograms of actin treated with cathepsin B. Actin (78Opg/ml) was incubated with cathepsin B (1.2 mu/ml) at 37°C and pH 5.5 for the period indicated below each lane in 50 mM Tris-acetate buffer containing 3 mM dithiothreitol/l mM NaNJ0.3 mM pepstatin. Lane 6 was without added cathepsin B. The incubated actin (2.6pg) was applied to a 10% polyacrylamide gel containing 0.1% SDS. Fig. 7. Electrophoretograms of a-actinin treated with cathepsin B at various pHs. a-Actinin (77 pg/ml) was incubated with cathepsin B (1.2 mu/ml) at 37°C and the pH indicated below each lane for 17 hr in 50 mM sodium acetate-HCl buffer (lanes 2, 3 and 4) or 50 mM Tris-acetate buffer (lanes 1, 5, 6 and 7) containing 3 mM dithiothreitol/l mM NaNJO. mM pepstatin. Lane 1 was without added cathepsin B. The incubated a-actinin (1.5 pg) was applied to a 10% polyacrylamide gel containing 0.1% SDS. Fig. 8. Electrophoretograms of tropomyosin treated with cathepsin B at various pHs. Tropomyosin (136 pg/ml) was incubated with cathepsin B (1.2 mu/ml) at 37°C and the pH indicated below each lane for 17 hr in 50 mM sodium acetate-HCl buffer (lanes 2, 3 and 4) or 50 mM Tris-acetate buffer (lanes 1, 5, 6 and 7) containing 3 mM dithiothteitol/l mM NaNJO. mM pepstatin. Lane 1 was without added cathepsin B. The incubated tropomyosin (5~3 was applied to a 10% polyacrylamide gel containing 0.1% SDS. Fig. 9. Electrophoretograms of troponin treated with cathepsin Bat various pHs. Troponin (1256 &ml) was incubated with cathepsin B (1.2 mu/ml) at 37°C and the pH indicated below each lane for 17 hr in 50mM sodium acetate-HCl buffer (lanes 2, 3 and 4) or 50mM T&a-acetate buffer (lanes 1, 5, 6 and 7) containing 3 mM dithiothreitol/l mM NaNJO. mM pepstatin. Lane 1 was without added cathepsin B. The incubated troponin (8.37 pg) was applied to a 10% polyacrylamide gel containing 0.1% SDS.
Myofibrils
treated
with cathepsin
B
1971
(I1
Mr
Myosin heavy
__
chain wActinin
-
,200,000 r;;g; -
5.1
3.8
5.1 4.2
5.9 5.1
6.7
3456
12
Lane No.
95:ooo
7
(2) Myosin heavy chain a-Actinin
Actin Tropomyosin Troponin
hr
0
2
Lane No.
1
23
4
8 4
24 24 5
6
0 7
(3) Mr Myosin heavy chain
-
200,000 180,000 150,000 87,000 81,000 75,000 69,000
PH
5.1
3.9 4.3
5.1 5.1
Lane No.
1
2
34
Figs I-3-caplions
6.8 6.2
56 opposite.
7
MASANORIMATSUISHIet al.
1972
_
Mr
,200,000 -180,000 -150,000
Myosin heavy chain
hr
0
Lane No.
12
3
6
10 24
24
345
6
(5)
Mr
Actin
44 41 ::
PH
5.1
Lane No.
4.4 5.1 6.8 5.1 6.1 234
1
5
6
(6)
Mr
44,000 41,000 38,000 30,000
Actin
hr
0
3
6
10
24 24
Lane No.
1
2
3
4
5
Figs
46k-captions
6
on p. 1970.
Myofibrils treated with cathepsin B
1973 Mr
(7) cl-Actinin
PH
5.1
3.9
5.1 4.4
Lane No.
1
2
6.1 5.1
34
5
6.8 6
7
Mr
Tropomyosin
5.0
PH
3.8
5.1 4.3
Lane No.
1
6.0 5.1
2345
6.7 67
(9)
Troponin
Mr
:.h
T ,
-36,000 -
Troponin I -. Troponin C .-
30,000 21,000
-17,000 -12,000 -10,000
PH
5.1
3.8
5.1 4.4
Lane No.
1
23
Figs 7-9-captions
6.1 5.1
45 on p. 1970.
6.8 67
1974
MASANORI MATST.NSHI et al.
Mr
Troponin
T __i
Troponin Troponin
I 4 C -
36,000 30,000 21,000 17,000 12,000 10,000
hr
0
3
Lane No.
1
2345
Troponin T
6
10 24
24 6
-
,36,000 21,000
hr
0
2
Lane No.
1
2345
4
8
24
24 6
Mr
Troponin
I
Troponin
C
hr
0
2
Lane No.
7
2345
4
Figs lo-12-captions
8
24
24 6
opposite.
Myofibrils treated with cathepsin B
Fig. 13. Phase contrast micrographs of myofibrils incubated with or without cathepsin B. Myofibrils (1.5 mJm1) were incubated with (b) or without (a) cathepsin B (I.45 mu/ml) at 4°C for 9 days in the presence of 40 mM Tris-acetate buffer (pH .5.5)/0.1 M KCl/3 mM dith~othreitol~l mM EDTA/lO mM NaNJO. 1mM pepstatin. Magnification: 400 x .
