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Post-mortem glycolysis in rabbit Longissimus dorsi muscles following electrical stimulation
Meat Science 12 (1985) 225-241
Post-Mortem Glycolysis in Rabbit Longissimus dorsi Muscles Following Electrical Stimulation Douglas J. Horgan & Ronald Kuypers CS[RO Division of Food Research, Meat Research Laboratory, PO Box 12, Cannon Hill, Queensland, Australia 4170 (Received: 30 April, 1984)
S UMMA R Y The effects of both high voltage and low voltage electrical stimulation were studied in rabbit Longissimus dorsi muscles. The rate of fall of pH as well as the activities of phosphorylase a, phosphorylase kinase, and phosphorylase phosphatase were measured. The effects on the yield, A TPase activities, and calcium permeability of the sarcoplasmic reticulum (SR) were also measured. Although onO' high voltage stimulation increased the post-stimulation rate of pH fall, both types of electrical stimulation increased the phosphorvlase a activity, apparently by increasing the activio' of phosphorylase kinase and destroying a large part of the phosphorylase phosphatase activity. Electrical stimulation reduced the yieM of SR and increased its basal A TPase activity. It also increased the ability of the SR to retain accumulated calcium. We conclude that the different rates of pH fall observed following the two types of stimulation are due to the differential effects of these treatments on one of the A TPase activities, probably the myofibrillar A TPase.
INTRODUCTION Electrical stimulation (ES) appears to affect the rate of glycolysis in prerigor muscles in two stages. Firstly, during stimulation, the rate can be increased up to 150-fold resulting in a dramatic fall in pH (Chrystall & Devine, 1978). Secondly, after cessation o f stimulation, glycolysis rates ranging up to almost three times that of unstimulated muscles have been reported by various workers (see Table l). The very rapid rate o f 225 Meat Science 0309-1740/85/$03-30 © Elsevier Applied Science Publishers Ltd, England, 1985. Printed in Great Britain
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glycolysis observed during stimulation can be attributed to the activation of the enzyme glycogen phosphorylase. This is caused by calcium ions released from the SR activating phosphorylase kinase (Brostrom et al., 1971) which in turn activates phosphory!ase by converting the inactive b form to the active phosphorylase a (Krebs et al., 1958; Danforth et al., 1962). Recent work by Newbold & Small (1984) confirmed these findings in electrically stimulated sheep muscles. In addition they observed that the phosphorylase a level had peaked and returned to resting levels after 30 s of stimulation and did not increase again despite further stimulation for another 90 s. To explain the increased rates ofglycolysis and high energy phosphate turnover following the cessation of stimulation, Bendall (1976) suggested that damage to the SR or to the mitochondria caused by sustained electrical stimulation may allow increased calcium levels to persist in the sarcoplasm. However, Tume (1979) found no difference in calcium uptake ability of the SR isolated from control and electrically stimulated sheep muscles. This was despite the fact that the stimulated muscles showed a faster rate of pH fall than control muscles following the stimulation (Tume, 1980). Thus, the mechanism for the increased rate of glycolysis following stimulation remains unresolved. Recently, we showed that changes in the activities of phosphorylase kinase and phosphorylase phosphatase induced by pressure treatment of pre-rigor muscles can regulate phosphorylase activity, and explain the increased rates of glycolysis observed during pressure treatments (Horgan & Kuypers, 1983). Since electrical stimulation and pressure treatment show many similarities in their effects on the rates of glycolysis, the present study was undertaken to see if these enzymes play a similar role during and following electrical stimulation. The effects of two electrical stimulation systems, low and high voltage, on phosphorylase, phosphorylase kinase and phosphorylase phosphatase activities and on some properties of the SR were studied in rabbit muscles. MATERIALS A N D M E T H O D S Animals
Adult rabbits (male and female) of 2-3 kg liveweight were used. All animals were stunned by a blow to the head and killed by severing the
228
Douglas J. Horgan, Ronald Kuypers
blood vessels in the neck. The animals were skinned and the head removed before electrical stimulation. Electrical stimulation
Carcasses were hung by the Achilles tendon using insulated hooks. Electrodes were inserted in the Bicepsfemoris muscles and ventral to the Ligamentum nuchae in the neck region. Two systems of stimulation were used, each for 90 s duration.
Low voltage stimulation (L VS) A direct pulse with a frequency of 40 s- 1 and a pulse width of 2 ms was used, with 10 V for the first 10 s followed by 50, 75 and 100 V for 20, 30 and 30 s respectively. This is essentially the same low voltage system as used by Shaw & Walker (1977).
High voltage stimulation (HVS) Alternating half sinusoidal pulses with a frequency of 14.3 s - l , and a pulse width of 10 ms were used. Each pulse had a peak 500 V (300 V root mean square). Removal and treatment of muscles
In 15 animals the Longissimus dorsi (LD) muscle was removed from one side of the carcass before stimulation, to serve as a control. Immediately following stimulation the remaining LD muscle was removed. The muscles were placed in polyethylene bags and subsequently held at 25 °C in a water bath. Eleven animals were stimulated with the carcass intact. These muscles were also treated as above with control LD muscles being removed from unstimulated carcasses. Measurement of pH
About l g of muscle was homogenised in l 0 ml of neutral 5 mM iodoacetic acid in a Buhler homogeniser. The pH of the homogenate was determined at 25 °C using a combined electrode. Muscle extracts
These were prepared as reported previously (Horgan & Kuypers, 1983).
