J. Anim Sci. 2007. 85:2670-2681. doi:10.2527/jas.2007-0164
© 2007 American Society of Animal Science
Effect of postmortem storage on activity of µ- and m-calpain in five bovine muscles1
J. P. Camou2,
J. A. Marchello,
V. F. Thompson,
S. W. Mares and
D. E. Goll3
Muscle Biology Group, University of Arizona, Tucson 85721
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Abstract
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An in situ system involving incubation of 60- to 80-g pieces of muscle at 4°C under different conditions was used to determine the effects of time of postmortem storage, of pH, and of temperature on activities of µ- and m-calpain activity in bovine skeletal muscle. Casein zymograms were used to allow measurement of calpain activity with a minimum of sample preparation and to ensure that the calpains were not exposed to ionic strengths of 100 or greater before assay of their activities. In 4 of the 5 muscles (longissimus dorsi, lumbar; longissimus dorsi, thoracic; psoas major; semimembranosus; and triceps brachii) studied, µ-calpain activity decreased nearly to zero within 48 h postmortem. Activity of m-calpain also decreased in the in situ system used but at a much slower rate. Activities of both µ- and m-calpain decreased more slowly in the triceps brachii muscle than in the other 4 muscles during postmortem storage. Although previous studies have indicated that µ-calpain but not m-calpain is proteolytically active at pH 5.8, these studies have used calpains obtained from muscle at death. Both µ- and m-calpain are proteolytically inactive if their activities are measured at pH 5.8 and after incubating the muscle pieces for 24 h at pH 5.8. Western analysis suggested that neither the large 80-kDa subunit nor the small 28-kDa subunit of m-calpain was autolyzed during postmortem storage of the muscle pieces. As has been reported previously, the 80-kDa subunit of µ-calpain was autolyzed to 78- and then to a 76-kDa polypeptide after 7 d postmortem, but the 28-kDa small subunit was not autolyzed; hence, the autolyzed µ-calpain molecule in postmortem muscle is a 76-/28-kDa molecule and not a 76-/18-kDa molecule as previously assumed. Because both subunits were present in the postmortem calpains, loss of µ-calpain activity during postmortem storage is not due to dissociation of the 2 subunits and inactivation. Although previous studies have shown that the 76-/18-kDa µ-calpain molecule is completely active proteolytically, it is possible that the 76-/28-kDa µ-calpain molecule in postmortem muscle is proteolytically inactive and that this accounts for the loss of µ-calpain activity during postmortem storage. Because neither µ- nor m-calpain is proteolytically active at pH 5.8 after being incubated at pH 5.8 for 24 h, other proteolytic systems such as the caspases may contribute to postmortem proteolysis in addition to the calpains.
Key Words: µ-calpain • m-calpain • meat tenderness • zymogram • postmortem muscle
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INTRODUCTION
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Considerable evidence accumulated during the past 15 to 20 yr indicates that proteolytic degradation of cytoskeletal proteins by the Ca2+-dependent protease (calpain) system has an important role in postmortem tenderization (Koohmaraie, 1988
, 1992, 1996; Goll et al., 1992, 1998). High levels of calpastatin, the specific inhibitor of µ- and m-calpain, the 2 well-characterized proteases in the calpain system, are related to increased toughness. Because calpastatin inhibits both µ- and m-calpain, it remains unclear whether one or the other or both calpains are active in postmortem muscle. Some results have suggested that µ-calpain is responsible for postmortem proteolysis with very little contribution from m-calpain (Koohmaraie, 1996; Geesink and Koohmaraie, 1999a,b). This conclusion seems based, at least partly, on finding that skeletal muscle m-calpain is not autolyzed during postmortem storage (Boehm et al., 1998; Veiseth et al., 2001) and the assumption that unautolyzed m-calpain is not proteolytically active.
Despite earlier hypotheses to the contrary, however, studies have now shown that both unautolyzed µ- and unautolyzed m-calpain are proteolytically active (Cottin et al., 2001; Goll et al., 2003). Complicating the situation is that proteolytic activity of µ-calpain, when measured in in vitro assays, decreases rapidly during postmortem storage, whereas proteolytic activity of m-calpain decreases only slightly. Although the decreased activity of µ-calpain isolated from postmortem muscle has been suggested to be an artifact induced during isolation and assay, it remains unclear whether µ-calpain is capable of significant proteolytic activity in situ after 24 to 48 h postmortem.
We have used zymography, which allows assay of proteolytic activity with a minimum amount of experimental manipulation, to estimate activity of µ- and m-calpain in 60- to 80-g pieces of 5 different bovine skeletal muscles after storage at selected pH values and times.