(Figs IQ-12 opposite)
Fig. 10. Electrophoretograms of troponin treated with cathepsin B. Troponin (1256 pg/ml) was incubated with cathepsin B (2.84mU/ml) at 37°C and pH 5.5 in 50mM Tris-acetate buffer containing 3 mM dithiothreitol~l mM NaNJO. mM pepstatin. Lane 6 was without added cathepsin B. The incubated troponin (4 pg) was applied to a 10% polyacrylamide gel containing 0.1% SDS. Fig. 11. Electrophoretograms of troponin T treated with cathepsin B. Troponin T. (93pgg/ml) was incubated with cathepsin B (1.8 mu/ml) at 37°C and pH 5.5 in 50 mM Tris-acetate buffer containing 3 mM di~ioth~itol~i mM NaNJO. mM pepstatin. Lane 6 was without added cathepsin B. The incubated troponin T (0.62 fig) was applied to a 10% polyacrylamide gel containing 0.1% SDS. Fig. 12. Electrophoretograms of troponins I and C treated with cathepsin B. Troponins I and C (175 pg/ml) were incubated with cathepsin B (1.8 mu/ml) at 37°C and pH 5.5 in 50 mM Tris-acetate buffer containing 3 mM dithiothreitol/l mM NaNJO. mM pepstatin. Lane 6 was without added cathepsin B. The incubated troponins I and C (1.2 fig) were applied to a 10% polyacrylamide gel containing 0.1% SDS.
1975
1976
MASAN~RIMATSUISNIer al.
Fig;. 14. Electron micrographs of glycerinated muscle fibers incubated with or without cathepsin L B. G1:fcerinated muscle fibers were incubated with (b) or without (a) cathepsin B (5.4mU/ml) at 37°C for 4.5 hr in the presence of 40mM Tris-acetate buffer (pH 5.5)lO.l M KC1/3 mM dithiothreitoljl InM EDTAjlO mM NaN,fO.l mM pep&tin. Ma~~tion: 26,500 x .
Myofibrils treated with cathepsin B Furthermore, the ability to degrade actin is a common property of rabbit and monkey (Hirao et al., 1984) skeletal muscle cathepsins B. However, the degradation product produced by rabbit cathepsin B cannot be compared with that produced by monkey cathepsin B (Hirao et al., 1984), which was not identified. Rabbit cathepsin B hydrolyzed myosin and actin most remarkably at around pH 5, although monkey cathepsin B was reported to degrade these proteins most noticeably at around pH 3. This discrepancy may reflect the species specificity of cathepsin B. Rat liver cathepsin B (Noda et al., 1981) was reported to degrade myosin, actin, tropomyosin and troponin T, although no degradation product of actin was detected on SDS-polyacrylamide gel electrophoresis. On the contrary, rabbit skeletal muscle cathepsin B did not degrade tropomyosin, but degraded actin, a few degradation products being appreciably detected on electrophoresis. These discrepancies strongly suggest the tissue specificity of cathepsin B. Therefore, it is critical to apply the results obtained as to the action of liver cathepsin B towards muscle proteins in order to elucidate the action of skeletal muscle cathepsin B. It was reported that skeletal muscle cathepsin L degraded myosin, a-actinin, actin and troponin, but not tropomyosin (Matsukura et al., 1981). Skeletal muscle cathepsin D was reported to degrade myosin, cr-actinin, tropomyosin and troponin, but not actin (Matsumoto et al., 1983). Comparison of the present results with these reports indicates that cathepsin B is different from cathepsin L in that it does not degrade a-actinin, and from cathepsin D in that it degrades actin but not a-actinin or tropomyosin. Furthermore, it was reported that the optimum pHs for the hydrolysis of myosin, actin and troponin were around 3 for cathepsin D, and around 4 for cathepsin L, while it was around 5 for cathepsin B. These differences in the ability to degrade individual myofibrillar proteins and the optimum pH for hydrolysis seem to be useful as indicators for distinguishing cathepsin B from cathepsins D and L. The troponin preparation used in this study was shown to be contaminated by some proteinase, other than cathepsin B, which degraded troponin into 30,000 and 12,000 Da fragments (Figs 9 and 10). Skeletal muscle cathepsin D was reported to bind to troponin (Matsumoto et al., 1983). However, even if cathepsin D contaminated the troponin preparation, it was unlikely to degrade troponin, because in this experiment troponin was incubated in the presence of pepstatin, which is a specific inhibitor of cathepsin D. Cathepsin L and calpain were supposed to be the proteinases contaminating troponin, because both of them are cysteine proteinases which are not inhibited by pepstatin, and it was reported that cathepsin L (Matsukura et al., 1981) hydrolyzed troponin into 30,000 and 13,000 Da fragments, and calpain (Ishiura et al., 1979) digested troponin into 30,000 and
1917
14,000 Da fragments. In this study, the incubation mixtures did not contain Ca2+, which is an activator of calpain, while they did contain dithiothreitol, which is an activator of cathepsin L. Therefore, it is more possible that cathepsin L bound to troponin and acted on it, than that calpain did. The ability of cathepsin B to cause the fragmentation of myofibrils was demonstrated to be greater than that of cathepsin D (Matsukura et al., 1984), but lower than that of cathepsin L (Matsukura et al., 1984). Moreover, the changes in the ultrastructure of myofibrils caused by cathepsin B were much smaller than those by cathepsin L, which caused remarkable destruction of the Z-line and the M-line. Therefore, cathepsin B is assumed to be less responsible than cathepsin L for the weakening of the myofibrillar structure around the Z-line, which occurs during the postmortem storage of muscles. are grateful to Dr M. Yamaguchi (Department of Veterinary Anatomy and Cell Biology, Ohio State University, U.S.A.) for his help with the electron microscope observations. Acknowledgements-We
REFERENCES
Ebashi S., Wakabayashi T. and Ebashi, F. (1971) Troponin and its components. J. Biochem. 69, 44445. Gornall A. G., Bardawill C. J. and David M. M. (1949) Determination of serum-protein by means of the biuret reaction. J. biol. Chem. 177, 751-766. Hartshorne D. J. and Mueller H. (1969) The preparation of tropomyosin and troponin from natural actomyosin. Biochim.
biophys. Acta 175, 301-319.
