Journal of Muscle Research and Cell Motility 11, 41-47 (1990)

Deterioration induced by physiological concentration of calcium ions in skinned muscle fibres N. K A S U G A and Y. U M A Z U M E *

Department of Physiology, Jikei University School of Medicine. Minato-ku, Tokyo, 105, Japan Received 2 May 1989 and in revised form 2 August 1989

Summary The deteriorating effect of ~M order of Ca 2+ on skinned frog skeletal muscle fibres was studied from the view point of the digestion of proteins by calcium-activated neutral protease (CANP). Tension developed in solutions containing no MgATP (rigor solution) decreased irreversibly with the addition of Ca 2+ in quantities of more than 0.1 ~tM.Low temperature was seen to suppress (Q10 > 4), and neutral pli to maximize, this decrease in tension. In rigor solution containing Ca 2+, SDS electrophoresis indicated that a 95 k dalton component (oE-actinin) was released from the fibre; electron micrography showed the disappearance of Z-lines. These results suggest that one of the causes for decrease in rigor tension is the proteolytic activity of CANP, and its inhibitors were shown to be quite useful in experiments on skinned fibre.

Introduction Skinned fibres, that is, muscle fibres from which the cell m e m b r a n e s have b e e n r e m o v e d (Natori, 1954), have corne to be widely used in studies on the mechanisms of contraction as well as excitationcontraction coupling in skeletal, cardiac and smooth muscles. W h e n the Ca 2+ concentration of an incubating solution is increased 1-10 ~M, the skinned fibre generates similar force to intact fibres (Ebashi & Endo, 1968); h o w e v e r , the generated force gradually decreases with structural destruction, even in the presence of physiological concentrations of Ca 2+. This process is c o m m o n l y called 'deterioration', and it has troubled m a n y investigators. Although the causes of deterioration have not yet been clarified, some possible factors include: (1) since the skinned fibres have lost some intracellular as well as extracellular structures, which w o u l d normally support the regular a r r a n g e m e n t of sarcomeres against a contracting force, irreversible disorder easily occurs in structures located inside sarcomeres as well as the striation pattern d u r i n g contraction; (2) in artificial solutions, some proteases m a y digest such structural and cyto-skeletal proteins in skinned fibres as connectin (titin), which was s h o w n by Wang (1985) to be d e g r a d e d by Ca 2+. Increases in intracellular Ca 2+ concentrations in intact vertebrate skeletal muscles p r o m o t e d a m a g e of myofibrils (Publicover et al., 1978) and cause the removal of Z-lines (Busch et al., 1972). Furthermore, *To whom all correspondence should be sent. 0142-4319/90 $03.00 + .12 O 1990 Chapman and Hall Ltd.

several authors have r e p o r t e d finding higher concentrations of Ca 2+ in resting dystrophic muscle fibres with a selective loss of the Z-lines (Pearce, 1966; Cullen & Fulthorpe, 1975; Bodensteiner & Engel, 1978). These p h e n o m e n a have b e e n considered to be attributable to calcium-activated neutral protease, or CANP for short (McGowan et al., 1976; R e d d y et al., 1975) In the present investigation, we u n d e r t o o k to determine the causes of Ca2+-induced deterioration in the tension of skinned fibres, from the view point of the destruction of such structures as Z-lines and elastic elements.

Materials and methods

Experimental procedures Fibres from semitendinosus muscles of the frog (Rana catesbeinana) were mechanically skinned (Natori, 1954) in a standard relaxing solution. A segment of skinned fibre, about 5mm long, was mounted horizontally in a trough (3 x 3 x 20 mm 3) positioned between a tension transducer (Shinkoh, Tokyo, UL2-240) and micromanipulator (Narishige, Tokyo, MM-33). Solutions were changed by pouring the new one at one end of the trough whilst sucking out the old one at the other. The sarcomere length of fibres in the trough was measured using the laser optical diffraction method. The various solutions in which fibres had been incubated were examined by SDS gel electrophoresis. After a bundle

42

KASUGA a n d UMAZUME

of about 401 cm long mechanically skinned fibres had been incubated in 40 ~tl of a given solution for I h, 5 ~1 of 10% SDS and 10% 2-mercaptoethanol were added to that solution. The prepared solution was then incubated for 2min at 100~ and examined by SDS slab gel electrophoresis, after which 10% and 5% acrylamide gels were used as separation and stacking gels, respectively. The gels were stained with Commassie Blue R-250 and destained with 7% acetic acid. Molecular weights were determined with the use of a calibration curve obtained by plotting the electrophoretic mobilities of various highly purified proteins against the logarithm of their known polypeptide chain molecular weights (Weber & Osborn, 1969). Standards were run simultaneously with each set of unknowns.

