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Effect of pressure treatment on the sarcoplasmic reticulum of red and white muscles
Meat Science5 (1980-81) 297-305
EFFECT OF PRESSURE TREATMENT ON THE SARCOPLASMIC RETICULUM OF RED AND WHITE MUSCLES
DOUGLAS J. HORGAN
CSIRO Division o f Food Research, Meat Research Laboratory, PO Box 12, Cannon Hill, Queensland, 4170, Australia (Received: 26 May, 1980)
SUMMARY
The effects of high pressure (150 M Pa) on the sarcoplasmic reticulum of red and white muscles have been studied. When whole, muscle is pressurised either pre-rigor or postrigor the major effect on the SR is the loss of extra ATPase activity and the loss of several proteins including the 100,000 dalton A TPase and calsequestrin. When isolated SR is pressurised the extra A TPase activity is lost but there is no protein degradation. Measurements of muscle p H and the influence of pH on pressurisation effects indicate active proteolysis in white muscle when it is pressurised. In all these studies the basal A TPase activity was relatively unaffected. The effect of pressure treatment on the yield of SR protein varied with different muscles, being greatest in the muscles which had the highest concentration of extra A TPase and calcium uptake activities. These muscles also reached the lowest p H during pressurisation, thus favouring proteolysis.
INTRODUCTION
The sarcoplasmic reticulum (SR) has two adenosine triphosphatase (ATPase) activities. The basal ATPase is a Mg2+-dependent enzyme which is active in the absence of calcium ions, while the extra ATPase is activated when micromolar concentrations of calcium are added to the basal assay system. The extra ATPase provides the energy for calcium uptake by the SR (Hasselbach & Makinose, 1961 ; Ebashi, 1961) and is the major protein constituent of the SR from white skeletal muscle (Martonosi & Halpin, 1971). Unlike the Ca 2 +-dependent (extra) ATPase, the Ca 2 ÷-independent (basal) ATPase has no known function in the muscle. It has been suggested that the basal ATPase activity is due to contamination of the SR with 297 Meat Science 0309-1740[81/0005-0297/$02.50 © Applied Science Publishers Ltd, England, 1981 Printed in Great Britain
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DOUGLAS J. HORGAN
other membranes such as the T-tubules (Headon et al., 1977). Another theory is that the basal ATPase is a structurally altered form of the extra ATPase which is no longer calcium activated (Inesi et al., 1976; Masuda & de Meis, 1977). Macfarlane (1973) showed that when pre-rigor muscle is subjected to pressures > 1 0 0 M P a for short times at temperatures between 25 ° and 35 °, contraction occurs, the rate of glycolysis is enhanced and the onset of rigor is accelerated. It has recently been shown that the SR of muscles such as the rabbit M. longissmus dorsi (LD) and beef M. sternomandibuIaris are affected by this type of treatment. The SR from pressure-treated muscles loses its extra ATPase activity while the basal ATPase activity remains intact (Horgan, 1979). In this study a number of muscles of widely differing types and from different animals have been studied. The muscles used were rabbit longissimus dorsi (predominantly white fibres), rabbit soleus (red), beef sternomandibularis (intermediate), beef masseter and sheep masseter (red). They were pressurised either pre-rigor or post-rigor and the effects on ATPase activities, protein yields and compositions of the isolated SR have been measured. The changes measured range in severity from the fastest down to the slowest of the muscles studied and correlate with the pH changes occurring in the muscles.
MATERIALS AND METHODS
(a) Pressure treatment Muscles were excised immediately following slaughter, vacuum sealed in plastic packets and treated in pressure vessels as described previously (Macfarlane, 1973). Control samples were treated similarly but were not pressurised. (b) Sarcoplasmie reticulum preparation The 8000-28,000 g fraction was prepared from control and pressurised muscles essentially by the method of Martonosi et al. (1968). The SR pellet was washed twice with 0.6M KC1 to remove contaminating myofibrillar proteins. The validity of this procedure for pressurised muscle was discussed previously (Horgan, 1979). (c) Assay procedures ATPase activities were measured at pH 7.0 and 25 ° by a continuous spectrophotometric method (Horgan, 1974). Calcium uptake activities were measured at pH 6-4 and 25 ° in the presence of 50 mM phosphate by the method of Horgan et al. (1972). Protein concentrations of the isolated SR fractions were measured by the method of Lowry et al. (1951). SDS-polyacrylamide gel electrophoresis was carried out in slabs using the system o f Laemmli (1970). The gels were scanned with a Kipp and Zonen DD2 densitometer after staining with Coomassie Blue. Muscle pH's were measured after blending the muscle sample in 5 mM iodoacetic acid neutralised with 1M sodium hydroxide.