Electrical stimulation of glycolysis in rabbit muscle
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Enzyme assays
Phosphorylase activities were assayed by the method of Hedrick & Fischer (1965). Phosphorylase kinase was assayed by the method of Cohen (1973), and phosphorylase phosphatase by the procedure of Brandt et al. (1975). Sarcoplasmic reticulum preparation and ATPase activities
The 8000-28 000 g fraction was prepared from control and stimulated 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. ATPase activities were measured at pH 7-0 and 25 °C by a coupled-enzyme spectrophotometric method (Horgan, 1974). Sodium azide (5 mM) was present in order to inhibit any contaminating mitochondrial AT Pase. Protein concentrations
These were determined by the method of Lowry et al. (1951). Statistical treatment of results
Analysis of covariance was used to compare slopes and intercepts of the regression lines obtained for the data. For all other experimental data, significant differences were determined by a paired t-test.
RESULTS Physical response to stimulation
The first step of LVS (10 V) caused the muscles of the carcass to contract markedly with the forelegs extending at right angles to the axis of the carcass. The second step (to 50 V at 10 s) caused further contractions with the forelegs remaining at right angles. However, by 25s fatigue was evidenced by the forelegs starting to sag. The next two steps caused the forelegs to extend further but almost immediately they began to sag, almost reaching the rest position. When the current was finally turned off,
Douglas J. Horgan, Ronald Kuypers
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further relaxation was evident with the forelegs assuming their initial position. During HVS the muscles of the carcasses contracted violently with the forelegs again extending at right angles to the body. During the first 15-20 s of stimulation regular fibrillation of the muscles was observed. After 25 s fatique occurred with the forelegs gradually sagging until at about 60 s they were at only 10 ° or so to the carcass axis. When the power was switched off, the forelegs relaxed completely.
Muscle pH Since no significant differences were found between experiments where one LD muscle was removed before stimulation and those where the carcass was stimulated intact, the pH data from all experiments were pooled. The effects of LVS and HVS on the rate of pH fall in rabbit LD muscle are shown in Fig. 1. Statistical analyses indicated that HVS caused an increase in the rate of fall of pH following stimulation. The HVS slope ( - 0 - 3 9 ) units h - t) was significantly different (P < 0-01) from that of the control ( - 0 - 2 4 units h - t). On the other hand LVS had no effect on the rate of fall of pH following stimulation (slope, - 0 . 2 4 units h - t ) . The 7-00
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intercepts of the LVS and HVS lines are the same, pH 6-47, and are significantly different (P < 0-001) from that of the control, pH 6-76. Thus, both low and high voltage electrical stimulation produced the same pH fall (about 0.3 units) during stimulation but only the HVS produced a faster rate of pH fall (1.6 times) following stimulation. The rates of fall of pH were linear for 240, 180 and 120min for the control, LVS and HVS respectively. These points indicate the times when the muscles of the three groups began to enter rigor. Phosphorylase a activity Phosphorylase a activity is expressed as the ratio of the activity in the absence of AM P (phosphorytase a) to the activity in the presence of I mM AM P (phosphorylase a + b) (Hedrick & Fischer, 1965). Figure 2 shows the phosphorylase a activity in post-mortem LD muscle following LVS and HVS. The phosphorylase a activity was constant from 20-60 minpost mortem for each treatment (results not shown). After 60 min the activity increased in a linear manner. Activities determined after 4, 3 and 2 h post mortem for control, LVS, and HVS respectively showed no further increases but remained at the peak levels observed at these times (results not shown). The slopes of the line obtained for the stimulated systems 0-15
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(LVS and HVS) were significantly different (P < 0-001) from the control. The slope of the HVS line was significantly different (P < 0.05) from that of the LVS line. Figure 3 shows the results o f a series of paired experiments where one LD was removed as a control before stimulation o f the rest o f the carcass. Phosphorylase a activities were measured as soon as possible after stimulation which was carried out from the shortest possible time after slaughter up to approximately 20 rain post mortem. The results show that the phosphorylase a activities in stimulated muscle are always initially low, whereas in control muscles initial values can be 20~o or more of the total, declining rapidly with time post mortem. Together, Figs 2 and 3 show that immediately following electrical stimulation, phosphorylase a activity is quite low (about 3 ~o of total) and remains at this level until about ! hpos t mortem when the activity starts to increase at a linear rate. The phosphorylase a activities in LVS and HVS muscles increase at faster rates (2.3 times for LVS and 3"7 times for HVS) and reach higher values than the control activities. It is also of interest that the phosphorylase a activity in untreated muscle increases with time post mortem. In all cases the highest level of phosphorylase a activity was reached at the time the muscle began to enter rigor (as determined by pH measurements).