Solutions Free MgATP concentrations in relaxing and rigor solutions were prepared to be 4.0 and 0 mM, respectively. The total concentration of EGTA was 4.0 mM, and KC1 concentration was adjusted to yield an ionic strength of 0.15M in all solutions. In various p l i solutions, the following buffers of 10mM were used: 2-(N-morpholino)ethanesulphonic acid (MES), for p l i less than 6.0, piperadine-N, N'-bis(2-ethanesulphonic acid) (PIPES), for pli of 6.5 and 7.0; and N-2-hydroxyethyl piperadine-N'-3-propanesulphonic acid (EPPS) for p l i above 7.5. The pli values were adjusted with KOH. The C a 2+ concentration was varied by the addition of an appropriate amount of CaC12. The total amount of salts required to make up any given solution was obtained by multiequilibrium equation. Adopted association constants of pli buffers were: HPIPES/H.PIPES = 6.31, 7.67 and 8.47 X 10 6 M -1 at 20, 10 and 5~C, respectively; HMES-H-MES = 1.41 x 10 6 M - 1 at 20~ HEPPS/H.EPPS = 1.0 • 10s M-1 at 20~C. The apparent association c o n s t a n t Kap p o f C a 2+ and EGTA was that determined by Harafuji and Ogawa (1980): l o g Kap p -- 6.46 + 2(pli - 6.8) - [~,/I/(1 + ~/I) - 0.4.I] + 0.009 (T - 20), where I is ionic strength in M and T is temperature in ~ Other association constants used were those mentioned elsewhere (Umazume et al., 1986). Mixed concentrations of salts for the main solutions were as follows (mM): standard relaxing solution - - 90KC1, 5.2MGC12, 4.3 Na2ATP, 4 EGTA, 10 PIPES, pli 7.0 at 20~ standard rigor s o l u t i o n - 113 KC1, 1.2MGC12, 4 EGTA, 10 PIPES, pli 7.0 at 20~

Results

Deterioration induced by calcium ions To s t u d y the deteriorating effect of Ca 2+ on the tension of s k i n n e d fibres, we first o b s e r v e d the changes occurring in the tension of rigor skinned fibres, since t h e y p r e s e r v e o r d e r e d structures in a well defined a n d established state, t h e r e b y enabling us to c o m p a r e their structure c h a n g e s with those in tension. Figure 1 d e m o n s t r a t e s the e x p e r i m e n t a l p r o c e d u r e s for quantitative analysis of the Cai n d u c e d decline in tension. After the fibre, at sarcomere length of 2 . 5 ~ m , w a s s o a k e d in rigor solution at A, it w a s stretch b y 6 to 7% of fibre length at B to e m p h a s i z e the deteriorating effect of Ca 2+. The tension c o n t i n u e d to rise d u r i n g stretch a n d started to decay at the e n d of stretch to reach a n e w steady level higher t h a n the initial isometric force. (Although the cause of slow decline in tension following stretch h a s not b e e n clarified, Suzuki a n d Sugi (1983) r e p o r t e d that the extension of sarcomere structures m i g h t be elastic a n d reversible with stretch of less t h a n 5 to 8%.) W h e n the concentration of calcium ions w a s raised at C, a gradual decay in tension could be observed; the rate of d e c a y increased along w i t h the increase in the concentration of Ca 2+. The time course of the d e c a y w a s not affected b y destruction of internal m e m b r a n e s w i t h the addition of 0.5% Brij-58, s u g g e s t i n g that t h e y are not involved in this process. I h after a p p l y i n g Ca 2+, we obtained the values of P a n d Ap, P b e i n g the extrapolated tension of that p r e c e d i n g the addition of Ca 2+ ( d a s h e d line in Fig. 1), a n d AP b e i n g the difference b e t w e e n P a n d the actual tension. The ratio AP: P w a s a d o p t e d as an indicator of tension decline. Figure 2 s h o w s the relation b e t w e e n A P : P a n d Ca 2+ concentration. A significant decline w a s detected e v e n in low concentrations of Ca 2+, such as 0.1 ~M.