EFFECT OF PRESSURE ON THE S A R C O P L A S M I C R E T I C U L U M OF MUSCLES
299
RESULTS
Table 1 shows the effect ofpre-rigor pressure treatment on five different muscles. The muscles were all pressurised for 10 min at 150 M P a (35 °) as these conditions had been previously shown to produce maximum effects (Horgan, 1979). The effects o f pressure treatment ranged in severity from the large changes found for rabbit white muscles to the minor changes found for the m a s s e t e r muscles. A comparison of the two types of rabbit muscle shows that with both the extra ATPase activity is greatly reduced both in specific activity and in total number of units of activity (specific activity multiplied by the yield). In the case of the basal ATPase activity the total number of units of activity is relatively unchanged by pressure treatment but in the case of the white muscles, because of the large reduction in yield, the activity has been concentrated, giving a high specific activity. On the other hand, the yield of SR from the red muscle was reduced by about 10 ~ , and the SR showed a corresponding increase in specific activity of the basal ATPase. In the case of the two m a s s e t e r muscles the yield of SR was unaffected (beef), or slightly increased (sheep) by' pressure treatment. At the same time there was a slight decrease in the specific activity of the basal ATPase. Therefore, like the other muscles, the total amount of basal activity (specific activity × yield) was not affected by the pressure treatment. Beef neck muscle SR has ATPase activities which are intermediate between those of the white and the red muscles. Correspondingly, the effect of pressure is intermediate in severity, with the yield reduced to below half the control and the basal ATPase doubled in specific activity. The severity o f changes to SR, as indicated by SDS gel electrophoresis, also varied with the different types of muscle pressure-treated. With white muscles such as the rabbit p s o a s , loss of the 100,000 dalton band and an increase in the number and concentration of proteins in the 70,000 to 20,000 daltons range have been described TABLE 1 E F F E C T O N S R OF P R E S S U R E T R E A T M E N T a O F M U S C L E S
Muscle
Rabbit white
(longissimus dorsi) R a b b i t red
(so&us) Beef n e c k
(sternomandibularis) Beef red
(masseter) Sheep red
(masseter)
Treatment and number of samples Control Pressurised Control Pressurised Control Pressurised Control Pressurised Control Pressurised
(6) (6) (3) (3) (3) (3) (3) (3) (4) (4)
SR A TPase activity b Basal 0-116 1.657 0-994 1-222 0-263 0-502 0.209 0.181 0.263 0-227
+ 0-026 + 0-310 _ 0-026 __+0.067 ___0~080 _ 0.106 __+0.027 _ 0-017 ___0-050 ± 0-044
" S a m p l e s were pressurised for 10 m i n at 150 M P a (35°). b R e s u l t s expressed as m e a n ___s t a n d a r d error.
(#moles/min/mg of protein) Extra 0.772 0.014 0" 181 0.023 0'668 0:005 0-021
_+ 0-073 __. 0.010 +0.068 4- 0:023 _ 0-102 _ 0-005 _ 0.006 0 0-072 __. 0.010 0-018 4- 0.010
YieM of SR (mg/g of muscle) 1.450 + 0-041 0' 134 _ 0-011 0-697 4- 0-098 0.630 __+0.091 0"700 ___0.201 0.270 + 0.046 0.583 ___0.127 0 " 5 9 7 _ 0.052 0.455 + 0.072 0.560 4- 0-110
300
DOUGLAS J. HORGAN
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tVfr. 10 "3 Fig. 1. Densitometric scans o f SR preparations from beef masseter muscle run on SDS gel slabs. Top: SR from control muscle. B o t t o m : SR from muscle pressurised for 10min at 150 M P a ( 3 5 ° ) .
TABLE 2 E F F E C T O N S R OF P R E S S U R E T R E A T M E N T O F F R E S H A N D P O S T - R I G O R R A B B I T W H I T E M U S C L E
Treatment
(A) (B) (C) (D)
Control (pre-rigor) Pressurised ° fresh Post-rigor Pressurised ~ post-rigor
S R A TPase activity (#moles/rain/rag) Basal Extra Tritonstimulated extra b 0.087 1.840 0-300 1-870
0.950 0 1-080 0
°Samples were pressurised for 10 m i n at 150 M P a (35°). b Measured in the presence o f 0.05 ~o Triton-Xl00.
'
3.60 0 4-25 0
Yield (mg/g o f muscle) 1.91 0-18 1.13 0.20
EFFECT OF PRESSURE O N T H E S A R C O P L A S M I C R E T I C U L U M OF MUSCLES
301
previously ( H o r g a n , 1979). Since then it has been shown, with the aid o f g l y c o p r o t e i n staining, that the calsequestrin is also destroyed by pressure treatment. A n example o f the effect o f pressure treatment on the SDS gel pattern o f a slow red muscle is given in Fig. 1 where SR fractions f r o m control and pressurised b e e f m a s s e t e r muscles are shown. There a p p e a r to be no m a j o r changes caused by the pressure treatment. Table 2 shows the results typical o f an experiment where the L D muscles o f a rabbit were divided into f o u r and treated as follows: A. B. C. D.
C o n t r o l : N o treatment. Pressurised fresh: I m m e d i a t e l y after excision, the muscles were pressurised for 10min at 150 M P a (35°). Post-rigor: Muscles were stored at r o o m t e m p e r a t u r e for 4 h and at 1 ° for a further 20 h to allow full development o f rigor. Pressurised post-rigor: Muscle treated as in C a n d then pressurised as in B.