Hirao T., Hara K. and Takahashi K. (1984) Purification and characterization of cathepsin B from monkey skeletal muscle. J. Biochem. 95, 871-879. Huxley H. E. (1963) Electron microscope studies on the structure of natural and synthetic protein filaments from striated muscle. J. molec. Biol. 7, 28 I-305. Ishiura S., Sugita H., Suzuki K. and Imahori K. (1979) Studies of a calcium-activated neutral protease from chicken skeletal muscle. II. Substrate specificity. J. Biothem. 86, 579-581.
Lowry 0. H., Rosebrough N. J., Farr A. L. and Randall R. J. (1951) Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 265-275. Masaki T. and Takaiti 0. (1969) Some properties of chicken a-actinin. J. Biochem. 66, 637-643. Matsukura U., Okitani A, Nishimuro T. and Kato H. (1981) Mode of degradation of myofibrillar proteins by an endogeneous protease, cathepsin L. Biochim. biophys. Acta 662, 41-47.
Matsukura U., Matsumoto T., Tashiro Y., Okitani A. and Kato H. (1984) Morphological changes in myofibrils and glycerinated muscle fibers on treatment with cathepsins D and L. Inr. J. Biochem. 16, 957-962. Matsumoto T., Okitani A., Kitamura Y. and Kato H. (1983) Mode of degradation of myofibrillar proteins by rabbit muscle cathepsin D. Biochim. biophys. Acfa 755, 76-80.
Mommaerts W. F. H. M. (1951) Reversible polymerization and ultracentrifugal purification of actin. J. biol. Chem. 188, 559-565.
1978
MASANOIU MATSUISIUet al.
Mueller H. (1966) EGTA-sensitixing activity and molecular properties of tropomyosin prepared in the presence of a sulthydryl protecting agent. Biochem. Z. 345, 300-321. Noda T., Isogai K., Hayashi H. and Katunuma N. (1981) Susceptibilities of various myofibrillar proteins to cathepsin B and morphological alteration of isolated myofibrils by this enzyme. J. Biochem. 90, 371-379. Okitani A., Matsukura U., Kato H. and Fujimaki M. (1980) Purification and some properties of a myofibrillar proteindegrading protease, cathepsin L, from rabbit skeletal muscle. J. Biochem. 87, 1133-1143. Okitani A., Matsuishi M., Matsumoto T., Kamoshida E., Sato M., Matsukura U., Watanabe M., Kato H. and Fujimaki M. (1988) Purification and some properties of cathepsin B from rabbit skeletal muscle. Eur. J. Biochem. 171, 377-381. Perry S. V. (1955) Myosin adenosinetriphosphatase. Meth. Enzym. 2, 582-588.
Richards E. G., Chung C.-S., Menxel D. B. and Olcott H. S. (1967) Chromatography of myosin on diethylaminoethylSephadex A-50. Biochemirtry 6, 528-540. Schwartz W. N. and Bird J. W. C. (1977) Degradation of myofibrillar proteins by cathepsins B and D. Biochem. J. 167, 81 l-820. Suzuki A., Saito M., Sato H. and Nonami Y. (1978) Effects of materials released from myofibrils by CAF (Caz+-activated factor) on Zdisk reconstitution. Agric. Biol. Chem. 42, 2111-2116. Weber K. and Osborn M. (1969) The reliability of molecular weight determinations by dodecyl sulfate-polyacrylamide gel electrophoresis. J. biol. Chem. 244, 4406-4412. Woods E. F. (1969) Comparative physicochemical studies on vertebrate tropomyosins. Biochemistry 8, 4336-4344. Yang R., Okitani A. and Fujimaki M. (1970) Studies on myofibrils from the stored muscle. Part I. Post-mortem changes in adenosine triphosphatase activity of myofibrils from rabbit muscle. Agric. Biol. Chem. 34, 1765-1772.
Int. J. Biochem.Vol. 24, No. 12, Pp. 19674978, 1992 Printed in Great Britain
MODE OF ACTION OF RABBIT SKELETAL MUSCLE CATHEPSIN B TOWARDS MYOFIBRILLAR PROTEINS AND THE MYOFIBRILLAR STRUCTURE MASANORI MATSUISHI,~* TIBUYO MATSUMOT~,~* AKIHIRO OKITANI’ and HIROMICHI KATO~
‘Department of Food Science and Technology, Nippon Veterinary and Animal !kience University, Musashino-shi, Tokyo 180, Japan, *Department of Nutrition, Faculty of Home Economics, Tokyo Kasei University, Itabashi-ku, Tokyo 173, Japan and 3Department of Agricultural Chemistry, University of Tokyo, Bunkyo-ku, Tokyo 113, Japan (Received 22 April
1992)
Abstract-1. The mode of degradation of myofib~~r proteins and the structural changes in myofibrils due to the action of cathepsin B highly purified from rabbit skeletal muscle were studied. 2. Cathepsin B degraded myosin heavy chain, actin and troponin T, but not a-actinin, tropomyosin, troponin I or troponin C among myofibrillar proteins. 3. Cathepsin B optimally degraded myosin heavy chain,, actin and troponin T at around pH 5. Degradation of myosin heavy chain produced 6 fragments, 180,080, 150,080,87,000,81,000, 75,000 and 69,000 Da, respectively. Actin was hydrolyzed into fragments of 41,000, 38,008 and 30,080 Da. Troponin T was degraded into fra8rnents of 21,000, 12,008 and 10,000 Da. 4. Cathepsin B caused the fragmentation of myofibrils and disturbance of the lateral arrangement of myotibrils. 5. Cathepsin B partly disintegrated the Z-line and the M-line, and induced disordering of the arrangement of filaments in the I-band.