Temperature and pli dependence In order to s t u d y the n a t u r e of tension decline in rigor

I

t

A

B

C

Fig. 1. A representative trace of tension. The fibre was incubated in standard rigor solution at A, stretched at B and incubated in rigor solution containing 1 ~M C a 2+ at C. Vertical and horizontal bars indicate 0.3 mN and 10 min, respectively. Temperature was 20 ~

Calcium induced deterioration of skinned fibres

AP:P in 16[XM Ca 2+ at 20~ The optimum p l i for tension decline was around p l i 7.0. From these results, we came to suspect that the decline in tension was related to some enzymatic activity.

1-0

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0.E

(t4

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i

I 0 "~

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,

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[ Ca 2+] (M) Fig. 2. Relation between Ap:P and Ca 2+ concentration ai sarcomere length of 2.5 ~m. Point and bar indicate mean and S.D. respectively. skinned fibres, the dependence of AP:P on temperature and p l i was studied at a high concentration of Ca 2+. The AP:P values in the presence of 16 ~tM Ca 2+ were 0.03 + 0.01 (mean + s.d. n = 3), 0.20 + 0.05 (n = 3) and 0.91 + 0.02 (n = 6) ai 5, 10 and 20~ respectively. Figure 3 shows the pli dependence of

1-0

SDS gel electrophoresis It is well known that the digesting effect of CANP accompanies a release of OE-actinin from myofibrils (Reddy et al., 1975). We therefore checked to see what proteins are released from the skinned fibre in high concentrations of Ca 2+, in an effort to confirm the involvement of CANP in the deterioration of rigor skinned fibres. Solutions in which the skinned fibres were incubated for l h were analyzed by SDSpolyacrylamide gel electrophoresis (Fig. 5). No band was observed for the standard relaxing and rigor solutions (Fig. 5a, b), but a band corresponding to 95k dalton was observed for the rigor solution containing 16 ~M Ca 2+ (Fig. 5c).

0.8

0.6

0.4

0.2

0

Electron microscopy Electron micrographs of the skinned fibres incubated in rigor solution containing 16[~M Ca 2+, were obtained. I h after raising the Ca 2+ concentration of rigor solution, 1% glutaraldehyde was introduced into the solution and left standing for I h more. Little or no change was observed in tension or optical diffraction patterns during fixation, suggesting that the artifact was not serious. Then the central portion of the fibre segment was embedded in an Epon mixture and sectioned. Electron microscope (JEOL, JEM100S) was used at 60kV at 2000 to 20000x. In many sarcomeres, Z-lines had disappeared in high concentrations of Ca 2+ (Fig. 4b). Hattori and Takahashi (1982) reported that there are two types of Z-line deterioration which are clearly distinguished by their p l i dependency and can be observed with electron microscopy, Z-line weakening and Z-line removal. According to them, the former is a Ca2+-induced change in the nature of the Z-line itself, which is minimized ai p l i 6.5, whereas the latter is caused by proteolytic activity of CANP, which is maximized at p l i 7. The type of Z-line change and the p l i dependency found in the present study coincided with the latter, suggesting that the rigor tension decay obtained was related to the activities of CANP.

I

!

I

5.5

60

65

I

I

7.0 75 pH

I

8.0 8.5

Fig. 3. Relation between AP:P and pli obtained from fibres in rigor solution containing 16 ~M Ca 2+. Point and bar indicate mean and S.D. respectively.

Deterioration in stretched fibres The above results suggest that deterioration of Z-lines, caused by the proteolytic activity of CANP, is one of the causes of the decrease seen in the tension of rigor fibres associated with Ca 2+ application. If this is the case, the resting tension, which is ascribed to the force of elastic filaments connecting thick filaments to Z-lines (Higuchi & Umazume, 1985), should decline even in fibres stretched so as to

44

KASUGA and UMAZUME

Fig. 4. Electron micrographs of skinned fibers in longitudina9 section. (A): fibre incubated in standard rigor solution for I h; (B): fibre incubated in rigor solution containing 16 ~M C a 2+ for lh. Bar = 2 ~m.