Following the above treatments all samples were h o m o g e n i s e d and SR prepared in the usual m a n n e r . C o m p a r i n g satnples A and C shows that rigor reduced the yield o f SR and increased the basal A T P a s e activity. T h e increase is, however, small c o m p a r e d with the increase in basal A T P a s e activity caused by pressurisation. SR f r o m pressurised fresh and pressurised post-rigor samples had very similar basal A T P a s e activity. T h e densitometric scans o f the SR p r e p a r a t i o n s f r o m the f o u r samples described in Table 2 are shown in Fig. 2. C o m p a r i s o n o f samples A and C reveals only m i n o r differences in the region arotind 50,000 daltons. The two pressurised samples (B and D) a p p e a r to have nearly identical scan profiles. Pressure treatment destroyed the 100,000 dalton A T P a s e band. T h e effect o f pressure treatment on the p H o f the five different muscles investigated is shown in Table 3. The effect depends on the type o f muscle fibres. Fast TABLE 3 EFFECT OF PRESSURE ON MUSCLE pH
Muscle type Control
Rabbit white (longissimus dorsi)
Rabbit red (so&us)
Beef neck (sternomandibularis)
Beef red (masseter)
Sheep red (masseter)
Ultimate pH a Pressurised
5-75 + 0.12 (11) 6.42 ___0.02 (5) 6.15 _+0.04 (4) 6.42 4- 0-23 (3) 6.52 4- 0.09 (4)
5.43 4- 0.02 (6) 6.17 4- 0.01 (3) 5.86 4- 0.06 (4) 6.35 __0-01 (3) 6.73 ___0.08 (4)
Pressure-induced p H drop b
0.98 4- 0-09 (6) 0.44 4- 0.05 (5) 0-83 4- 0.06 (5) 0-46 4- 0.06 (3) 0.18 4- 0.04 (4)
a Measured after 24 h. Results expressed as mean + standard error with number of observations in parentheses. bThis is the fall in pH during the pressure treatment. Muscles were pressurised for 10min at 150 MPa (35 °) and the pH measured immediately before and after pressurisation.
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30
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Fig. 2. Densitometric scans of SR preparations from rabbit L. dorsi muscle that had been treated as described in Table 2. (A) Control, no treatment. (B) Pressurised fresh. (C) Post-rigor. (D) Pressurised post-rigor. Full details of treatments are in 'Results' section.
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616
i 6"4
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610
5"8
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Fig. 3. Effect of p H on the extra A T P a s e activity o f isolated rabbit white muscle SR preparations pressurised for 10 rain at 150 M Pa (35 °). The combined results o f three separate experiments are shown with the extra A T P a s e activity of the pressurised SR expressed as a percentage of a control where the SR was suspended in the same p H buffer (50 m ~ KCI, 50 mM PIPES) but not pressurised.
EFFECT OF PRESSURE ON THE SARCOPLASMIC RETICULUM OF MUSCLES
303
muscles such as the rabbit white have low ultimate pH's, both under control and pressurised conditions and showed a large pressure-induced pH drop. Slow muscles, such as the rabbit red and the m a s s e t e r muscles, on the other hand, have much higher ultimate pH's and showed a small pressure-induced pH drop. When isolated SR was pressure treated, the basal ATPase activity was stable. The effect on the extra ATPase activity was pH dependent with the reduction in activity greatest at lower pH's (Fig. 3). SDS gels of the SR preparations after pressurisation (not shown) did not indicate any proteolysis at any of the pH values tested.
DISCUSSION
This study shows that dramatic changes to the SR can be effected by pressurising muscles both pre-rigor and post-rigor. The SR preparations of all muscles tested were affected by the pressure treatment but the severity of the changes induced depended on the type of muscle. The changes in the SR explain the observed changes in muscle glycolysis and contraction upon pressurisation (Macfarlane, 1973) and shed some light on the relationship between the two major ATPase activities in the SR (Horgan, 1979). The results in Table 1 and Figs 1 and 2, showing the effect ofpressurisation on the AT Pase activities, protein yields and SDS gel patterns of SR, extendthe results of a previous study (Horgan, 1979). The SR preparations from all the muscles studied were affected by pressure treatment. The principal effect, common to all muscles, appears to be the destruction of the extra ATPase. Besides the loss of enzyme activity, the SDS gels show that the protein itself (the 100,000 dalton band) is lost during pressurisation of the muscles (Fig. 2). The effect of pressure treatment on the protein yield of SR varies greatly from muscle to muscle. Fast muscles such as the rabbit white are severely affected, whilst slow muscles such as the masseters, are hardly affected. It is well known that the calcium stimulated ATPase activity and associated calcium uptake activity vary with type of muscle. White muscles contain far more of these activities than do red muscles (Margreth et al., 1972; Sreter & Gergely, 1964; Sreter, 1969). Besides the extra ATPase protein, calsequestrin, which is also associated with calcium pumping, is also destroyed by pressurisation. It follows, therefore, that if the reduction in protein yield is caused mainly by loss of proteins associated with calcium uptake then the white muscle SR will be more severely affected than the red muscle SR. The SDS gels show that after pressure treatment the relatively simple protein pattern of white muscle SR (Fig. 2, A and C) is changed to resemble the complex pattern of slow muscle SR (compare Figs 1 and 2, B and D). The destruction of the SR by pressurisation would cause the release of Ca 2 ÷ into the sarcoplasm which would, in turn, stimulate both contraction and glycolysis. The effect of rigor on SR properties and on pressure treatment is shown in Table 2
304
DOUGLAS J. H O R G A N
and Fig. 2. The results are of interest for three reasons. The first is the relatively slight effect of rigor development on the isolated SR. Comparison of treatments A and C in Table 2 shows that the post-rigor preparation has a strong ATPase activity which is stimulated by the addition of Triton-Xl00. This indicates calcium uptake activity and in fact, when tested, the post-rigor preparation had a calcium uptake activity that was 65 ~ that of the fresh control. Greaser et al. (1969) reported that SR preparations from 24 hour post mortem porcine muscles had only 5 ~o o f the calcium uptake activity o f fresh preparations. A second point of interest is that pressure treatment of post-rigor muscle has the same effect on SR preparations as does pressure treatment of fresh muscle. This shows that the changes seen on pressurisation of fresh muscle are not due solely to the drop in p H o f the muscle caused by the increase in glycolysis. The third point o f interest is that the basal ATPase activity of the SR from postrigor muscle is increased compared with the fresh muscle control. This result is quite reproducible and shows that ageing of the muscle partially produces some of the effects of pressurisation. The question that arises in all these experiments is whether the increase in specific activity of the basal ATPase is solely due to purification by removal of other proteins from the SR or whether there is an increase in the amount of basal ATPase activity. In several experiments the yield of basal ATPase activity from pressurised and aged muscles is greater than that from control muscle. This could indicate synthesis of new activity or could simply be due to an improved yield o f SR from the treated muscle. The first possibility would support the hypothesis of Inesi et al. (1976) that the basal and extra ATPases are interconvertible forms o f the same protein. However, the selective and complete destruction o f the extra A T Pase activity and the 100,000 dalton protein band indicate that the basal and extra ATPase are separate proteins associated with different parts of the SR membrane. The role of p H in the pressurisation of muscle and isolated SR is illustrated in Table 3 and Fig. 3. The yields of SR from muscles with a high ultimate pH, such as the masseters, are not reduced much by pressurisation whereas those from muscles with low ultimate pH, such as the rabbit white muscles, are. This is consistent with the idea that a protease is responsible for the breakdown of the SR. The nature of the protease is not known. One possibility.is that pressure treatment disrupts lysosomal membranes, thus releasing proteolytic enzymes. Penny & Ferguson-Pryce (1979), in their study o f autolysis in beef muscle homogenates, concluded that there were at least two proteolytic enzyme systems involved---cathepsin B at pH's below 6.0 and the calcium activated factor (CAF) at higher pH's. The results with isolated SR show that the basal ATPase activity is resistant to pressurisation at all pH's tested, whereas, as shown in Fig. 3, the extra A T P a s e activity is labile, especially at low pH. Berman et al. (1977) found that the basal A T P a s e activity of SR from rabbit white muscles was more acid-resistant at 37 ° and atmospheric pressure than the extra ATPase activity. However, SDS gels show no sign o f any proteolytic breakdown o f the pressurised SR at any pH tested. The loss o f extra ATPase activity thus appears
EFFECT OF PRESSURE ON THE SARCOPLASMIC RETICULUM OF MUSCLES
305
to be due to denaturation of the active site, rather than to general proteolysis. During the isolation of the SR there are several washing steps (including two with 0"6M KC1) which would probably remove contaminating proteases. In the whole muscle, pressurisation may therefore affect the SR both by denaturing the extra ATPase and by proteolytic digestion of some of the SR proteins, including the extra ATPase and calsequestrin.
ACKNOWLEDGEMENTS
This work was supported in part by the Australian Meat Research Committee. The skilled technical assistance of Mr R. Kuypers is gratefully acknowledged.