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MATERIALS AND METHODS
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Materials
Acrylamide (99.9%) was from ICN Biomedicals Inc. (Aurora, OH); bisacrylamide (99.99%) was from Swartz/ Mann Biotech (Cleveland, OH); SDS (99%) was from BioRad (Hercules, CA); Tris (ultrapure, 99.8%) and 2-(N-morpholino)ethane sulfonic acid (MES), free acid, ultrapure, were from Mallinckrodt Baker (Phillipsburg, NJ); EDTA (free acid, 99%) was from EMD Biosciences (Gibbstown, NJ); 2-mercaptoethanol (MCE), fluorescein isothiocyanate (FITC), and the protease inhibitors (except for E-64) used in calpain-homogenizing buffers (Edmunds et al., 1991; Thompson and Goll, 2000) were from Sigma Chemical (St. Louis, MO); E-64 was from Peptides International (Louisville, KY); and BODIPY-FL was from Invitrogen Corp. (D-2184, Carlsbad, CA). Hammersten casein was purchased from US Biochemical Corp. (Cleveland, OH).
A monoclonal antibody for µ-calpain was from Affinity BioReagents (9A4H8D3, Golden, CO), a monoclonal antibody for the large subunit of m-calpain (C3989) was from Sigma, and a monoclonal antibody for the small subunit common to both calpains was from Calbiochem (208730, San Diego, CA). The secondary antibody for the µ-calpain and small subunit antibodies was a goat anti-mouse immunoglobulin G coupled to horseradish peroxidase from Calbiochem (La Jolla, CA). The secondary antibody for the m-calpain antibody was a goat anti-rabbit immunoglobulin G coupled to horseradish peroxidase from American Qualex (San Clemente, CA).
All other chemicals used were analytical reagent grade or purer. Unless indicated otherwise, all protein isolations and purifications were done at 4°C using pre-cooled instruments and solutions, and all experiments used doubly deionized water that had been passed through a filter to remove organic material and then through a 0.45-µm filter.
Muscle Sampling and Experimental Design
Use of animal tissues in these studies was approved by the University of Arizona Institutional Animal Care and Use Committee. All muscle samples were removed within 30 to 45 min after exsanguination, immediately placed on ice, and transported to the laboratory. None of the muscle samples in these experiments were frozen, and all postmortem times for each animal were assayed before a second animal was sampled. Because of the small sample sizes, the muscle undoubtedly cooled more rapidly than if it had remained on the carcass. That the different buffers used in the soaking solutions had marked effects on calpain activity in these tissues demonstrates that the buffers penetrated through the sample already during the first 24 h. Three kinds of studies were done, as described in the subsequent sections.
Effect of Postmortem Storage Time on Calpain Activity in 5 Different Muscles.
Seven animals were used in these experiments. A 60- to 80-g muscle sample was removed from each of 5 muscles: longissimus dorsi, thoracic (10th to 12th rib); longissimus dorsi, lumbar; semimembranosus; triceps brachii; and psoas major. Immediately after arrival in the laboratory, a 2-g sample was removed for processing (the at-death sample). The remainder of the 60- to 80-g sample was placed in a zippered moisture-proof bag with 5 mL of 0.1 M NaN3 to prevent drying and was stored at 4°C. Two-gram pieces were then taken from each muscle sample after 7, 24, 31, 48, 72, 96, 120, and 144 h. The 2-g samples were diced into small pieces with a scalpel. The diced samples were homogenized in 
6 vol (wt/wt) of 20 mM Tris-HCl, pH 7.5; 5 mM EDTA; 0.1% MCE; 1 mM Pefabloc; and 2.5 µM E-64 by using a Brinkmann Polytron homogenizer at 25,000 rpm for 20 s followed by a 30-s pause and then a second 20-s homogenization. The homogenate was centrifuged at 10,000 x gmax for 20 min, and the supernatant was filtered through glass wool and then used for zymogram assay of calpain activity. The pH of the homogenates from postmortem muscle was approximately 6.5 to 6.6, so the buffering capacity of the 6 volumes of 20 mM Tris-HCl was sufficient to maintain the pH at a level at which the calpains were extracted.
Effect of Temperature and pH on Calpain Activity During Postmortem Storage.