abolish the overlap b e t w e e n thick and thin filaments. The effect of Ca 2+ was, therefore, examined in highly stretched fibres. After the fibre was stretched u p to sarcomere length of 5~m, in standard relaxing solution, it was incubated in the standard rigor solution. The tension started to decay at the end of stretch to reach a steady level after 20--30 min. W h e n the a m o u n t of Ca 2+ was increased, a decline in tension was again observed above Ca 2+ concentrations of 0.1 ~tM. 1 h after applying Ca 2+, we obtained the value of p and Ap,p being the extrapolated tension of that preceding the addition of Ca 2+ and Ap being the difference b e t w e e n p and actual tension. The relation b e t w e e n Ap,p and Ca 2+ concentration was seen to be very similar to that of non-stretched rigor fibres (Fig. 6).

Effect of inhibitors If deterioration is attributable to the activities of CANP, an inhibitor of C A N P w o u l d be a s s u m e d to p r e v e n t it. The effects of inhibitors were, therefore, examined in rigor fibres at slack length. The AP:P was seen to be r e d u c e d with increasing concentrations of inhibitors, and almost completely s u p p r e s s e d with 5 mM monoiodoacetic acid and 0.5 mM leupeptin (Fig. 7). Antipain and E-64 of 0.5 mM were also f o u n d to be effective.

Effects of leupeptin on con tractingfibres We studied the effect of leupeptin on contracting skinned fibres in a solution containing MgATP and Ca 2+. The 0.5 mM l e u p e p t i n did hot affect the Ca 2+ sensitivity to force generation; the Ca 2+ concentration of the mechanical threshold was 1 ~M and that of saturation was 10 ~M. Next, we observed the decrease of tension generation in successive contractions. A given skinned fibre in this experiment was exposed to 16~M Ca 2+ for l m i n , at 3 m i n intervals, at 20~ Tension was seen to decrease gradually to 30% of the initial level in eight contractions. O n the other hand, in a fibre incubated in 0.5 mM leupeptin, the tension decreased b y only 15%, even in eight contraction (Fig. 8). W h e n the fibre was continuously contracted for a long period of time in 0.5mM leupeptin, it maintained almost steady tension for more than 20 min, but tore apart within 3 min in the solution without leupeptin. Discussion In the p r e s e n t study, it was clearly d e m o n s t r a t e d that the tension in rigor fibres decreased even in the p h y s i o l o g i c a l range of Ca 2+ concentration. The d e c r e m e n t d e p e n d e d on the t e m p e r a t u r e with Q10 > 4, and was minimized at a p l i of 7.0. These results

Calcium i n d u c e d deterioration of skinned fibres

45

1.0

0.6 6~

0.4

0 0

.

2 1 2 3 4 (mM or 0.1 mM)

5

Fig. 7. Effect of CANP inhibitors on AP: P, measured in rigor solution containing 16 ~M C a 2+ and various amounts of monoiodoacetic acid (open circle, in mM units) and leupeptin (closed circle, in 0.1mM units). Each symbol indicates mean value for 3 fibres.

Fig. 5. SDS-polyacrylamide gel electrophoretic patterns. A, B and C: solutions in which fibres had been incubated for lh. (A), standard relaxing solutions; (B), standard rigor solution; (C), rigor solution containing 16~tM Ca 2+. (D) fibers incubated in the solution described in (C). Numbers indicate molecular weights in dalton.

1.0

suggest that Ca2§ decline in tension is related to enzymatic activity activitated by Ca 2+. It has been r e p o r t e d that C A N P breaks d o w n intact myofibrils during the normal metabolic t u r n o v e r of myofibrila r proteins a n d causes the myofilament to deteriorate at neutral p l i (Reddy et al., 1975). The sarne authors also r e p o r t e d that C A N P causes a selective loss of the Z-lines associated with the removal of er In the p r e s e n t study, SDS-gel electrophoresis s h o w e d a release of a 95k dalton

0.8

1.0 0.6

'ar 0.8

.
o6

0.4

~

OE0.4

0.2

0.2

0

0 10-~o

]0 .7

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Cca87 ( M ) Fig. 6. Relation between AP:P and Ca 2+ concentration at sarcomere length of 5 ~m. Point and bar indicate mean and S.D., respectively.

Number of order Fig. 8. Changes in tension on successive contractions at 20~ without (closed circle) and with (open circle) leupeptin. Each syumbol indicates mean value for 3 fibres.