REFERENCES BERMAN, M. C., MCINTOSr~, D. E. & KENCH, J. E. (1977). J. Biol. Chem., 252, 994. EaAsrn, S. (1961). J. Biochem., 511, 236. GREASER, M., CASSENS, R. G., HOEKSTRA, W. G. &z BRISKLY, E. J. (1969). J. Fd. Sci., 34, 633. HASSELBACH, W. t~ MAKINOSE, M. (1961). 'Biochem. Z., 333, 518. HEADON, D. R., BARRETT,E. J., JOYCE, N. M. & O'FLAHERTY, J. (1977). Mol. Cell. Biochem., 17, 117. HORGAN, D. J. (1974). Arch. Biochem. Biophys., 162, 16. HORGAN, D. J. (1979). J. Food Ski., 44, 492. HORGAN, D. J., TUME, R. K. d~. NEWBOLD, R. P. (1972). Anal. Biochem., 48, 147. INESl, G., COHEN, J. A. & COAN, C. R. (1976). Biochemistry, 15, 5293. LAEMMLI, U. K. (1970). Nature, 227, 680. LOWRY, O. H., ROSEBROUGH, N. J., FARR, A. L. • RANDALL, R. J. (1951). J. Biol. Chem., 193, 265. MACFARLANE, J. J. (1973). J. Food Sci., 38, 294. MARGRETH, A., SALVIATI,G., DI MAURO, S. & TURATI, G. (1972). Biochem. J., 126, 1099. MARTONOSI, A., DONLEY, J. & HALPIN, R. A. (1968). J. BID1. Chem., 243, 6-1. MARTONOSI, A. d~ HALPIN, R. A. (1971). Arch. Biochem. Biophys., 144, 66. MASUDA, H. t~ DE MEIS, L. (1977). J. Biol. Chem., 252, 8567. PENNY, I. F. & FERGUSON-PRYCE, R. (1979). Meat Science, 3, 121. SRETER, F. (1969). Arch. Biochem. Biophys., 134, 25. SRETER, F. A. & GERGELY, J. (1964). Biochem. Biophys. Res. Commun., 16, 438.
EFFECT OF PRESSURE TREATMENT ON THE SARCOPLASMIC RETICULUM OF RED AND WHITE MUSCLES
DOUGLAS J. HORGAN
CSIRO Division o f Food Research, Meat Research Laboratory, PO Box 12, Cannon Hill, Queensland, 4170, Australia (Received: 26 May, 1980)
SUMMARY
The effects of high pressure (150 M Pa) on the sarcoplasmic reticulum of red and white muscles have been studied. When whole, muscle is pressurised either pre-rigor or postrigor the major effect on the SR is the loss of extra ATPase activity and the loss of several proteins including the 100,000 dalton A TPase and calsequestrin. When isolated SR is pressurised the extra A TPase activity is lost but there is no protein degradation. Measurements of muscle p H and the influence of pH on pressurisation effects indicate active proteolysis in white muscle when it is pressurised. In all these studies the basal A TPase activity was relatively unaffected. The effect of pressure treatment on the yield of SR protein varied with different muscles, being greatest in the muscles which had the highest concentration of extra A TPase and calcium uptake activities. These muscles also reached the lowest p H during pressurisation, thus favouring proteolysis.
INTRODUCTION
The sarcoplasmic reticulum (SR) has two adenosine triphosphatase (ATPase) activities. The basal ATPase is a Mg2+-dependent enzyme which is active in the absence of calcium ions, while the extra ATPase is activated when micromolar concentrations of calcium are added to the basal assay system. The extra ATPase provides the energy for calcium uptake by the SR (Hasselbach & Makinose, 1961 ; Ebashi, 1961) and is the major protein constituent of the SR from white skeletal muscle (Martonosi & Halpin, 1971). Unlike the Ca 2 +-dependent (extra) ATPase, the Ca 2 ÷-independent (basal) ATPase has no known function in the muscle. It has been suggested that the basal ATPase activity is due to contamination of the SR with 297 Meat Science 0309-1740[81/0005-0297/$02.50 © Applied Science Publishers Ltd, England, 1981 Printed in Great Britain
298
DOUGLAS J. HORGAN
other membranes such as the T-tubules (Headon et al., 1977). Another theory is that the basal ATPase is a structurally altered form of the extra ATPase which is no longer calcium activated (Inesi et al., 1976; Masuda & de Meis, 1977). Macfarlane (1973) showed that when pre-rigor muscle is subjected to pressures > 1 0 0 M P a for short times at temperatures between 25 ° and 35 °, contraction occurs, the rate of glycolysis is enhanced and the onset of rigor is accelerated. It has recently been shown that the SR of muscles such as the rabbit M. longissmus dorsi (LD) and beef M. sternomandibuIaris are affected by this type of treatment. The SR from pressure-treated muscles loses its extra ATPase activity while the basal ATPase activity remains intact (Horgan, 1979). In this study a number of muscles of widely differing types and from different animals have been studied. The muscles used were rabbit longissimus dorsi (predominantly white fibres), rabbit soleus (red), beef sternomandibularis (intermediate), beef masseter and sheep masseter (red). They were pressurised either pre-rigor or post-rigor and the effects on ATPase activities, protein yields and compositions of the isolated SR have been measured. The changes measured range in severity from the fastest down to the slowest of the muscles studied and correlate with the pH changes occurring in the muscles.