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Fig. 4. Lossof phosphorylase phosphatase activity followingelectrical stimulation. O, non-stimulated control (regression coefficient=0.93); A, low voltage stimulation (regression coefficient= 0-93); W, high voltage stimulation (regression coefficient= 0.93). Each point is the mean of three separate experiments. Bars indicate + SE. Phosphorylase phosphatase activity The activities of phosphorylase phosphatase following LVS and HVS are shown in Fig. 4. The correlation coefficient obtained for each line was 0-93. Analysis showed that the slopes obtained from the LVS and HVS data were not significantly different from that o f the control. The intercepts from the stimulated lines were significantly different from the control (P < 0-001). The results show that regardless o f treatment, phosphorylase phosphatase activity falls in a linear m a n n e r with t i m e p o s t m o r t e m and that electrical stimulation causes an immediate loss o f 50 o f the activity. Comparison o f Figs 4 and 1 reveals m a n y similarities suggesting that fall in pH may be responsible for the loss o f phosphatase activity with time post m o r t e m . This hypothesis was tested by preparing an extract from an untreated LD muscle 10 min after slaughter. Aliquots of the extract were adjusted to a range o f p H from 6-7-5-90, incubated for 10min at 25°C, and then diluted for assay at pH 7-0 in the normal manner. The results (Fig. 5) show that phosphatase activity is indeed lost with decreasing pH. Thus the losses of activity shown in Fig. 4 can be explained by the fall of pH that occurs post m o r t e m and the 50 ~ loss
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Phosphorylase kinase activity The effects of LVS and HVS on phosphorylase kinase activities are shown in Fig. 7. The results are expressed as relative activities because of the large variations found between individual experiments (e.g. in control muscles a range of 0-2-0.8 units g - i muscle was observed in eight experiments. However, the results are consistent in that each experiment showed the same relative effects. Figure 7 shows that the phosphorylase
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kinase activity in control muscles remains constant in post-mortem muscle for the first three hours and then begins to fall rapidly. The loss of activity after three hours is due to acid denaturation o f the enzyme as the pH falls below 6-0. This was demonstrated previously using purified phosphorylase kinase (Horgan & Kuypers, 1983). With all stimulated muscles a peak of activity was observed. The peak was greatest for the HVS muscles and also occurred earlier. The earlier loss of activity in the stimulated muscles is due to the lower pH o f the muscles at these times.
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DISCUSSION The increase in the rate of glycolysis (as measured by the fall in pH) following ES is very small compared to the near maximum rate that occurs during stimulation, i.e. an increase of approximately two-fold (Table 1) compared with up to a 300-fold increase during the first 30 s of stimulation (Bendall et al., 1976). Therefore, any persistent changes in the biochemistry of stimulated muscle would be expected to be relatively small. The rates of pH fall following ES found by various workers are listed in chronological order in Table 1. In general it appears that LVS has a smaller effect than HVS and in some cases no effect at all. Our results for glycolytic rates following stimulation (Fig. 1) are within the range of those in Table 1. The effects of time post m o r t e m on the phosphorylase a activities of both stimulated and non-stimulated muscles can be divided into two parts. The first part from zero time to approximately 30 min post m o r t e m is shown in Fig. 3. The high initial values for unstimulated muscles are sampling effects caused by the irritability of the muscle which disappears after approximately 25 min (Newbold & Small, 1984). Stimulation, both HVS and LVS, removes this sampling effect resulting in low and constant activities of phosphorylase a over this initial period. The increases in the phosphorylase a activities in unstimulated muscles that start 60-90 min post m o r t e m (Fig. 2) may explain some observed peculiarities of the pH vs. time curves. For example, Bendall (1978) found an inflection point in the curve for beef muscle with the rate of pH fall below pH 6-6 being twice that from pH 7.0 to 6-6. We have also observed an inflection point in some experiments where the rate of pH fall in unstimulated rabbit LD muscle doubled after 120min post m o r t e m (results not shown). We have found that phosphorylase kinase from unstimulated muscles, displays, in the absence of calcium ions, 3-5 ~o of its potential activity (results not shown). Thus the conversion of phosphorylase b to phosphorylase a can occur in post-mortem muscle but would at first be reversed by phosphorylase phosphatase. However, after 90 min or so, as the phosphatase activity falls, due to decreasing pH (Figs 4, 5 and 6), the levels ofphosphorylase a would be expected to rise, as long as A T P was available. The faster rates of increase of phosphorylase a activities in stimulated muscles (compared to unstimulated) (Fig. 2) can be explained by a combination of the loss of phosphorylase phosphatase activity (Fig. 4)
Electrical stimulation o f glycolysis in rabbit muscle
239