Sixty-eighty grams of diaphragm muscle were removed and taken to the laboratory as described before. The 60- to 80-g samples were divided into small pieces of approximately 3 x 1 x 0.5 cm by slicing with a scalpel, and the small pieces were placed in 250-mL beakers containing 200 mL of a soaking solution consisting of 50 mM Tris-MES, pH 7.5, 7.0, 6.5, or 5.8; 2 mM ethylene glycol tetraacetic acid (EGTA); and 5 mM NaN3. The beakers were covered with parafilm and stored at 4°C. Combinations of Tris-MES were used to prepare buffers of different pH values but all having the same cations and anions. Buffers were made by adjusting a 0.5 M stock solution of Tris to the desired pH by adding MES except for the pH 5.8 buffer, in which a stock solution of 0.25 M Tris was titrated to pH 5.8 by adding 0.5 M MES. The stock solutions were then diluted to a final concentration of 50 mM Tris in the assay (hence, the concentration of MES varied depending on the pH). In the experiments using the BODIPY assay, activities were monitored over a 30-min period rather than using a single time-point measurement. In some experiments that used the semimembranosus muscle, the EGTA was omitted or was replaced with 225 or 450 µM leupeptin or 10 mM NaF, an inhibitor of the glycolytic pathway (Jerez et al., 2003) and of phosphatases (Jaumont and Hancock, 2001) that does not affect calpain activity (Thompson and Mares, personal communication). A 2-g sample was removed immediately (0-time sample) before placing the small muscle slices in beakers containing the specified pH. Two-gram samples (weighed after blotting on a paper towel to remove excess moisture) were then removed at 24, 48, and 120 h. The samples were homogenized as described before for the postmortem storage experiments, except that 50 mM Tris-MES, pH 7.5, 7.0, 6.5, or 5.8, depending on the pH of the soaking solution used in that experiment, and 2 mM EGTA was used as the homogenizing solution. The supernatants were used for zymogram assay of calpain activity; the zymograms were initially electrophoresed at pH 8.8, but then incubation with Ca2+ was done at pH 7.5, 7.0, 6.5, or 5.8, depending on the experiment, and at 22 to 24°C. Seven animals were used in these experiments. The small thickness of the slices (0.5 mm) allowed rapid penetration of the incubation buffer into the fibers, as documented by the effects that the different buffers had on calpain activity (see results section). The calpains seem to be absorbed to subcellular structures in muscle (Reville et al., 1976), and autolyzed µ-calpain is absorbed to myofibrils in postmortem muscle (Delgado et al., 2001), so calpains are not lost during soaking. That activity of both µ- and m-calpain remained constant for 120 h when the muscles were soaked in EGTA-containing buffers confirms that no calpain activity was lost from the muscle samples during soaking. Most of these experiments used diaphragm muscle, but similar results were obtained when using semimembranosus muscle, so there was no indication that muscle fiber type had any effect on the results of these experiments, at least within the range of fiber types used in this study.
Effect of Temperature and pH on Proteolytic Activity of µ- and m-Calpain Purified from At-Death Muscle.
Although the ability of µ-calpain purified from at-death muscle to degrade proteins in bovine myofibrils at pH 5.6 and 4°C has been determined (Huff-Lonergan et al., 1996), studies determining the effects of pH and temperature in the ranges that muscle experiences during postmortem storage at 2°C on the proteolytic activities of calpains have been limited. Therefore, the activity of µ- or m-calpain purified from at-death muscle was assayed at all possible combinations of pH values of 5.8, 6.5, 7.0, and 7.5 (Tris-MES buffers) and temperatures of 4, 10, and 25°C to determine the effects of pH and temperature on proteolytic activity of the calpains. The purified µ- or m-calpain was obtained from human placenta or bovine skeletal muscle by using the procedures described by Thompson and Goll (2000a) or Thompson et al. (2002). Assays were done by using the FITC-casein assay (Wolfe et al., 1989) or the BOD-IPY microplate assay (Thompson et al. 2000b). The assays were run for 30 min, as reported in Wolfe et al. (1989) or Thompson et al. (2000b). These experiments used 3 preparations of purified calpains; results using human and bovine calpain were identical.