MATERIALS AND METHODS
(a) Pressure treatment Muscles were excised immediately following slaughter, vacuum sealed in plastic packets and treated in pressure vessels as described previously (Macfarlane, 1973). Control samples were treated similarly but were not pressurised. (b) Sarcoplasmie reticulum preparation The 8000-28,000 g fraction was prepared from control and pressurised muscles essentially by the method of Martonosi et al. (1968). The SR pellet was washed twice with 0.6M KC1 to remove contaminating myofibrillar proteins. The validity of this procedure for pressurised muscle was discussed previously (Horgan, 1979). (c) Assay procedures ATPase activities were measured at pH 7.0 and 25 ° by a continuous spectrophotometric method (Horgan, 1974). Calcium uptake activities were measured at pH 6-4 and 25 ° in the presence of 50 mM phosphate by the method of Horgan et al. (1972). Protein concentrations of the isolated SR fractions were measured by the method of Lowry et al. (1951). SDS-polyacrylamide gel electrophoresis was carried out in slabs using the system o f Laemmli (1970). The gels were scanned with a Kipp and Zonen DD2 densitometer after staining with Coomassie Blue. Muscle pH's were measured after blending the muscle sample in 5 mM iodoacetic acid neutralised with 1M sodium hydroxide.
EFFECT OF PRESSURE ON THE S A R C O P L A S M I C R E T I C U L U M OF MUSCLES
299
RESULTS
Table 1 shows the effect ofpre-rigor pressure treatment on five different muscles. The muscles were all pressurised for 10 min at 150 M P a (35 °) as these conditions had been previously shown to produce maximum effects (Horgan, 1979). The effects o f pressure treatment ranged in severity from the large changes found for rabbit white muscles to the minor changes found for the m a s s e t e r muscles. A comparison of the two types of rabbit muscle shows that with both the extra ATPase activity is greatly reduced both in specific activity and in total number of units of activity (specific activity multiplied by the yield). In the case of the basal ATPase activity the total number of units of activity is relatively unchanged by pressure treatment but in the case of the white muscles, because of the large reduction in yield, the activity has been concentrated, giving a high specific activity. On the other hand, the yield of SR from the red muscle was reduced by about 10 ~ , and the SR showed a corresponding increase in specific activity of the basal ATPase. In the case of the two m a s s e t e r muscles the yield of SR was unaffected (beef), or slightly increased (sheep) by' pressure treatment. At the same time there was a slight decrease in the specific activity of the basal ATPase. Therefore, like the other muscles, the total amount of basal activity (specific activity × yield) was not affected by the pressure treatment. Beef neck muscle SR has ATPase activities which are intermediate between those of the white and the red muscles. Correspondingly, the effect of pressure is intermediate in severity, with the yield reduced to below half the control and the basal ATPase doubled in specific activity. The severity o f changes to SR, as indicated by SDS gel electrophoresis, also varied with the different types of muscle pressure-treated. With white muscles such as the rabbit p s o a s , loss of the 100,000 dalton band and an increase in the number and concentration of proteins in the 70,000 to 20,000 daltons range have been described TABLE 1 E F F E C T O N S R OF P R E S S U R E T R E A T M E N T a O F M U S C L E S
Muscle
Rabbit white
(longissimus dorsi) R a b b i t red
(so&us) Beef n e c k
(sternomandibularis) Beef red
(masseter) Sheep red
(masseter)
Treatment and number of samples Control Pressurised Control Pressurised Control Pressurised Control Pressurised Control Pressurised
(6) (6) (3) (3) (3) (3) (3) (3) (4) (4)
SR A TPase activity b Basal 0-116 1.657 0-994 1-222 0-263 0-502 0.209 0.181 0.263 0-227
+ 0-026 + 0-310 _ 0-026 __+0.067 ___0~080 _ 0.106 __+0.027 _ 0-017 ___0-050 ± 0-044
" S a m p l e s were pressurised for 10 m i n at 150 M P a (35°). b R e s u l t s expressed as m e a n ___s t a n d a r d error.
(#moles/min/mg of protein) Extra 0.772 0.014 0" 181 0.023 0'668 0:005 0-021
_+ 0-073 __. 0.010 +0.068 4- 0:023 _ 0-102 _ 0-005 _ 0.006 0 0-072 __. 0.010 0-018 4- 0.010
YieM of SR (mg/g of muscle) 1.450 + 0-041 0' 134 _ 0-011 0-697 4- 0-098 0.630 __+0.091 0"700 ___0.201 0.270 + 0.046 0.583 ___0.127 0 " 5 9 7 _ 0.052 0.455 + 0.072 0.560 4- 0-110
300
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TABLE 2 E F F E C T O N S R OF P R E S S U R E T R E A T M E N T O F F R E S H A N D P O S T - R I G O R R A B B I T W H I T E M U S C L E
Treatment
(A) (B) (C) (D)
Control (pre-rigor) Pressurised ° fresh Post-rigor Pressurised ~ post-rigor
S R A TPase activity (#moles/rain/rag) Basal Extra Tritonstimulated extra b 0.087 1.840 0-300 1-870
0.950 0 1-080 0
°Samples were pressurised for 10 m i n at 150 M P a (35°). b Measured in the presence o f 0.05 ~o Triton-Xl00.
'
3.60 0 4-25 0
Yield (mg/g o f muscle) 1.91 0-18 1.13 0.20
EFFECT OF PRESSURE O N T H E S A R C O P L A S M I C R E T I C U L U M OF MUSCLES
301
previously ( H o r g a n , 1979). Since then it has been shown, with the aid o f g l y c o p r o t e i n staining, that the calsequestrin is also destroyed by pressure treatment. A n example o f the effect o f pressure treatment on the SDS gel pattern o f a slow red muscle is given in Fig. 1 where SR fractions f r o m control and pressurised b e e f m a s s e t e r muscles are shown. There a p p e a r to be no m a j o r changes caused by the pressure treatment. Table 2 shows the results typical o f an experiment where the L D muscles o f a rabbit were divided into f o u r and treated as follows: A. B. C. D.