and the increase in phosphorylase kinase activity in stimulated muscle (Fig. 7). The increase in phosphorylase kinase activity is probably due to limited proteolysis. Cohen (1980) demonstrated that proteolysis of phosphorylase kinase increased its activity 20-fold at saturating levels of calcium ions, and several hundred fold at low calcium levels ( l #M). It has been claimed that there is an increased release of proteolytic enzyme activities from the lysosomal membranes following ES of lamb muscles (Dutson et aL, 1980). The effects of ES on the properties of the SR (Table 2) suggest two possible mechanisms by which increased ATPase activity could persist in post-mortem muscle, following ES. First, the reduced yield of SR from stimulated muscle may cause an increase in cytoplasmic calcium concentrations and so activate a number of calcium-dependent ATPases (e.g. myofibrillar, SR, mitochondrial). This possibility is supported by the observation (P. V. Harris, personal communication) that HVS (800 V, 14"3 pulse s-1) of beef sides causes a 20-25 ~ shortening in several muscles compared to corresponding controls in unstimulated sides. Attempts were made in this study to measure the effect of ES on length changes in rabbit psoas muscles but they gave inconsistent results probably due to the small size of the muscles and the rapid onset of rigor. However, the reduced yield of SR may be due only to a difference in the separability of the SR from stimulated muscle compared to untreated muscle. In this case the increase in the basal ATPase activity of the SR from HVS muscles (Table 2) may account for the increased rate of glycolysis (Fig. 1). However, although the basal ATPase activity of SR from LVS muscles is not significantly increased it is also not significantly different to that from HVS muscles. Therefore, the different rates of glycolysis following HVS and LVS cannot be explained on this basis. Also of interest is the effect of ES on the calcium ion permeability of the SR as determined by the enhancement of the extra ATPase by the ionophore A23187. The larger enhancements found for the stimulated muscle SR suggest that it is less leaky to accumulated calcium. Tume (1979) and Joseph et al. (1980) came to the same conclusion by measuring calcium uptake activities in stimulated and control muscles from sheep and beef respectively. Scopes (1974) using a reconstituted glycolytic system showed that the rate of glycolysis in post-mortem muscle was dependent on the levels of both ATPase and phosphorylase a activities. In line with this, our results show that both HVS and LVS significantly increase (P < 0.001) the
240
Douglas J. Horgan, RonaM Kuypers
phosphorylase a activities through the combined effects of (1) increasing phosphorylase kinase activity, probably due to limited proteolysis, and (2) a 50 ~o loss o f phosphorylase phosphatase activity during stimulation. Although the effect of HVS is slightly greater (P < 0-05) than LVS both treatments should increase glycolysis. As only HVS increases the rate of glycolysis, the supply of A D P , i.e. the A T P a s e activity, must be the rate limiting step. It is unlikely that increases in the basal ATPase activity of the SR could account for the different effects o f HVS and LVS (see above). Therefore it seems that another A T P a s e activity, probably the myofibrillar ATPase, is involved and is responsible for the different rates of glycolysis observed.
ACKNOWLEDGEMENT This work was supported in part by the Australian Meat Research Committee.
REFERENCES Bendall, J. R. (1976). J. Sci. Fd Agric., 27, 819. Bendall, J. R. (1978). Meat Sci., 2, 91. Bendall, J. R., Ketteridge, C. C. & George, A. R. (1976). J. Sci. Fd Agric., 27, !123. Brandt, H., Capulong, Z. L. & Lee, E. Y. C. (1975). J. Biol. Chem., 250, 8038. Brostrom, C. O., Hunkeler, F. L. & Krebs, E. G. (1971). J. Biol. Chem., 246, 1961. Chrystall, B. B. & Devine, C. E. (1978). Meat Sci., 2, 49. Chrystall, B. B. & Devine, C. E. (1983a). Meat Sci., 8, 83. Chrystall, B. B. & Devine, C. E. (1983b). N.Z.J. Agr. Res., 26, 89. Chrystall, B. B., Devine, C. E. & Davey, C. L. (1980). In: Fibrous proteins: scientific, industrial and medical aspects, Vol. 2 (Parry, D. A. D. & Creamer, L. K. (Eds)). Academic Press, London, p. 67. Cohen, P. (1973). Eur. J. Biochem., 34, 1. Cohen, P. (1980). Eur. J. Biochem., !11,563. Danforth, W. H., Helmreich, E. & Cork C. F. (1962). Proc. N.A.S., 48, 1191. Dutson, T. R., Smith, G. C. & Carpenter, Z. L. (1980). J. Fd Sci., 45, 1097. Hallund, O. & Bendall, J. R. (1965). J. Fd Sci., 30, 296. Hedrich, J. L. & Fischer, E. H. (1965). Biochemistry, 4, 1337. Horgan, D. J. (1974). Arch. Biochem. Biophys., 162, 16. Horgan, D. J. & Kuypers, R. (1983). Meat Sci., 8, 65.
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Joseph, A. L., Dutson, T. R. & Carpenter, Z. L. (1980). Proc. 26th European Meat Res. Wkrs Conf., Colorado Springs. Paper J.3. Krebs, E. G., Kent, A. B. & Fischer, E. H. (1958). J. Biol. Chem., 231, 73. Lowry, O. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. (1951). J. Biol. Chem., 193, 265. Martonosi, A., Donley, J. & Halpin, R. A. (1968). J. Biol. Chem., 243, 61. Morton, H. C. & Newbold, R. P. (1982). Meat Sci., 7, 285. Newbold, R. P. & Small, L. M. (1985). Meat Sci., 12, 1. Scopes, R. K. (1974). Biochem. J., 142, 79. Shaw, F. D. & Walker, D. J. (1977). J. Fd Sci., 42, 1140. Tume, R. K. (1979). Aust. J. Biol. Sci., 32, 163. Tume, R. K. (1980). Aust. J. Biol. Sci., 33, 43. Weber, A. (1971). J. Gen. Physiol., 57, 50.