INTRODUCITON
Cathepsin B, which is considered to be one of the major proteinases involved in the lysosomal pathway in the proteolytic system of animal cells, has been purified from various tissues and its properties have been investigated. Skeletal muscle cathepsin B was highly purified from monkey (Hirao et al., 1984) and rabbit (Okitani et uf., 1988). In addition, there have been a few studies on the mode of degradation of myofibrillar proteins by cathepsin B in order to clarify its role in the degradation of the proteins in skeletal muscle cells. Hirao et al. (1984) investigated the action of cathepsin B highly purified from monkey skeietal muscle on skeletal muscle myosin and cardiac muscle myofibrils, and reported that myosin and actin were degraded. However, they did not determine whether or not regulatory proteins such as troponin and tro~myos~ were degraded. Schwartz and Bird (1977) reported that rat skeletal muscle cathepsin B degraded myosin. However, it was not certain that such degradation was due to cathepsin B alone, since there was no evidence that their cathepsin B preparation was really free from cathepsin L, which was later reported to be similar to cathepsin B in several properties (Okitani t-2 al., *To whom correspondence and reprint requests should be addressed. *Deceased.
1980). On the other hand, Noda et al. (1981) demonstrated that myosin, actin, tropomyosin and troponin were degraded when various myofibrillar proteins of rat skeletal muscle were treated with cathepsin B higbly purified from rat liver. However, the results obtained for the liver enzyme do not imply that the same results will be- obtained for the skeletal muscle enzyme, because it has not been examined whether this enxyme exhibits tissue specificity or not. As mentioned above, information on the mode of degradation of myofibrillar proteins by skeletal muscle cathepsin B is not sufficient yet. Therefore, we purified cathepsin B from rabbit skeletal muscle, definitely eliminating cathepsin L, according to the method reported previously (Okitani et al., 1988), and investigated its action towards various myofibrillar proteins. Moreover, we revealed structural changes in myofib~ls treated with cathepsin B. MATRRUIS AND METHOBS Materials
Rabbit muscles (longissimus dorsi and psoas) were obtained from carcasses immediately after death. The longissirnus dorsi muscle was minced and used to prepare cathepsin B, and myofibrils and their constituent proteins. The psoas muscle was used to prepare glycerinated muscle fibers. Pepstatin A was purchased from the Peptide Institute, Osaka, Japan. Bovine serum albumin, ovalbumin, chymotrypsinogcn and cytochrome c were purchased from
1967
Boehringer Mannheim GmbH. DEAE-Sephadex A-SO, Sephadex G-75, Sephadex G-100 and CNBr-activated Sepharose 4B were products of Pharmacia Pine Chemicals, Sweden. Phosph~~~o~ was obtained from Seikagaku Kogyo. DEAE-Toyopearl was obtained from Toyosoda Kogyo. Prewration of cathepsinB Cathepsin
B was prepared
according
to the method
Enzyme treatmentof myofibrifsfor observationof morphofogicaf changes Myofibrils (1.5 mgjml) were incubated with cathepsin B (1.45 mu/ml) at 4°C for 9 days in the presence of 40 mM T&-acetate buiTer (pH 5.5)lO.l M KCl/3 mM dithiothreitol/l mM EDTA/O. 1 mM pepstatin/lO mM NaN, . The morphological changes of myofibrils were observed under a phase-contrast microscope.
reported previously (Okitani et al., 1988). The crude enzyme
Enzyme tre&nent of gfycerinatedmusclefibers
extracted from the muscle homo~nate at pH 3.7 for 2hr was fractionated with @H&SO,. The precipitate between 25 and 45% saturation was applied to a Sephadex G-75 column and then to a phosphocellulose column. The active fractions obtained were purified on columns of APSepharose (an affinity adsorbent containing, as ligands, peptides carrying arginine at their C termini), DEAEToyopearl and Sephadex G-100. The purified enzyme was shown to be homogeneous on SD~poly~~la~de gel electrophoresis. The amount, in units, of cathepsin B activity was determined by incubation with 0.5mM N-abenzoyl-DL-arginine-@-naphthylamide at 37°C in 50 mM potassium phosphate buffer (pH 6.2)/0.4mM EDTA/ 0.3 mM dithiothreitol. One unit of activity represents the hydrolysis of 1 pmol substrate/~n,
Glycerinated muscle fibers were washed with 6.7mM potassium phosphate buffer (pH 7.01fO.lM KCl/l mM MgCl, and then cut into small pieces (ca 1cm in length) with a razor. Ten pieces were incubated with cathepsin B (54mU/ml) in 1 ml of 40mM Tris-acetate buffer (pH 55)/O. 1 M KC1/3 mM dithiothreitol/l mM EDTA/O.l mM pcpstatin at 37°C for 4.5 hr.
Preparationof myojbrffs Myofibrils were prepared as described by Yang et al. (1970). Preparationof gfy~erfnotedmuscfefibers Glycerinated muscle fibers were prepared from psoas muscle according to the procedure of Huxley (1963). Preparationof myojibriffarproteins Myosin was prepared as described by Perry (1955) and purified further by DEAE-Sephadex A-50 column chromatography as described by Richards et al. (1967). Actin was prepared according to the method of Mommaerts (1951). a-Actinin was prepared by the method of Masaki and Takaiti (1969). Preparation of troponin and its subunits was carried out according to the method of Ebashi et al. (1971). Tropomyosin was prepared from the residue obtained on lithium chlorideextraction of troponin by the method of Ebasi et 41. (1971), according to the methods of Mueller
(1966), Hartshome and Mueller (1969) and Woods (1969). Enzyme treatmentof myojibrifsandtheirconstituentproteins for efectrophoreticonafysis The substrate proteins (2.Omg/ml of myofibrils or 77-f256&nl of each of the constituent proteins) were incubated with cathepsin B (1.2-2.84 mU/ml) at 37°C in 5OmM sodium acetate-HCl buffer or Tris-acetate buffer/3 mM dithiothreitol/0.2-0.3 mM pepstatin/l mM NaN,, in a total volmne of 0.3 ml. The enzyme concentrations employed for the treatment of myofibrils and the constituent proteins were comparable to the values expected for the muscle homogenates, on the basis of the purification data previously reported (Okitani et al., 1988). Pep&tin was added to block cathepsin D type activity, if any, bound to the substrate proteins. At a given time, 0. IS ml of 60 mM sodium phosphate butl’er (pH 7.2)/5.24% SDS/32.4% 2-mercaptoethanol/20.2% glycerol/O.O29% bromophenol blue was added to the incubated mixtures, followed by boiling for 5 min to stop the enzymatic reaction.