C o n t r o l : N o treatment. Pressurised fresh: I m m e d i a t e l y after excision, the muscles were pressurised for 10min at 150 M P a (35°). Post-rigor: Muscles were stored at r o o m t e m p e r a t u r e for 4 h and at 1 ° for a further 20 h to allow full development o f rigor. Pressurised post-rigor: Muscle treated as in C a n d then pressurised as in B.
Following the above treatments all samples were h o m o g e n i s e d and SR prepared in the usual m a n n e r . C o m p a r i n g satnples A and C shows that rigor reduced the yield o f SR and increased the basal A T P a s e activity. T h e increase is, however, small c o m p a r e d with the increase in basal A T P a s e activity caused by pressurisation. SR f r o m pressurised fresh and pressurised post-rigor samples had very similar basal A T P a s e activity. T h e densitometric scans o f the SR p r e p a r a t i o n s f r o m the f o u r samples described in Table 2 are shown in Fig. 2. C o m p a r i s o n o f samples A and C reveals only m i n o r differences in the region arotind 50,000 daltons. The two pressurised samples (B and D) a p p e a r to have nearly identical scan profiles. Pressure treatment destroyed the 100,000 dalton A T P a s e band. T h e effect o f pressure treatment on the p H o f the five different muscles investigated is shown in Table 3. The effect depends on the type o f muscle fibres. Fast TABLE 3 EFFECT OF PRESSURE ON MUSCLE pH
Muscle type Control
Rabbit white (longissimus dorsi)
Rabbit red (so&us)
Beef neck (sternomandibularis)
Beef red (masseter)
Sheep red (masseter)
Ultimate pH a Pressurised
5-75 + 0.12 (11) 6.42 ___0.02 (5) 6.15 _+0.04 (4) 6.42 4- 0-23 (3) 6.52 4- 0.09 (4)
5.43 4- 0.02 (6) 6.17 4- 0.01 (3) 5.86 4- 0.06 (4) 6.35 __0-01 (3) 6.73 ___0.08 (4)
Pressure-induced p H drop b
0.98 4- 0-09 (6) 0.44 4- 0.05 (5) 0-83 4- 0.06 (5) 0-46 4- 0.06 (3) 0.18 4- 0.04 (4)
a Measured after 24 h. Results expressed as mean + standard error with number of observations in parentheses. bThis is the fall in pH during the pressure treatment. Muscles were pressurised for 10min at 150 MPa (35 °) and the pH measured immediately before and after pressurisation.
D
B 2
J
®1 u \
i
f
i
100
50
30
C
A
1
100
5~0
30 ~r.10-3
Fig. 2. Densitometric scans of SR preparations from rabbit L. dorsi muscle that had been treated as described in Table 2. (A) Control, no treatment. (B) Pressurised fresh. (C) Post-rigor. (D) Pressurised post-rigor. Full details of treatments are in 'Results' section.
30
o
mO o
0
QI •
20
<~ 10
7-2
7"0
i 6"8
616
i 6"4
i 6"2
610
5"8
pH
Fig. 3. Effect of p H on the extra A T P a s e activity o f isolated rabbit white muscle SR preparations pressurised for 10 rain at 150 M Pa (35 °). The combined results o f three separate experiments are shown with the extra A T P a s e activity of the pressurised SR expressed as a percentage of a control where the SR was suspended in the same p H buffer (50 m ~ KCI, 50 mM PIPES) but not pressurised.
EFFECT OF PRESSURE ON THE SARCOPLASMIC RETICULUM OF MUSCLES
303
muscles such as the rabbit white have low ultimate pH's, both under control and pressurised conditions and showed a large pressure-induced pH drop. Slow muscles, such as the rabbit red and the m a s s e t e r muscles, on the other hand, have much higher ultimate pH's and showed a small pressure-induced pH drop. When isolated SR was pressure treated, the basal ATPase activity was stable. The effect on the extra ATPase activity was pH dependent with the reduction in activity greatest at lower pH's (Fig. 3). SDS gels of the SR preparations after pressurisation (not shown) did not indicate any proteolysis at any of the pH values tested.