Post-Mortem Glycolysis in Rabbit Longissimus dorsi Muscles Following Electrical Stimulation Douglas J. Horgan & Ronald Kuypers CS[RO Division of Food Research, Meat Research Laboratory, PO Box 12, Cannon Hill, Queensland, Australia 4170 (Received: 30 April, 1984)
S UMMA R Y The effects of both high voltage and low voltage electrical stimulation were studied in rabbit Longissimus dorsi muscles. The rate of fall of pH as well as the activities of phosphorylase a, phosphorylase kinase, and phosphorylase phosphatase were measured. The effects on the yield, A TPase activities, and calcium permeability of the sarcoplasmic reticulum (SR) were also measured. Although onO' high voltage stimulation increased the post-stimulation rate of pH fall, both types of electrical stimulation increased the phosphorvlase a activity, apparently by increasing the activio' of phosphorylase kinase and destroying a large part of the phosphorylase phosphatase activity. Electrical stimulation reduced the yieM of SR and increased its basal A TPase activity. It also increased the ability of the SR to retain accumulated calcium. We conclude that the different rates of pH fall observed following the two types of stimulation are due to the differential effects of these treatments on one of the A TPase activities, probably the myofibrillar A TPase.
INTRODUCTION Electrical stimulation (ES) appears to affect the rate of glycolysis in prerigor muscles in two stages. Firstly, during stimulation, the rate can be increased up to 150-fold resulting in a dramatic fall in pH (Chrystall & Devine, 1978). Secondly, after cessation o f stimulation, glycolysis rates ranging up to almost three times that of unstimulated muscles have been reported by various workers (see Table l). The very rapid rate o f 225 Meat Science 0309-1740/85/$03-30 © Elsevier Applied Science Publishers Ltd, England, 1985. Printed in Great Britain
Douglas J. Horgan, Ronald Kuypers
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glycolysis observed during stimulation can be attributed to the activation of the enzyme glycogen phosphorylase. This is caused by calcium ions released from the SR activating phosphorylase kinase (Brostrom et al., 1971) which in turn activates phosphory!ase by converting the inactive b form to the active phosphorylase a (Krebs et al., 1958; Danforth et al., 1962). Recent work by Newbold & Small (1984) confirmed these findings in electrically stimulated sheep muscles. In addition they observed that the phosphorylase a level had peaked and returned to resting levels after 30 s of stimulation and did not increase again despite further stimulation for another 90 s. To explain the increased rates ofglycolysis and high energy phosphate turnover following the cessation of stimulation, Bendall (1976) suggested that damage to the SR or to the mitochondria caused by sustained electrical stimulation may allow increased calcium levels to persist in the sarcoplasm. However, Tume (1979) found no difference in calcium uptake ability of the SR isolated from control and electrically stimulated sheep muscles. This was despite the fact that the stimulated muscles showed a faster rate of pH fall than control muscles following the stimulation (Tume, 1980). Thus, the mechanism for the increased rate of glycolysis following stimulation remains unresolved. Recently, we showed that changes in the activities of phosphorylase kinase and phosphorylase phosphatase induced by pressure treatment of pre-rigor muscles can regulate phosphorylase activity, and explain the increased rates of glycolysis observed during pressure treatments (Horgan & Kuypers, 1983). Since electrical stimulation and pressure treatment show many similarities in their effects on the rates of glycolysis, the present study was undertaken to see if these enzymes play a similar role during and following electrical stimulation. The effects of two electrical stimulation systems, low and high voltage, on phosphorylase, phosphorylase kinase and phosphorylase phosphatase activities and on some properties of the SR were studied in rabbit muscles. MATERIALS A N D M E T H O D S Animals
Adult rabbits (male and female) of 2-3 kg liveweight were used. All animals were stunned by a blow to the head and killed by severing the
228
Douglas J. Horgan, Ronald Kuypers
blood vessels in the neck. The animals were skinned and the head removed before electrical stimulation. Electrical stimulation
Carcasses were hung by the Achilles tendon using insulated hooks. Electrodes were inserted in the Bicepsfemoris muscles and ventral to the Ligamentum nuchae in the neck region. Two systems of stimulation were used, each for 90 s duration.
Low voltage stimulation (L VS) A direct pulse with a frequency of 40 s- 1 and a pulse width of 2 ms was used, with 10 V for the first 10 s followed by 50, 75 and 100 V for 20, 30 and 30 s respectively. This is essentially the same low voltage system as used by Shaw & Walker (1977).
High voltage stimulation (HVS) Alternating half sinusoidal pulses with a frequency of 14.3 s - l , and a pulse width of 10 ms were used. Each pulse had a peak 500 V (300 V root mean square). Removal and treatment of muscles
In 15 animals the Longissimus dorsi (LD) muscle was removed from one side of the carcass before stimulation, to serve as a control. Immediately following stimulation the remaining LD muscle was removed. The muscles were placed in polyethylene bags and subsequently held at 25 °C in a water bath. Eleven animals were stimulated with the carcass intact. These muscles were also treated as above with control LD muscles being removed from unstimulated carcasses. Measurement of pH
About l g of muscle was homogenised in l 0 ml of neutral 5 mM iodoacetic acid in a Buhler homogeniser. The pH of the homogenate was determined at 25 °C using a combined electrode. Muscle extracts
These were prepared as reported previously (Horgan & Kuypers, 1983).