SD~-~fy~ry~~
gel efectrophores~
SDS-polyacrylamide gel electrophoresis was carried out as described by Weber and Osbom (1969) using 5 or 10% gels containing 0.1% SDS. The gels were stained with Coomassie brilliant blue R-250. The molecular masses of myofibrillar proteins and their degradation products were determined by comparing their mobilities with those of myosin heavy chain (2OOkDa), a-actinin (95 kDa), bovine serum albumin (68 kDa), ovalbumin (45 kDa), chymotrypsinogen (25 kDa) and cytochrome c (12.5 kDa). Electronmicroscopy Double fixation in 3.0% glutaraldehyde and 1.0% osmium tetraoxide was followed by dehydration through a graded ethanol and embedding in an Epon mixture. Thin sections were stained with saturated many1 acetate in water and poststained with lead citrate (Suzuki et al., 1978). Specimens were examined under a Hitachi H 300 electron microscope operated at 75 kV. Protein ~te~~~tion Protein concentrations of myofibrils and their constituent proteins were determined by the biuret method (Gomall et al., 1949) or the method of Lowry et al. (1951), using bovine serum albumin as a standard. RESULTS
Myofibrils were incubated with cathepsin 3 at 37°C and pH 3.8-6.7 for 17 hr, and then their changes were investigated by SDS-polyacrylamide gel electrophoresis. As shown in Fig. 1, myosin heavy chain was degraded at pH 3.8-5.1, two bands of 180,000 and 15O,~Da, which seemed to represent the degradation products of myosin heavy chain, being produced. These changes were most remarkable at pH 5.1. The intensity of the band corresponding to actin decreased, and a 30,000 Da band appeared at pH 4.2 and 5.1. Figure 2 shows the time-course of changes in myofibrils treated with cathepsin B at pH 5.0 and 37°C for up to 24 hr. The intensity of the bands of myosin heavy chain and actin decreased with the elapse of the time up to 24 hr. The 180,000 and 150,000 Da bands appeared after 2 hr incubation, and their intensity increased up to 8 ht, but decreased after 24 hr incubation. 87,000, 81,000,75,000,69,000
Myofibrils treated with cathepsin B and 30,000 Da bands appeared after 4 hr incubation, but disappeared after 24 hr incubation. These degradation products were thought to be further degraded into smaller polypeptides before the elapse of 24 hr. Action towards donated myo~bri~~arprotein In order to determine the origins of various degradation products observed on the treatment of myofibrils with cathepsin B and to clarify the susceptibility of troponin T and/or tropomyosin, both of which comigrated on the electrophoresis of myofibrils, the changes in various isolated myofibrillar proteins on treatment with cathepsin B were examined by SDS-polyacrylamide gel electrophoresis. Action towards myosin. Figure 3 shows the changes in myosin on Trident with cathepsin B at 37°C and pH 3.9-6.8 for 17 hr. The degradation of myosin heavy chain and the generation of a 150,000 Da fragment were observed at pH 4.3-6.8. These changes were most notable at pH 5.1-6.2. In this pH range, 87,000, 81,000, 75,000 and 69,000 Da fragments also appeared. Figure 4 shows the time-course of changes in myosin on treatment with cathepsin B at pH 5.5 for up to 24 hr. The reason why pH 5.5 was used for the treatment is that it is close to the ultimate pH of postmortem muscles, i.e. pH 5.6 and the optimum pH for the cathepsin B action towards myosin. Thus, treatment at this pH also provided information on the autolysis of postmortem muscles. In the following experiments, pH 5.5 was used for the same reason. After 3 hr incubation, 180,000 and 150,000 Da fragments emerged, the amounts of which were almost constant until 24 hr. Fragments of 87,000, 81,000, 75,000 and 69,000 Da also appeared after 3 hr incubation. After 24hr incubation, myosin heavy chain decreased considerably, These results demonstrated that myosin heavy chain was easily hydroly~d almost completely into the 150,OOf1Da fragment, which was relatively resistant to further degradation. Action towarak actin. The changes in actin on treatment with cathepsin B at 37°C and pH 4.4-6.8 for 17 hr are shown in Fig. 5. Fragments of 41,000, 38,000 and 30,000 Da appeared, and these changes were most remarkable at pH 5.1. Figure 6 shows the time+ourse of changes in actin on treatment with cathepsin B at pH 5.5 for up to 24 hr. The 38,000 Da fragment emerged after 3 hr incubation and the 41,000 Da fragment after 6 hr incubation. The results in Figs 5 and 6 reveafed that the major degradation product of actin was the 38,000 Da fragment. Moreover, the combination of these results and the following results demonstrated that the 30,000 Da fragment was generated only from actin, suggesting that actin was the origin of the 30,000 Da fragment observed on treatment of myofibrils with cathepsin B. Action towards a-actinin and tropomyosin. As shown in Figs 7 and 8, a-actinin and tropomyosin did not undergo any change on treatment with cathepsin B at 37°C for 17 hr, at pH 3.9-6.8 or pH 3.8-6.7, respectively.