DISCUSSION
This study shows that dramatic changes to the SR can be effected by pressurising muscles both pre-rigor and post-rigor. The SR preparations of all muscles tested were affected by the pressure treatment but the severity of the changes induced depended on the type of muscle. The changes in the SR explain the observed changes in muscle glycolysis and contraction upon pressurisation (Macfarlane, 1973) and shed some light on the relationship between the two major ATPase activities in the SR (Horgan, 1979). The results in Table 1 and Figs 1 and 2, showing the effect ofpressurisation on the AT Pase activities, protein yields and SDS gel patterns of SR, extendthe results of a previous study (Horgan, 1979). The SR preparations from all the muscles studied were affected by pressure treatment. The principal effect, common to all muscles, appears to be the destruction of the extra ATPase. Besides the loss of enzyme activity, the SDS gels show that the protein itself (the 100,000 dalton band) is lost during pressurisation of the muscles (Fig. 2). The effect of pressure treatment on the protein yield of SR varies greatly from muscle to muscle. Fast muscles such as the rabbit white are severely affected, whilst slow muscles such as the masseters, are hardly affected. It is well known that the calcium stimulated ATPase activity and associated calcium uptake activity vary with type of muscle. White muscles contain far more of these activities than do red muscles (Margreth et al., 1972; Sreter & Gergely, 1964; Sreter, 1969). Besides the extra ATPase protein, calsequestrin, which is also associated with calcium pumping, is also destroyed by pressurisation. It follows, therefore, that if the reduction in protein yield is caused mainly by loss of proteins associated with calcium uptake then the white muscle SR will be more severely affected than the red muscle SR. The SDS gels show that after pressure treatment the relatively simple protein pattern of white muscle SR (Fig. 2, A and C) is changed to resemble the complex pattern of slow muscle SR (compare Figs 1 and 2, B and D). The destruction of the SR by pressurisation would cause the release of Ca 2 ÷ into the sarcoplasm which would, in turn, stimulate both contraction and glycolysis. The effect of rigor on SR properties and on pressure treatment is shown in Table 2
304
DOUGLAS J. H O R G A N
and Fig. 2. The results are of interest for three reasons. The first is the relatively slight effect of rigor development on the isolated SR. Comparison of treatments A and C in Table 2 shows that the post-rigor preparation has a strong ATPase activity which is stimulated by the addition of Triton-Xl00. This indicates calcium uptake activity and in fact, when tested, the post-rigor preparation had a calcium uptake activity that was 65 ~ that of the fresh control. Greaser et al. (1969) reported that SR preparations from 24 hour post mortem porcine muscles had only 5 ~o o f the calcium uptake activity o f fresh preparations. A second point of interest is that pressure treatment of post-rigor muscle has the same effect on SR preparations as does pressure treatment of fresh muscle. This shows that the changes seen on pressurisation of fresh muscle are not due solely to the drop in p H o f the muscle caused by the increase in glycolysis. The third point o f interest is that the basal ATPase activity of the SR from postrigor muscle is increased compared with the fresh muscle control. This result is quite reproducible and shows that ageing of the muscle partially produces some of the effects of pressurisation. The question that arises in all these experiments is whether the increase in specific activity of the basal ATPase is solely due to purification by removal of other proteins from the SR or whether there is an increase in the amount of basal ATPase activity. In several experiments the yield of basal ATPase activity from pressurised and aged muscles is greater than that from control muscle. This could indicate synthesis of new activity or could simply be due to an improved yield o f SR from the treated muscle. The first possibility would support the hypothesis of Inesi et al. (1976) that the basal and extra ATPases are interconvertible forms o f the same protein. However, the selective and complete destruction o f the extra A T Pase activity and the 100,000 dalton protein band indicate that the basal and extra ATPase are separate proteins associated with different parts of the SR membrane. The role of p H in the pressurisation of muscle and isolated SR is illustrated in Table 3 and Fig. 3. The yields of SR from muscles with a high ultimate pH, such as the masseters, are not reduced much by pressurisation whereas those from muscles with low ultimate pH, such as the rabbit white muscles, are. This is consistent with the idea that a protease is responsible for the breakdown of the SR. The nature of the protease is not known. One possibility.is that pressure treatment disrupts lysosomal membranes, thus releasing proteolytic enzymes. Penny & Ferguson-Pryce (1979), in their study o f autolysis in beef muscle homogenates, concluded that there were at least two proteolytic enzyme systems involved---cathepsin B at pH's below 6.0 and the calcium activated factor (CAF) at higher pH's. The results with isolated SR show that the basal ATPase activity is resistant to pressurisation at all pH's tested, whereas, as shown in Fig. 3, the extra A T P a s e activity is labile, especially at low pH. Berman et al. (1977) found that the basal A T P a s e activity of SR from rabbit white muscles was more acid-resistant at 37 ° and atmospheric pressure than the extra ATPase activity. However, SDS gels show no sign o f any proteolytic breakdown o f the pressurised SR at any pH tested. The loss o f extra ATPase activity thus appears
EFFECT OF PRESSURE ON THE SARCOPLASMIC RETICULUM OF MUSCLES
305
to be due to denaturation of the active site, rather than to general proteolysis. During the isolation of the SR there are several washing steps (including two with 0"6M KC1) which would probably remove contaminating proteases. In the whole muscle, pressurisation may therefore affect the SR both by denaturing the extra ATPase and by proteolytic digestion of some of the SR proteins, including the extra ATPase and calsequestrin.
ACKNOWLEDGEMENTS
This work was supported in part by the Australian Meat Research Committee. The skilled technical assistance of Mr R. Kuypers is gratefully acknowledged.
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