Electrical stimulation of glycolysis in rabbit muscle
229
Enzyme assays
Phosphorylase activities were assayed by the method of Hedrick & Fischer (1965). Phosphorylase kinase was assayed by the method of Cohen (1973), and phosphorylase phosphatase by the procedure of Brandt et al. (1975). Sarcoplasmic reticulum preparation and ATPase activities
The 8000-28 000 g fraction was prepared from control and stimulated 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. ATPase activities were measured at pH 7-0 and 25 °C by a coupled-enzyme spectrophotometric method (Horgan, 1974). Sodium azide (5 mM) was present in order to inhibit any contaminating mitochondrial AT Pase. Protein concentrations
These were determined by the method of Lowry et al. (1951). Statistical treatment of results
Analysis of covariance was used to compare slopes and intercepts of the regression lines obtained for the data. For all other experimental data, significant differences were determined by a paired t-test.
RESULTS Physical response to stimulation
The first step of LVS (10 V) caused the muscles of the carcass to contract markedly with the forelegs extending at right angles to the axis of the carcass. The second step (to 50 V at 10 s) caused further contractions with the forelegs remaining at right angles. However, by 25s fatigue was evidenced by the forelegs starting to sag. The next two steps caused the forelegs to extend further but almost immediately they began to sag, almost reaching the rest position. When the current was finally turned off,
Douglas J. Horgan, Ronald Kuypers
230
further relaxation was evident with the forelegs assuming their initial position. During HVS the muscles of the carcasses contracted violently with the forelegs again extending at right angles to the body. During the first 15-20 s of stimulation regular fibrillation of the muscles was observed. After 25 s fatique occurred with the forelegs gradually sagging until at about 60 s they were at only 10 ° or so to the carcass axis. When the power was switched off, the forelegs relaxed completely.
Muscle pH Since no significant differences were found between experiments where one LD muscle was removed before stimulation and those where the carcass was stimulated intact, the pH data from all experiments were pooled. The effects of LVS and HVS on the rate of pH fall in rabbit LD muscle are shown in Fig. 1. Statistical analyses indicated that HVS caused an increase in the rate of fall of pH following stimulation. The HVS slope ( - 0 - 3 9 ) units h - t) was significantly different (P < 0-01) from that of the control ( - 0 - 2 4 units h - t). On the other hand LVS had no effect on the rate of fall of pH following stimulation (slope, - 0 . 2 4 units h - t ) . The 7-00
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Electrical stimulation of glycolysis in rabbit muscle
231
intercepts of the LVS and HVS lines are the same, pH 6-47, and are significantly different (P < 0-001) from that of the control, pH 6-76. Thus, both low and high voltage electrical stimulation produced the same pH fall (about 0.3 units) during stimulation but only the HVS produced a faster rate of pH fall (1.6 times) following stimulation. The rates of fall of pH were linear for 240, 180 and 120min for the control, LVS and HVS respectively. These points indicate the times when the muscles of the three groups began to enter rigor. Phosphorylase a activity Phosphorylase a activity is expressed as the ratio of the activity in the absence of AM P (phosphorytase a) to the activity in the presence of I mM AM P (phosphorylase a + b) (Hedrick & Fischer, 1965). Figure 2 shows the phosphorylase a activity in post-mortem LD muscle following LVS and HVS. The phosphorylase a activity was constant from 20-60 minpost mortem for each treatment (results not shown). After 60 min the activity increased in a linear manner. Activities determined after 4, 3 and 2 h post mortem for control, LVS, and HVS respectively showed no further increases but remained at the peak levels observed at these times (results not shown). The slopes of the line obtained for the stimulated systems 0-15
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Douglas J. Horgan, RonaM Kuypers
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(LVS and HVS) were significantly different (P < 0-001) from the control. The slope of the HVS line was significantly different (P < 0.05) from that of the LVS line. Figure 3 shows the results o f a series of paired experiments where one LD was removed as a control before stimulation o f the rest o f the carcass. Phosphorylase a activities were measured as soon as possible after stimulation which was carried out from the shortest possible time after slaughter up to approximately 20 rain post mortem. The results show that the phosphorylase a activities in stimulated muscle are always initially low, whereas in control muscles initial values can be 20~o or more of the total, declining rapidly with time post mortem. Together, Figs 2 and 3 show that immediately following electrical stimulation, phosphorylase a activity is quite low (about 3 ~o of total) and remains at this level until about ! hpos t mortem when the activity starts to increase at a linear rate. The phosphorylase a activities in LVS and HVS muscles increase at faster rates (2.3 times for LVS and 3"7 times for HVS) and reach higher values than the control activities. It is also of interest that the phosphorylase a activity in untreated muscle increases with time post mortem. In all cases the highest level of phosphorylase a activity was reached at the time the muscle began to enter rigor (as determined by pH measurements).