1969
Action towura!s troponin. Figure 9 shows the changes in troponin on treatment with cathepsin B at 37°C and pH 3.8-6.8 for 17 hr: 30,000 and 12,000 Da fragments emerged in the case of incubation at pH 5.1 without cathepsin B (lane 1). This phenomenon was also seen in the case of incubation at pH 5.5 without cathepsin B, as shown in Fig. 10 (lane 6). These changes were considered to be caused by some proteinase, other than cathepsin B, contaminating the troponin (see Discussion). Taking into account the phenomena observed in Fig. 9, the degradation product with cathepsin B alone is conceivably the 10,000 Da fragment which was observed in the case of incubation at pH 5.1-6.1 with cathepsin B (lanes 4, 5 and 6), but not in a control (lane 1). Since this fragment was generated most noticeably at pH 5.1, this pH was judged to be the optimum pH for the action of cathepsin B towards troponin. Figure 10 shows the time-course of the changes in troponin on treatment with cathepsin B at pH 5.5 for up to 24 hr. With the elapse of time, troponin T decreased, and the fragments of 12,000 and 10,000 Da increased. Since the generation of the 12,000 Da fragment after 24 hr incubation was more remarkable in the mixture with cathepsin B (lane 5) than in that without cathepsin B (lane 6), a part of the fragment was suggested to be generated by cathepsin B. It was clear that the 10,000 Da fragment was produced by cathepsin B. The density of the 30,000 Da band observed on all treatments with cathepsin B for more than 3 hr (lanes 2, 3, 4 and 5) was lower than without cathepsin B (lane 6). Moreover, its density after 24 hr incubation was lower than that after 10 hr incubation. Therefore, this fragment was thought to be produced by unknown proteinases, other than cathepsin B, contaminating the troponin and then further degraded by cathepsin B. The density of the band of troponin I on all treatments for more than 3 hr was much higher than at 0 hr, which might be due to the appearance of the degradation product of troponin T at the same place as troponin I. Therefore, whether or not troponin I was degraded cannot be determined from these results. No change in the band of troponin C was observed. Troponin T, and a mixture of troponins I and C, which were isolated from the troponin complex, were treated with cathepsin B at pH 5.5 in order to clarify the su~ptibility of troponin I, and the origin of the fragments observed in Figs 9 and 10. As shown in Fig. 11, troponin T was degraded into 21,000, 12,000 and 10,008 Da fragments. On the other hand, troponins I and C were not degraded at all, as shown in Fig. 12. Structural changes in myofibrils induced by cathepsin B treatment The changes in myofibrils treated with cathepsin B at pH 5.5 and 4°C for 9 days were examined under a phase-contrast microscope. As shown in Fig. 13,
1970
MASANORIMATSUIS?~ et al.
more fragmented myofibrils were observed in the presence of cathepsin B than in its absence. This indicated that cathepsin B possesses the ability to cause the fragmentation of myofibrils. Figure 14 shows the changes in the ultrastructure of the glycerinated muscle fibers incubated with cathepsin B at pH 5.5 and 37°C for 4.5 hr. The disturbance of the lateral arrangement of myofibrils occurred in the muscle fibers incubated with cathepsin B, as compared to without cathepsin B. Furthermore, the Z-line was destroyed in places, the H-zone partly disappeared and the arrangement of the filaments at the I-band was disturbed.
DISCUSSION
Rabbit skeletal muscle cathepsin B degraded myosin heavy chain, actin and troponin, but not a-actinin or tropomyosin. The susceptibility of troponin, a-actinin and tropomyosin to skeletal muscle cathepsin B was demonstrated first in this work. Rabbit skeletal muscle cathepsin B mainly produced a 150,000 Da fragment from myosin heavy chain, which is similar to the fact that monkey (Hirao et al., 1984) and rat (Schwartz et al., 1977) skeletal muscle cathepsins B hydrolyze myosin heavy chain mainly into 130,000-150,000 Da fragments.
(Figs I-3--+pposite)
Fig. 1. Eleetrophoretograms of myofibrils treated with cathepsin B at various pHs. Myofibrils (2 mg/ml) were incubated with cathepsin B (1.8 mu/ml) at 37°C and the pH indicated below each lane for 17 hr in 50 mM sodium acetate-HCl buffer (lanes 2, 3 and 4) or 50 mM Tris-acetate buffer (lanes 1, 5, 6 and 7) containing 3 mM dithiothreitol/l mM NaNJO. mM pepstatin. Lane 1 was without added cathepsin B. The incubated myofibrils (13 pg) were applied to a 5% polyacrylamide gel containing 0.1% SDS. Fig. 2. Electrophoretograms of myofibrils treated with cathepsin B. Myofibrils (2 mg/ml) were incubated with cathepsin B (1.8 mu/ml) at 37°C and pH 5.0 for the period indicated below each lane in 50 mM Tris-acetate buffer containing 3 mM dithiothreitol/l mM NaN,/0.25 mM pepstatin. Lanes 6 and 7 were without added cathepsin B. The incubated myofibrils (13 p(g) were applied to a 5% polyacrylamide gel containing 0.1% SDS. Fig. 3. Electrophoretograms of myosin treated with cathepsin B at various pHs. Myosin (400 yg/ml) was incubated with cathepsin B (1.2 mu/ml) at 37°C and the pH indicated below each lane for 17 hr in 50 mM sodium acetate-HCl buffer (lanes 2, 3 and 4) or 50 mM Tris-acetate buffer (lanes 1, 5,6 and 7) containing 3 mM dithiothreitol/l mM NaNJO. mM pepstatin. Lane 1 was without added cathepsin B. The incubated myosin (2.7 pg) was applied to a 5% polyacrylamide gel containing 0.1% SDS. (Figs 4-9-on