Zymogram Assays of Calpain Activity
Zymography was done, as described by Arthur and Mykles (2000), using Hammersten casein without FITC labeling. Three to 8 µL of muscle homogenate containing 15 µg of protein were loaded in each well of a 4% polyacrylamide stacking gel; the resolving gel was a 10% polyacrylamide gel (8 x 7-cm [width x height] and 0.75-mm thick). All experiments were standardized at 15-µg loads to allow comparisons among the different treatments used. The E-64 in the sample buffer does not inhibit the calpains in the absence of Ca2+ (Thompson and Goll, 2000a), and because it has no net charge in the pH range from 5.8 to 8.8, it does not enter the gel and so does not interfere with activity of the calpains. The gel was prerun for 30 min, and the sample was then loaded. After electrophoresis for 3 h at 4°C (electrophoresis was done at 125 V and pH 8.8), the gel was removed, rinsed with deionized H2O, and then incubated with shaking for 1 h at room temperature (22 to 24°C) in 50 mM Tris-HCl, pH 7.5 (for those experiments involving different pH values, incubation was done in 37.5 mM Tris-MES, pH 7.5, 7.0, 6.5, or 5.8), 5 mM CaCl2, 0.1% MCE. The Ca2+-containing incubation buffer was replaced, and the gel was incubated for another 1 h with shaking, and the same pH buffer was used for that experiment. The Ca2+-containing buffer was replaced a third time, and the gel was incubated overnight at room temperature and at the selected pH with shaking. Hence, assay of proteolytic activity was done at 22 to 24°C, although the muscle samples were stored at 4°C. The Ca2+-containing buffer was removed, and the gels were fixed and then stained with Coomassie Brilliant Blue R250. Size of the clear areas denoting calpain activity were quantitated by using a UVP Epi Chemi II bioimaging system, and these measurements were used to estimate relative changes in calpain activities for a particular muscle or treatment. Preliminary experiments showed that homogenates of samples taken at death and on d 1 to 4 could be stored for 5 d in an ice bucket with no detectable loss of proteolytic activity. An average activity for µ-calpain as measured in a zymogram assay for the 5 muscles used in this study was 1.98 ± 0.17 integrated optical density (IOD; mean ± the SE measured as IOD units obtained with the UVP Epi Chemi II system) for at-death samples and 2.17 ± 0.14 IOD for the same samples after 144 h. The small SE indicate that all 5 muscles had similar proteolytic activities at death. Therefore, all samples taken from an individual animal were stored until the d-5 sample was taken and then all were assayed in a single zymogram. This allowed a direct analysis of the changes that occurred during postmortem storage.
Other Procedures
Protein concentration was determined by using the Coomassie Brilliant Blue G-250 assay as described by Bradford (1976) and BSA calibrated by Kjeldahl analysis to prepare calibration curves. The FITC casein was made according to Wolfe et al. (1989), and BODIPY-FL casein was prepared as described in Thompson et al. (2000b). Sodium dodecyl sulfate-PAGE was done as described by Laemmli (1970) using mini-gels (Wolfe et al., 1989). Western analysis was done according to Towbin et al. (1979) using a semidry transfer as described by Taylor et al. (1995). Dilutions used for the antibodies were as follows: 1:10,000 for both the primary and secondary antibodies used in Western blotting of the 80-kDa subunits of µ- and m-calpain and 1:2,000 for the primary antibody and 1:50,000 for the secondary antibody used in Western blotting of the small 28-kDa subunit of the calpains. Detection was done by using chemiluminescence.
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RESULTS
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Zymography
Examples of the zymograms obtained for µ- and m-calpain at the different times of postmortem storage are shown in Figure 1. Zymogram assays make it possible to estimate both µ- and m-calpain activity in crude muscle extracts without having to remove calpastatin. Also, as indicated in the materials and methods section, the muscle extracts are not salted out or subjected to column chromatography but are homogenized in 6 vol of a solution that contains no salt, so the 120 to 150 mM salt that exists in skeletal muscle is diluted immediately to 20 to 25 mM. Previous studies have shown that autolysis of the calpains weakens the interaction between the large and the small subunits, so exposure to ionic strengths greater than 300 mM in in vitro assays results in dissociation of the subunits and irreversible loss of 50% of their catalytic activity within 20 min of exposure (Li et al., 2004). Loss of catalytic activity of autolyzed calpains also occurs at 100 mM ionic strength in in vitro experiments but at a much slower rate than at 300 mM, and activity of the autolyzed calpains is stable for some time at ionic strengths below 100 mM (Li et al., 2004). Hence, ionic strength should not be a factor contributing to inactivation of µ-calpain in this study. Proteolytic activity of the unautolyzed calpains is stable for several hours at ionic strengths as high as 1,000 mM (Li et al., 2004).
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Figure 1. Zymogram showing changes in µ- and m-calpain activity in the longissimus dorsi, lumbar muscle during 144 h of postmortem storage at 4°C. The upper row is µ-calpain activity, and the bottom row is m-calpain activity. There was little µ-calpain activity after 48 h of postmortem storage. The faint bands below the µ-calpain band at 24, 31, and 48 h postmortem represent autolyzed µ-calpain activity.
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In addition to separating µ- and m-calpain activity in a single assay, the autolyzed forms of the 2 calpains are also separated in zymograms (Figure 2). Thus, it is possible by using quantitative bioimaging to estimate the activities of autolyzed µ-calpain, unautolyzed µ-calpain, autolyzed m-calpain, and unautolyzed m-calpain in a single assay without removing calpastatin.
Although purified calpain standards can be used to calibrate the bioimaging, making it possible to express zymogram results in units of calpain activity per milligram of protein, the purpose of this study was to measure changes in the activities of µ- and m-calpain and the autolyzed forms of these 2 enzymes during postmortem storage. Therefore, activities at different times of postmortem storage are expressed as percentage of the activity measured for the respective at-death calpain. In experiments measuring the effects of incubating muscle in solutions of different pH values on calpain activity, calpain activity is expressed as IOD obtained from scanning the zymogram gel.