233
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Fig. 4. Lossof phosphorylase phosphatase activity followingelectrical stimulation. O, non-stimulated control (regression coefficient=0.93); A, low voltage stimulation (regression coefficient= 0-93); W, high voltage stimulation (regression coefficient= 0.93). Each point is the mean of three separate experiments. Bars indicate + SE. Phosphorylase phosphatase activity The activities of phosphorylase phosphatase following LVS and HVS are shown in Fig. 4. The correlation coefficient obtained for each line was 0-93. Analysis showed that the slopes obtained from the LVS and HVS data were not significantly different from that o f the control. The intercepts from the stimulated lines were significantly different from the control (P < 0-001). The results show that regardless o f treatment, phosphorylase phosphatase activity falls in a linear m a n n e r with t i m e p o s t m o r t e m and that electrical stimulation causes an immediate loss o f 50 o f the activity. Comparison o f Figs 4 and 1 reveals m a n y similarities suggesting that fall in pH may be responsible for the loss o f phosphatase activity with time post m o r t e m . This hypothesis was tested by preparing an extract from an untreated LD muscle 10 min after slaughter. Aliquots of the extract were adjusted to a range o f p H from 6-7-5-90, incubated for 10min at 25°C, and then diluted for assay at pH 7-0 in the normal manner. The results (Fig. 5) show that phosphatase activity is indeed lost with decreasing pH. Thus the losses of activity shown in Fig. 4 can be explained by the fall of pH that occurs post m o r t e m and the 50 ~ loss
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Phosphorylase kinase activity The effects of LVS and HVS on phosphorylase kinase activities are shown in Fig. 7. The results are expressed as relative activities because of the large variations found between individual experiments (e.g. in control muscles a range of 0-2-0.8 units g - i muscle was observed in eight experiments. However, the results are consistent in that each experiment showed the same relative effects. Figure 7 shows that the phosphorylase
Electrical stimulation o f glycolysis in rabbit muscle
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kinase activity in control muscles remains constant in post-mortem muscle for the first three hours and then begins to fall rapidly. The loss of activity after three hours is due to acid denaturation o f the enzyme as the pH falls below 6-0. This was demonstrated previously using purified phosphorylase kinase (Horgan & Kuypers, 1983). With all stimulated muscles a peak of activity was observed. The peak was greatest for the HVS muscles and also occurred earlier. The earlier loss of activity in the stimulated muscles is due to the lower pH o f the muscles at these times.
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DISCUSSION The increase in the rate of glycolysis (as measured by the fall in pH) following ES is very small compared to the near maximum rate that occurs during stimulation, i.e. an increase of approximately two-fold (Table 1) compared with up to a 300-fold increase during the first 30 s of stimulation (Bendall et al., 1976). Therefore, any persistent changes in the biochemistry of stimulated muscle would be expected to be relatively small. The rates of pH fall following ES found by various workers are listed in chronological order in Table 1. In general it appears that LVS has a smaller effect than HVS and in some cases no effect at all. Our results for glycolytic rates following stimulation (Fig. 1) are within the range of those in Table 1. The effects of time post m o r t e m on the phosphorylase a activities of both stimulated and non-stimulated muscles can be divided into two parts. The first part from zero time to approximately 30 min post m o r t e m is shown in Fig. 3. The high initial values for unstimulated muscles are sampling effects caused by the irritability of the muscle which disappears after approximately 25 min (Newbold & Small, 1984). Stimulation, both HVS and LVS, removes this sampling effect resulting in low and constant activities of phosphorylase a over this initial period. The increases in the phosphorylase a activities in unstimulated muscles that start 60-90 min post m o r t e m (Fig. 2) may explain some observed peculiarities of the pH vs. time curves. For example, Bendall (1978) found an inflection point in the curve for beef muscle with the rate of pH fall below pH 6-6 being twice that from pH 7.0 to 6-6. We have also observed an inflection point in some experiments where the rate of pH fall in unstimulated rabbit LD muscle doubled after 120min post m o r t e m (results not shown). We have found that phosphorylase kinase from unstimulated muscles, displays, in the absence of calcium ions, 3-5 ~o of its potential activity (results not shown). Thus the conversion of phosphorylase b to phosphorylase a can occur in post-mortem muscle but would at first be reversed by phosphorylase phosphatase. However, after 90 min or so, as the phosphatase activity falls, due to decreasing pH (Figs 4, 5 and 6), the levels ofphosphorylase a would be expected to rise, as long as A T P was available. The faster rates of increase of phosphorylase a activities in stimulated muscles (compared to unstimulated) (Fig. 2) can be explained by a combination of the loss of phosphorylase phosphatase activity (Fig. 4)
Electrical stimulation o f glycolysis in rabbit muscle
239