46 protein, which may fairly be presumed to be er from skinned fibres incubated in 16~tM Ca 2+. Moreover, electron microscopic observation of the disappearence of Z-lines upon the addition of Ca 2+ confirmed the involvement of CANP in the Ca2+-induced decline in tension in rigor skinned fibres. The concentration of Ca 2+ at which tension was reported herein to begin to decrease is much lower than those reported previously. Several reports bave asserted that the concentration of Ca 2+ necessary to cause myofibrils to deteriorate ranges from 10 ~tM to 1 mM (Busch et al., 1972; Ishiura et al., 1978; Hattori & Takahashi, 1982). The present study showed that the threshold concentration of Ca 2+ for decreasing tension was 0.1~M. The possible reasons for such sensitivity of fibres to Ca 2+ include the following: (1) the measured tension can detect the mechanical changes in any sarcomeres, because they arranges in a series along the long axis of the fibre, that is, even if only a few sarcomeres were destroyed with all others left intact, the tension detected from both ends of the fibre should decrease; (2) Z-lines in an isometricallyheld fibre are much more susceptible to deterioration than those in freely suspended myofibrils, because the mechanical stress imposed by thin filaments tends to pull Z-lines apart; (3) some of the isozymes of CANP, such as calpain I, activated in a low concentration of Ca 2+ exist in skinned fibres but are lost in the myofibrils. Actually, Mellgren (1980) and Kawashima et al. (1984) reported that CANP was activated by a ~M order of Ca 2+ in the dog heart muscle and in the rabbit and chicken skeletal muscles. Recently, elastic filaments have been reported to exist in sarcomeres and to connect Z-lines, or Z-line and thick filaments, bearing most of the resting tension in mechanically skinned fibres (Higuchi & Umazume, 1985). In highly-stretched muscle fibres, in which thick and thin filaments had no overlap, a

KASUGA and UMAZUME decline in resting tension was observed in the present study. This decline could be ascribed to a deanchoring of these elastic filaments because of the destruction of Z-lines. There also exists the possibility of disconnection of the elastic filaments themselves. Recent studies bave shown that structures consisting of such elastic proteins as connectin (Maruyama, et al., 1984) maintain a regular arrangement of sarcomere structure. Haeberle and co-workers (1985) found that CANP was able to produce striking effects on the contractile mechanics and ultrastructures of skinned uterine smooth muscle, and described that these effects appeared to result from the loss of cytoskeletal structures such as intermediate filaments. Moreover, Wang (1985) reported that connectin (titin) from skeletal muscle was degraded by C a 2+ and suggested that this effect could be ascribed to the activity of CANP. It is, therefore, possible that the elastic filaments are also digested by CANP and that this process is also involved in the decline of tension. In the physiological state, why does the muscle contract at a ~tM order of intracellular Ca 2+ without deterioration? The reasons are not clear, but some possibilities can be considered; CANP inhibitors may exist inside intact muscles, or perhaps the composition of intracellular solutions may be suitable for the inhibition of CANP activity. Indeed, Mellgren & Carr (1983) suggested the existence of endogenious inhibitors of Ca2+-dependent proteases. In the present study, it was shown that one of the causes of deterioration in the tension of skinned fibres is the proteolytic activity of CANP. CANP inhibitors are very useful to prevent this deterioration.

Acknowledgement We are grateful to Dr Takemori for his valuable comments on this manuscript.

References BUSH, W. A., STROMER~ M. H . , GOLLf D. E. & SUZUKI, A.

(1972) Ca2+-specificremoval of Z-lines from rabbit skeletal muscle. J. Cell. Biol. 52, 367-81. BODENSTEINER, J. B. & ENGEL, A. G. (1978) Intracellular calcium accumulation in Duchenne dystrophy and other myopaties: A study of 567,000 muscle fibers in 114 biopsies. Neurology 28, 439-46. CULLEN, M. J. & FULTHORPE, J. J. (1975) Stages in fibre breakdown in Duchenne muscular dystrophy. J. Neurol. Sci. 24, 179-200. EBASHI, S. & E N D O , M. (1968) Calcium and muscle contraction. Prog. Biophys. Mol. Biol. 18, 123-83. HARAFUJI, H. & O G A W A , Y. (1980) Re-examination of the apparent binding constant of ethylene glycol bis