Effect of Postmortem Storage on Calpain Activity
Proteolytic activity of µ-calpain decreases rapidly during postmortem storage, and very little µ-calpain activity (less than 4% of the original at-death activity) can be detected in 4 of the 5 muscles included in this study after 48 h of postmortem storage (Figures 1
, 3). Activity of µ-calpain declined much more slowly during postmortem storage in the triceps brachii muscle than in the other 4 muscles studied (Figure 3). It should be stressed that the assays in Figure 1 to 3 were done at 22 to 24°C and pH 7.5, and calpain activity in situ in skeletal muscle that is 2 to 4°C and pH 5.8 after 24 h postmortem would be substantially lower than that shown in Figure 3. Hence, the results suggest that there would be very little proteolytic activity of µ-calpain in situ in bovine muscle after 48 h postmortem. The zymograms in Figure 1 indicate that there is some autolysis of µ-calpain beginning 24 h postmortem, but there is little proteolytic activity due to either autolyzed or un-autolyzed µ-calpain after 48 h postmortem.
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Figure 3. Changes in the proteolytic activity of µ- and m-calpain in longissimus dorsi, thoracic (LDT); longissimus dorsi, lumbar (LDL); semimembranosus (SM); triceps brachii (TB); and psoas major (PM) bovine muscles during 144 h of postmortem storage at 4°C. Activities are expressed as percentage of the activity in at-death muscle and are means ± SE of 5 gels (animals) for each muscle. The activities at different postmortem times for each muscle were measured from the same gel, so differences in staining or destaining the gels did not influence the numbers.
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Proteolytic activity of m-calpain also decreased during postmortem storage, and only 10 to 20% of m-cal-pain activity remained after 144 h postmortem under the conditions of our study. The decrease in m-calpain activity observed in this study was more extensive and occurred more rapidly postmortem than the decrease that has been reported in previous studies (Vidalenc et al., 1983; Ducastaing et al., 1985; Koohmaraie et al., 1987; Boehm et al., 1998). The reason for this difference is not clear; the small sample size used in the current study undoubtedly resulted in a more rapid decrease in temperature than would have occurred if the muscle had been left on the carcass, but this more rapid decrease in temperature would be expected to preserve calpain activity, not increase its rate of loss. Again, m-calpain activity in the triceps brachii decreased at a much slower rate during postmortem storage than it did in the other 4 muscles used in this study. There is no evidence in the zymograms that any autolysis of m-calpain occurred during the first 144 h postmortem (Figure 1).
Effect of Muscle pH and Assay pH on Calpain Activity
Several previous studies have reported that purified µ-calpain retains significant proteolytic activity at low pH values, 5.8 or less (Koohmaraie et al., 1986; Huff-Lonergan et al., 1996), whereas purified m-calpain has little or no activity at pH values below 6.0 (Maddock et al., 2005, 2006). All of these previous studies used calpains purified from at-death muscle. In some of the studies (Maddock et al., 2005, 2006), the assays evidently were done at ambient temperature. In the other 2 studies, the assays were done at 25°C and also at a single temperature of 5°C (Koohmaraie et al., 1986) or 4°C (Huff-Lonergan et al., 1996); both these latter studies included only µ-calpain. When attempting to ascertain the relative roles of µ- and m-calpain in postmortem proteolytic tenderization, it is important to know the effect of pH on the proteolytic activities of µ-calpain and m-calpain present in the muscle tissue at different times of postmortem storage. Therefore, a set of experiments was done to determine the effects of pH on activities of µ- and m-calpain in muscle at different times of postmortem storage. Diaphragm muscle and zymogram assays were used in these experiments. To achieve designated and definite pH values, muscle pieces were soaked in Tris-MES solutions of differing pH as described in the materials and methods section. Two important features of these experiments were as follows: 1) all soaking solutions contained 2 mM EGTA, which we had found in preliminary studies preserved calpain activity during postmortem storage (see next section); inclusion of EGTA was necessary so the µ-calpain had some activity that could be used to determine the effects of pH on it activity; and 2) the zymograms were done at the same pH as that in which the muscle samples were incubated and were done at room temperature. A pH in the range of 6.5 to 7.5 has only a small effect on the proteolytic activity of µ-calpain and almost no effect on activity of m-calpain during the first 24 h postmortem (Figure 4). Proteolytic activity of µ-calpain decreases to approximately 30 to 60% of its activity in at-death muscle after 120 h when muscle is both stored at pH 7.5, 7.0, or 6.5, and the µ-calpain is then assayed at this same pH (Figure 4). At pH 5.8, however, activity of µ-calpain decreases to nearly zero after 24 h postmortem (actually after storage of an at-death sample for 24 h at the selected pH) and remains nearly zero for 144 h postmortem (Figure 4). Proteolytic activity of m-calpain remains virtually unchanged during 144 h of postmortem storage at pH values in the range of 6.5 to 7.5 (decreases slightly at pH 7.5), but m-calpain in muscle incubated at pH 5.8 has no detectable (at least with the zymogram assay) proteolytic activity if assayed at pH 5.8; this m-calpain activity remains zero during postmortem storage at this pH (Figure 4). Hence, proteolytic activity of m-calpain is affected more than activity of µ-calpain at pH values below 6.5, but even µ-calpain in muscle stored at pH 5.8 for 24 h is proteolytically inactive if it is also assayed at pH 5.8. Because skeletal muscle pH decreases to 5.8 or less in most bovine, ovine, or porcine animals within 24 h postmortem, the results in Figure 4, to the extent that they mimic conditions that occur in situ during postmortem storage, indicate that there would be very little proteolytic activity of either µ- or m-calpain in postmortem skeletal muscle after 24 h (again, recall that these zymogram assays are being done at 22 to 24°C, whereas in situ muscle temperature after 24 h postmortem is likely near 2 to 4°C).