and the increase in phosphorylase kinase activity in stimulated muscle (Fig. 7). The increase in phosphorylase kinase activity is probably due to limited proteolysis. Cohen (1980) demonstrated that proteolysis of phosphorylase kinase increased its activity 20-fold at saturating levels of calcium ions, and several hundred fold at low calcium levels ( l #M). It has been claimed that there is an increased release of proteolytic enzyme activities from the lysosomal membranes following ES of lamb muscles (Dutson et aL, 1980). The effects of ES on the properties of the SR (Table 2) suggest two possible mechanisms by which increased ATPase activity could persist in post-mortem muscle, following ES. First, the reduced yield of SR from stimulated muscle may cause an increase in cytoplasmic calcium concentrations and so activate a number of calcium-dependent ATPases (e.g. myofibrillar, SR, mitochondrial). This possibility is supported by the observation (P. V. Harris, personal communication) that HVS (800 V, 14"3 pulse s-1) of beef sides causes a 20-25 ~ shortening in several muscles compared to corresponding controls in unstimulated sides. Attempts were made in this study to measure the effect of ES on length changes in rabbit psoas muscles but they gave inconsistent results probably due to the small size of the muscles and the rapid onset of rigor. However, the reduced yield of SR may be due only to a difference in the separability of the SR from stimulated muscle compared to untreated muscle. In this case the increase in the basal ATPase activity of the SR from HVS muscles (Table 2) may account for the increased rate of glycolysis (Fig. 1). However, although the basal ATPase activity of SR from LVS muscles is not significantly increased it is also not significantly different to that from HVS muscles. Therefore, the different rates of glycolysis following HVS and LVS cannot be explained on this basis. Also of interest is the effect of ES on the calcium ion permeability of the SR as determined by the enhancement of the extra ATPase by the ionophore A23187. The larger enhancements found for the stimulated muscle SR suggest that it is less leaky to accumulated calcium. Tume (1979) and Joseph et al. (1980) came to the same conclusion by measuring calcium uptake activities in stimulated and control muscles from sheep and beef respectively. Scopes (1974) using a reconstituted glycolytic system showed that the rate of glycolysis in post-mortem muscle was dependent on the levels of both ATPase and phosphorylase a activities. In line with this, our results show that both HVS and LVS significantly increase (P < 0.001) the
240
Douglas J. Horgan, RonaM Kuypers
phosphorylase a activities through the combined effects of (1) increasing phosphorylase kinase activity, probably due to limited proteolysis, and (2) a 50 ~o loss o f phosphorylase phosphatase activity during stimulation. Although the effect of HVS is slightly greater (P < 0-05) than LVS both treatments should increase glycolysis. As only HVS increases the rate of glycolysis, the supply of A D P , i.e. the A T P a s e activity, must be the rate limiting step. It is unlikely that increases in the basal ATPase activity of the SR could account for the different effects o f HVS and LVS (see above). Therefore it seems that another A T P a s e activity, probably the myofibrillar ATPase, is involved and is responsible for the different rates of glycolysis observed.
ACKNOWLEDGEMENT This work was supported in part by the Australian Meat Research Committee.
REFERENCES Bendall, J. R. (1976). J. Sci. Fd Agric., 27, 819. Bendall, J. R. (1978). Meat Sci., 2, 91. Bendall, J. R., Ketteridge, C. C. & George, A. R. (1976). J. Sci. Fd Agric., 27, !123. Brandt, H., Capulong, Z. L. & Lee, E. Y. C. (1975). J. Biol. Chem., 250, 8038. Brostrom, C. O., Hunkeler, F. L. & Krebs, E. G. (1971). J. Biol. Chem., 246, 1961. Chrystall, B. B. & Devine, C. E. (1978). Meat Sci., 2, 49. Chrystall, B. B. & Devine, C. E. (1983a). Meat Sci., 8, 83. Chrystall, B. B. & Devine, C. E. (1983b). N.Z.J. Agr. Res., 26, 89. Chrystall, B. B., Devine, C. E. & Davey, C. L. (1980). In: Fibrous proteins: scientific, industrial and medical aspects, Vol. 2 (Parry, D. A. D. & Creamer, L. K. (Eds)). Academic Press, London, p. 67. Cohen, P. (1973). Eur. J. Biochem., 34, 1. Cohen, P. (1980). Eur. J. Biochem., !11,563. Danforth, W. H., Helmreich, E. & Cork C. F. (1962). Proc. N.A.S., 48, 1191. Dutson, T. R., Smith, G. C. & Carpenter, Z. L. (1980). J. Fd Sci., 45, 1097. Hallund, O. & Bendall, J. R. (1965). J. Fd Sci., 30, 296. Hedrich, J. L. & Fischer, E. H. (1965). Biochemistry, 4, 1337. Horgan, D. J. (1974). Arch. Biochem. Biophys., 162, 16. Horgan, D. J. & Kuypers, R. (1983). Meat Sci., 8, 65.
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Joseph, A. L., Dutson, T. R. & Carpenter, Z. L. (1980). Proc. 26th European Meat Res. Wkrs Conf., Colorado Springs. Paper J.3. Krebs, E. G., Kent, A. B. & Fischer, E. H. (1958). J. Biol. Chem., 231, 73. Lowry, O. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. (1951). J. Biol. Chem., 193, 265. Martonosi, A., Donley, J. & Halpin, R. A. (1968). J. Biol. Chem., 243, 61. Morton, H. C. & Newbold, R. P. (1982). Meat Sci., 7, 285. Newbold, R. P. & Small, L. M. (1985). Meat Sci., 12, 1. Scopes, R. K. (1974). Biochem. J., 142, 79. Shaw, F. D. & Walker, D. J. (1977). J. Fd Sci., 42, 1140. Tume, R. K. (1979). Aust. J. Biol. Sci., 32, 163. Tume, R. K. (1980). Aust. J. Biol. Sci., 33, 43. Weber, A. (1971). J. Gen. Physiol., 57, 50.