pp. 19724973)
Fig. 4. Electrophoretograms of myosin treated with cathepsin B. Myosin (400 pg/ml) was incubated with cathepsin B (1.2 mu/ml) at 37°C and pH 5.5 for the period indicated below each lane in 50mM Tris-acetate buffer containing 3 mM dithiothreitol/l mM NaNJO. mM pepstatin. Lane 6 was without added cathepsin B. The incubated myosin (3pg) was applied to a 5% polyacrylamide gel containing 0.1% SDS. Fig. 5. Electrophoretograms of actin treated with cathepsin B at various pHs. Actin (780pg/ml) was incubated with cathepsin B (1.2 mu/ml) at 37°C and the pH indicated below each lane for 17 hr in 50 mM sodium acetate-HCl buffer (lanes 2 and 3) or 50 mM Tris-acetate buffer (lanes 1,4, 5 and 6) containing 3 mM dithiothreitol/l mM NaNJO. mM pepstatin. Lane 1 was without added cathepsin B. The incubated actin (5.2pg) was applied to a 10% polyacrylamide gel containing 0.1% SDS. Fig. 6. Electrophoretograms of actin treated with cathepsin B. Actin (78Opg/ml) was incubated with cathepsin B (1.2 mu/ml) at 37°C and pH 5.5 for the period indicated below each lane in 50 mM Tris-acetate buffer containing 3 mM dithiothreitol/l mM NaNJ0.3 mM pepstatin. Lane 6 was without added cathepsin B. The incubated actin (2.6pg) was applied to a 10% polyacrylamide gel containing 0.1% SDS. Fig. 7. Electrophoretograms of a-actinin treated with cathepsin B at various pHs. a-Actinin (77 pg/ml) was incubated with cathepsin B (1.2 mu/ml) at 37°C and the pH indicated below each lane for 17 hr in 50 mM sodium acetate-HCl buffer (lanes 2, 3 and 4) or 50 mM Tris-acetate buffer (lanes 1, 5, 6 and 7) containing 3 mM dithiothreitol/l mM NaNJO. mM pepstatin. Lane 1 was without added cathepsin B. The incubated a-actinin (1.5 pg) was applied to a 10% polyacrylamide gel containing 0.1% SDS. Fig. 8. Electrophoretograms of tropomyosin treated with cathepsin B at various pHs. Tropomyosin (136 pg/ml) was incubated with cathepsin B (1.2 mu/ml) at 37°C and the pH indicated below each lane for 17 hr in 50 mM sodium acetate-HCl buffer (lanes 2, 3 and 4) or 50 mM Tris-acetate buffer (lanes 1, 5, 6 and 7) containing 3 mM dithiothteitol/l mM NaNJO. mM pepstatin. Lane 1 was without added cathepsin B. The incubated tropomyosin (5~3 was applied to a 10% polyacrylamide gel containing 0.1% SDS. Fig. 9. Electrophoretograms of troponin treated with cathepsin Bat various pHs. Troponin (1256 &ml) was incubated with cathepsin B (1.2 mu/ml) at 37°C and the pH indicated below each lane for 17 hr in 50mM sodium acetate-HCl buffer (lanes 2, 3 and 4) or 50mM T&a-acetate buffer (lanes 1, 5, 6 and 7) containing 3 mM dithiothreitol/l mM NaNJO. mM pepstatin. Lane 1 was without added cathepsin B. The incubated troponin (8.37 pg) was applied to a 10% polyacrylamide gel containing 0.1% SDS.
Myofibrils
treated
with cathepsin
B
1971
(I1
Mr
Myosin heavy
__
chain wActinin
-
,200,000 r;;g; -
5.1
3.8
5.1 4.2
5.9 5.1
6.7
3456
12
Lane No.
95:ooo
7
(2) Myosin heavy chain a-Actinin
Actin Tropomyosin Troponin
hr
0
2
Lane No.
1
23
4
8 4
24 24 5
6
0 7
(3) Mr Myosin heavy chain
-
200,000 180,000 150,000 87,000 81,000 75,000 69,000
PH
5.1
3.9 4.3
5.1 5.1
Lane No.
1
2
34
Figs I-3-caplions
6.8 6.2
56 opposite.
7
MASANORIMATSUISHIet al.
1972
_
Mr
,200,000 -180,000 -150,000
Myosin heavy chain
hr
0
Lane No.
12
3
6
10 24
24
345
6
(5)
Mr
Actin
44 41 ::
PH
5.1
Lane No.
4.4 5.1 6.8 5.1 6.1 234
1
5
6
(6)
Mr
44,000 41,000 38,000 30,000
Actin
hr
0
3
6
10
24 24
Lane No.
1
2
3
4
5
Figs
46k-captions
6
on p. 1970.
Myofibrils treated with cathepsin B
1973 Mr
(7) cl-Actinin
PH
5.1
3.9
5.1 4.4
Lane No.
1
2
6.1 5.1
34
5
6.8 6
7
Mr
Tropomyosin
5.0
PH
3.8
5.1 4.3
Lane No.
1
6.0 5.1
2345
6.7 67
(9)
Troponin
Mr
:.h
T ,
-36,000 -
Troponin I -. Troponin C .-
30,000 21,000
-17,000 -12,000 -10,000
PH
5.1
3.8
5.1 4.4
Lane No.
1
23
Figs 7-9-captions
6.1 5.1
45 on p. 1970.
6.8 67
1974
MASANORI MATST.NSHI et al.
Mr
Troponin
T __i
Troponin Troponin
I 4 C -
36,000 30,000 21,000 17,000 12,000 10,000
hr
0
3
Lane No.
1
2345
Troponin T
6
10 24
24 6
-
,36,000 21,000
hr
0
2
Lane No.
1
2345
4
8
24
24 6
Mr
Troponin
I
Troponin
C
hr
0
2
Lane No.
7
2345
4
Figs lo-12-captions
8
24
24 6
opposite.
Myofibrils treated with cathepsin B
Fig. 13. Phase contrast micrographs of myofibrils incubated with or without cathepsin B. Myofibrils (1.5 mJm1) were incubated with (b) or without (a) cathepsin B (I.45 mu/ml) at 4°C for 9 days in the presence of 40 mM Tris-acetate buffer (pH .5.5)/0.1 M KCl/3 mM dith~othreitol~l mM EDTA/lO mM NaNJO. 1mM pepstatin. Magnification: 400 x .