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Figure 4. Effect of pH and time of postmortem storage on proteolytic activity of µ- and m-calpain in bovine diaphragm that had been stored in Tris-morpholineethanes-ulfonic acid (MES) solutions of different pH values for 0, 24, 48, and 120 h at 4°C. Muscles were incubated in the Tris-MES buffers at the pH indicated and then were assayed by using zymography at the same pH. Activities are expressed as integrated optical density (IOD) and are means ± SE for 3 animals.
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Effect of Temperature and pH on Proteolytic Activity of Purified Calpains
To obtain additional information on the relative activities of µ- and m-calpain at different temperatures and pH values, we have used µ- and m-calpain purified from at-death bovine skeletal muscle and the FITC-casein assay, which is more sensitive than the zymogram assay (Wolfe et al., 1989; Thompson et al., 2000b), to measure the activities of µ- and m-calpain at all combinations of pH 5.8, 6.5, 7.0, and 7.5 and temperatures of 5, 15, and 25°C (Figure 5). Under these conditions, there is little difference in the activities of µ- and m-calpain when assayed at pH 5.8 and 15 or 5°C, although purified µ-calpain is approximately 2-fold more active at pH 5.8 than purified m-calpain when both are assayed at 25°C. The relative activities of µ- and m-cal-pain at pH 5.8 when assayed at 25°C likely accounts for previous reports indicating that µ-calpain is more active than m-calpain at low pH values (Koohmaraie et al., 1986, 1987; Maddock et al., 2005, 2006). Notably, activities of both at-death µ- and m-calpain when assayed at pH 5.8 and 5°C are only 5 to 7% as high as their activities when assayed at pH 7.5 and 25°C (Figure 5).
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Figure 5. Effect of temperature and pH on the proteolytic activity of purified µ- and m-calpain. Either µ- or m-calpain purified from bovine diaphragm muscle were assayed at the temperatures indicated and in Trismor-pholineethanesulfonic acid buffers at the pH values indicated as described in the materials and methods section. Proteolytic assays were done by using the fluorescein isothiocyanate (FITC)-casein assay (Wolfe et al., 1989). Calpain activity is given in fluorescence units (FU) in the FITC-casein assay. Numbers are means ± SE of 3 experiments.
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Effect of EGTA and Leupeptin on µ-Calpain Activity
It is unclear why µ-calpain loses proteolytic activity so rapidly during postmortem storage; Western analysis indicates that the 80-kDa subunit of µ-calpain is autolyzed to a 76/78-kDa form during the first 7 d of postmortem storage (Boehm et al., 1998), but numerous studies have shown that the 76/78-kDa autolyzed form of µ-calpain has the same specific proteolytic activity as the intact unautolyzed form (Edmunds et al., 1991). Therefore, several experiments were done to learn whether the proteolytic activity of µ-calpain could be retained during postmortem storage. We learned that including a Ca2+ chelator such as EDTA or EGTA in the incubation-soaking solution significantly slowed loss of µ-calpain activity and resulted in m-calpain activity remaining nearly constant up to 5 d postmortem (Figure 6, 7). It was not clear, however, whether Ca2+ was having some unusual, direct effect on the µ-calpain molecule or whether preventing proteolytic activity of µ-calpain and hence also preventing its autolysis to a 76/78-kDa form was preserving its activity. Therefore, leupeptin, a reversible inhibitor of the calpains, was included in the incubation-soaking solution in the absence of a Ca2+ chelator. Leupeptin decreases the rate of loss of µ-calpain activity (Figure 1, 7). Hence, loss of the proteolytic activity of µ-calpain during postmortem storage seems to be associated with its autolysis to a 78-/76-kDa form, a form that normally is completely active proteolytically.