(~-aminoethyl ether)-N, N, N', N'-tetraacetic acid with calcium around neutral pli. ]. Biochem. 87, 1305-12. HATTORI, A. & T A K A H A S H I , K. (1982) Calcium-induced weakening of skeletal muscle Z-disks. J. Biochem. 92, 381-90. HAEBERLE, J. R., C O O L I C A N , S. A. r EVAN, A. &

HATHAWAY, D. R. (1985) The effect of calcium dependent protease on the ultrastructure and contractile mechanics of skinned uterine smooth muscle. J. Musc. Res. Cell Motil. 6, 347-63. H I G U C H I , H. & U M A Z U M E , Y. (1985) Localization of the parallel elastic components in frog skinned muscle fibers studied by the dissociation of the A- and I-bands. Biophys. J. 48, 137-47.

C a l c i u m i n d u c e d d e t e r i o r a t i o n of s k i n n e d fibres ISHIURAr S. / MUROFUSHI, H. I SUZUKI, K. & IMAHORI I K. (1978) Studies of a calcium-activated neutral protease from chicken skeletal muscle. ]. Biochem. 84, 225-30. KAWASHIMA/ S., NOMOTO, M. / HAYASHI/ M. / INOMATA, M., NAKAMURA1 M. & IMAHORI1 K. (1984) Comparison of calcium-activated neutral proteases from skeletal muscle of rabbit and chicken. J. Biochem. 95, 95-101. MARUYAMA/ K./ SAWADA/ H., KIMURAf S. / OHASHI, K.~ HIGUCHI, H. & UMAZUME1 Y. (1984) Connectin filaments in stretched skinned fibers of frog skeletal muscle. J. Cell. Biol. 99, 1391-7. McGOWAN, E. B., SHAFIQ, S. A. & STRACHER, A. (1976) Delayed degeneration of dystrophic and normal muscle cell cultures treated with pepstatin, leupeptin, and antipain. Exp. Neurol. 50, 649-57. MELLGREN, R. L. (1980) Canine cardiac calcium-dependent proteases: Resolution of two fo,ms with different requirements for calcium. FEBS Letts. 109, 129-33. MELLGREN1 R. L. & CARR, T. C. (1983) The protein inhibitor of calcium-dependent proteases: Purification from bovine heart and possible mechanisms of regulation. Arch. Biochem. Biophys. 225, 779-86. NATORI, R. (1954) The role of myofibrilsl sarcoplasma and sarcolemma in muscle-contraction, likeikai Med. l. 11 18-28. PEARCE, G. W. (1966) Electron microscopy in the study of muscular dystrophy. Ann. N.Y. Acad. Sci. 138, 138-50.

47 PUBLICOVER, S. J., DUNCAN, C. J. & SMITH, J. L. (1978) The use of A23187 to demonstrate the role of intracellular calcium in causing ultrastructural damage in mammalian muscle. J. Neuropath. Exp. Neurol. 37, 544-57. REDDY, M. K., ETLINGERr J. D., RABINOWITZ r M., FISCHMAN, D. A. & ZAK, R. (1975) Removal of Z-lines and 0~-actinin from isolated myofibrils by a calciumactivated neutral protease. J. Biol. Chem. 250, 4278-84. SUZUKI, S. & SUGI, H. (1983) Extensibility of the myofilaments in vertebrate skeletal muscle as revealed by stretching rigor muscle fibers. J. Gen. Physiol. 81, 531-546. UMAZUME, Y., ONODERA, S. & HIGUCHI, H. (1986) Width and lattice spacing in radially compressed frog skinned muscle fibers at various p l i values, m a g n e s i u m ion concentrations and ionic strengths. J. Musc. Res. Cell Motil. 7, 251-8. WANG, K. (1985) Sarcomere-associated cytoskeletal lattices in striated muscle. In Cell and Muscle Motility, Vol. 6. (edited by SHAY, J. W.) pp. 315-69. Plenum Publishing Corp. WEBER, K. & OSBORN, M. (1969) The reliability of molecular weight determinations by dodecyl sulfatepolyacrylamide gel electrophoresis. J. Biol. Chem. 244, 4406-12.

Deterioration induced by physiological concentration of calcium ions in skinned muscle fibres.

The deteriorating effect of microM order of Ca2+ on skinned frog skeletal muscle fibres was studied from the view point of the digestion of proteins b...
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