(Figs IQ-12 opposite)
Fig. 10. Electrophoretograms of troponin treated with cathepsin B. Troponin (1256 pg/ml) was incubated with cathepsin B (2.84mU/ml) at 37°C and pH 5.5 in 50mM Tris-acetate buffer containing 3 mM dithiothreitol~l mM NaNJO. mM pepstatin. Lane 6 was without added cathepsin B. The incubated troponin (4 pg) was applied to a 10% polyacrylamide gel containing 0.1% SDS. Fig. 11. Electrophoretograms of troponin T treated with cathepsin B. Troponin T. (93pgg/ml) was incubated with cathepsin B (1.8 mu/ml) at 37°C and pH 5.5 in 50 mM Tris-acetate buffer containing 3 mM di~ioth~itol~i mM NaNJO. mM pepstatin. Lane 6 was without added cathepsin B. The incubated troponin T (0.62 fig) was applied to a 10% polyacrylamide gel containing 0.1% SDS. Fig. 12. Electrophoretograms of troponins I and C treated with cathepsin B. Troponins I and C (175 pg/ml) were incubated with cathepsin B (1.8 mu/ml) at 37°C and pH 5.5 in 50 mM Tris-acetate buffer containing 3 mM dithiothreitol/l mM NaNJO. mM pepstatin. Lane 6 was without added cathepsin B. The incubated troponins I and C (1.2 fig) were applied to a 10% polyacrylamide gel containing 0.1% SDS.
1975
1976
MASAN~RIMATSUISNIer al.
Fig;. 14. Electron micrographs of glycerinated muscle fibers incubated with or without cathepsin L B. G1:fcerinated muscle fibers were incubated with (b) or without (a) cathepsin B (5.4mU/ml) at 37°C for 4.5 hr in the presence of 40mM Tris-acetate buffer (pH 5.5)lO.l M KC1/3 mM dithiothreitoljl InM EDTAjlO mM NaN,fO.l mM pep&tin. Ma~~tion: 26,500 x .
Myofibrils treated with cathepsin B Furthermore, the ability to degrade actin is a common property of rabbit and monkey (Hirao et al., 1984) skeletal muscle cathepsins B. However, the degradation product produced by rabbit cathepsin B cannot be compared with that produced by monkey cathepsin B (Hirao et al., 1984), which was not identified. Rabbit cathepsin B hydrolyzed myosin and actin most remarkably at around pH 5, although monkey cathepsin B was reported to degrade these proteins most noticeably at around pH 3. This discrepancy may reflect the species specificity of cathepsin B. Rat liver cathepsin B (Noda et al., 1981) was reported to degrade myosin, actin, tropomyosin and troponin T, although no degradation product of actin was detected on SDS-polyacrylamide gel electrophoresis. On the contrary, rabbit skeletal muscle cathepsin B did not degrade tropomyosin, but degraded actin, a few degradation products being appreciably detected on electrophoresis. These discrepancies strongly suggest the tissue specificity of cathepsin B. Therefore, it is critical to apply the results obtained as to the action of liver cathepsin B towards muscle proteins in order to elucidate the action of skeletal muscle cathepsin B. It was reported that skeletal muscle cathepsin L degraded myosin, a-actinin, actin and troponin, but not tropomyosin (Matsukura et al., 1981). Skeletal muscle cathepsin D was reported to degrade myosin, cr-actinin, tropomyosin and troponin, but not actin (Matsumoto et al., 1983). Comparison of the present results with these reports indicates that cathepsin B is different from cathepsin L in that it does not degrade a-actinin, and from cathepsin D in that it degrades actin but not a-actinin or tropomyosin. Furthermore, it was reported that the optimum pHs for the hydrolysis of myosin, actin and troponin were around 3 for cathepsin D, and around 4 for cathepsin L, while it was around 5 for cathepsin B. These differences in the ability to degrade individual myofibrillar proteins and the optimum pH for hydrolysis seem to be useful as indicators for distinguishing cathepsin B from cathepsins D and L. The troponin preparation used in this study was shown to be contaminated by some proteinase, other than cathepsin B, which degraded troponin into 30,000 and 12,000 Da fragments (Figs 9 and 10). Skeletal muscle cathepsin D was reported to bind to troponin (Matsumoto et al., 1983). However, even if cathepsin D contaminated the troponin preparation, it was unlikely to degrade troponin, because in this experiment troponin was incubated in the presence of pepstatin, which is a specific inhibitor of cathepsin D. Cathepsin L and calpain were supposed to be the proteinases contaminating troponin, because both of them are cysteine proteinases which are not inhibited by pepstatin, and it was reported that cathepsin L (Matsukura et al., 1981) hydrolyzed troponin into 30,000 and 13,000 Da fragments, and calpain (Ishiura et al., 1979) digested troponin into 30,000 and
1917
14,000 Da fragments. In this study, the incubation mixtures did not contain Ca2+, which is an activator of calpain, while they did contain dithiothreitol, which is an activator of cathepsin L. Therefore, it is more possible that cathepsin L bound to troponin and acted on it, than that calpain did. The ability of cathepsin B to cause the fragmentation of myofibrils was demonstrated to be greater than that of cathepsin D (Matsukura et al., 1984), but lower than that of cathepsin L (Matsukura et al., 1984). Moreover, the changes in the ultrastructure of myofibrils caused by cathepsin B were much smaller than those by cathepsin L, which caused remarkable destruction of the Z-line and the M-line. Therefore, cathepsin B is assumed to be less responsible than cathepsin L for the weakening of the myofibrillar structure around the Z-line, which occurs during the postmortem storage of muscles. are grateful to Dr M. Yamaguchi (Department of Veterinary Anatomy and Cell Biology, Ohio State University, U.S.A.) for his help with the electron microscope observations. Acknowledgements-We
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