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Figure 6. Zymogram showing the effects of including ethylene glycol tetraacetic acid (EGTA) or leupeptin in the Tris·MES soaking solution on proteolytic activity of µ- and m-calpain during storage at 4°C for 0, 24, 48, or 120 h. The control samples were incubated in Tris-morpholineethanesulfonic acid buffer at pH 7.0, and µ-calpain loses its proteolytic activity within 48 h of storage. Inclusion of EGTA or leupeptin, however, prevents loss of activity. Notice that EGTA and leupeptin also prevent autolysis of the µ-calpain. Muscle samples were from bovine semimembranosus muscle.
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Figure 7. Effect of ethylene glycolbis (2-aminoethylether)-N-N-N-N-tetraacetic acid (EGTA) or leupeptin on proteolytic activity of µ-calpain at different times of postmortem storage. Bovine semimembranosus muscle was soaked at 2 to 4°C for the times indicated in a solution containing Trismorpholineethanesulfonic acid (MES) at pH 7.0 and either 2 mM EGTA or 225 µM leupeptin. The control samples were soaked in Tris-MES alone. Activities are expressed as integrated optical density (IOD) obtained by scanning zymogram gels. Numbers are means ± SE for 4 animals. Neither EGTA nor leupeptin had any large effect on changes in m-calpain activity during postmortem storage but significantly stabilized µ-calpain activity during the postmortem times tested.
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Western analysis of the calpains in the samples that had been soaked in EGTA showed that the 80-kDa subunit of µ-calpain was not autolyzed to any appreciable extent in those samples even after 120 h of soaking (Figure 8, E in Ba). The µ-calpain in samples soaked in leupeptin autolyzed much more slowly and to a lesser extent than in samples soaked in buffer alone (Figure 8, compare C, E, and L in Ba). In those samples soaked in buffer alone, however, the 80-kDa subunit of µ-calpain was autolyzed to a mixture of 78- and 76-kDa forms within 24 h postmortem and then was nearly completely autolyzed to a 76-kDa form within 120 h postmortem (Figure 8, Aa). Hence, autolysis of the 80-kDa subunit of µ-calpain parallels the loss of proteolytic activity of this calpain during postmortem storage. Autolysis of the 80-kDa subunit of m-calpain cannot be detected in mini-gels (Figure 8, C in Bb), because the 78-kDa autolyzed form and the 80-kDa unautolyzed form of this calpain migrate very closely in SDS-PAGE (Goll et al., 2003). Autolysis of the 28-kDa small subunit of the calpains during postmortem storage has not been studied, and it evidently has been assumed that autolysis of this subunit would parallel autolysis of the large subunit as it does when autolysis is done in vitro (Edmunds et al., 1991). The 28-kDa subunit of either µ- or m-calpain is not autolyzed in the samples incubated in EGTA or leupeptin even after 120 h of postmortem storage (Figure 8, E and L in Bc). To our surprise, there also is very little autolysis of the 28-kDa subunit in the control samples, although small amounts of the autolyzed 18-kDa fragment can be detected in the control samples incubated for 24, 48, or 120 h. Interestingly, the amount of autolysis of the 28-kDa subunit does not seem to increase after 24 h of postmortem storage (Figure 8, C in Bc). Hence, the autolyzed µ-calpain in those samples incubated in buffer alone is primarily a 76-/28-kDa molecule, not a 76-/18-kDa molecule as it would be if autolyzed in vitro. Inclusion of fluoride, an inhibitor of glycolysis and of phosphatases, in the incubation solutions had no effect on either the loss of activity or the autolysis of µ-calpain (results not shown here).
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Figure 8. Western blots of the large, 80-kDa, and small, 28-kDa, subunits of µ- and m-calpain in diaphragm muscle after soaking in different solutions during postmortem storage. The general protocol was to slice 60- to 80-g samples of diaphragm muscle that had been removed within 45 min after exsanguination into 3 x 1 x 0.5-cm pieces and place the pieces into beakers containing 200 mL of 1 of the following solutions: C. control sample: 120 mM KCl, 50 mM Tris-morpholineethanesulfonic acid (MES), pH 7.0; E. ethylene glycol tetraacetic acid (EGTA) sample: 120 mM KCl, 50 mM Tris-MES (pH 7.0), and 7.5 mM EGTA; L. Leupeptin sample: 120 mM KCl, 50 mM Tris-MES (pH 7.0), and 450 µM leupeptin. Incubations were at 2°C and for 0, 24, 48, or 120 h as indicated. After the designated time, a 2-g sample was removed and was homogenized as described in the materials and methods section. The supernatants were applied to a 1.0 x 3.5 cm DEAE-TSK column to remove a polypeptide that was labeled by all the secondary antibodies that we used. After flushing with 20 mM Tris-HCl (pH 7.5), 1 mM EDTA, and 0.1% 2-mercaptoethanol to remove the interfering polypeptide, the calpains were batch-eluted from the DEAE-TSK columns by using 500 mM KCl in the buffer used for that experiment. The eluted calpains were concentrated 6-fold by using Amicon concentrators, and the concentrated eluate was subjected to Western analysis. For all blots, µ and aµ = purified µ-calpain and purified autolyzed µ-calpain, respectively, and m and am = purified m-calpain and purified autolyzed m-calpain, respectively. Numbers on the sides of the blots are approximate molecular weights of polypeptides that have migrated that far. A. Western blots of the 80-kDa subunit of µ-calpain (Aa) and of the 28-kDa small subunit common to µ- and m-calpain of samples that had been soaked in control buffers for the times indicated. The blots shown in Aa and Ab are from the same samples but probed with an antibody to the 80-kDa subunit of µ-calpain (Aa) or an antibody to the 28-kDa subunit (Ab). Thus, the 80-kDa subunit of µ-calpain is completely autolyzed to a 76-kDa subunit in the 120-h sample, but the 28-kDa subunit in the same sample is not autolyzed. B. Western blots of the 80-kDa subunit of µ-calpain (Ba), the 80-kDa subunit of m-calpain (Bb), and the 28-kDa subunit (Bc) common to both µ- and m-calpain after soaking for the times indicated in either EGTA or leupeptin as indicated. For comparison, samples of µ-calpain, autolyzed µ-calpain, m-calpain, autolyzed m-calpain, and a control sample taken at 0 time (C0) are included. Blots in panels Ba, Bb, and Bc are from corresponding samples; for example, the E 48-h sample in Ba is the same sample as the E 48-h sample in Bb except probed with an antibody to 80-Da subunit of m-calpain and is the same as the E 48-h sample in Bc except probed with an antibody to the 28-kDa subunit. Hence, the completely autolyzed µ-80-kDa of the C120 sample (lane 6 in panel Ba) is from the same sample as the unautolyzed small subunit of the C120 sample in lane 6, panel Bc.
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DISCUSSION
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The results of this study are puzzling in several aspects. µ-Calpain loses most of its proteolytic activity within the first 48 to 72 h of postmortem storage even when it is assayed directly in muscle homogenates under conditions that prevent its exposure to ionic strengths greater than 150 mM; exposure to ionic strengths of 300 mM or greater have been reported to cause loss of catalytic activity of autolyzed µ-calpain (Geesink and Koohmaraie, 1999a,b, 2000). Moreover, assays in this study were done at 25°C, so proteolytic activity of µ-calpain in postmortem bovine muscle in situ where the temperature is 2 to 4°C would be even less than reported in this study. The results make it difficult to understand how µ-calpain could be responsible for postmortem proteolysis, at least beyond 2 to 3 d postmortem.
m-Calpain retains much of its proteolytic activity up to 6 d postmortem when assayed at 25°C and pH 7.5 but has virtually no activity when assayed at pH 5.8, the pH of postmortem muscle, regardless of temperature of the assay and the time postmortem. µ-Calpain retains a small amount of proteolytic activity when assayed at pH 5.8 during the first 24 h postmortem but then also has no activity when assayed at pH 5.8 after 24 h of storage at pH 5.8. Exposure to pH 5.8 would not occur until 12 to 24 h postmortem in situ, but after 12 to 24 h, pH 5.8 would likely have the same effect on µ-calpain activity as if the exposure had begun at death.
Together, these 2 results suggest that neither µ- nor m-calpain would be active proteolytically in bovine muscle after 24 h postmortem despite the wealth of evidence accumulated over the past 30 yr indicating that these 2 proteases are responsible for most of the proteolytic degradation of cytoskeletal proteins during postmortem storage. Some of the most compelling evidence indicating that µ- and possibly m-calpain are involved in postmortem proteolytic tenderization includes the following findings. Transgenic mice overex-pressing calpastatin, the specific inhibitor of the calpains, lost only 1, 3, and 17 % of their desmin, a calpain substrate, after 1, 3, and 7 d of postmortem storage, whereas control mice lost 6, 78, and 91 % of their at-death desmin after the same times of postmortem storage (Kent et al., 2004). Calpastatin inhibits both µ-and m-calpain, so the Kent et al. (2004) study does not provide any evidence as to whether µ- or m-calpain, or both, are involved, but it strongly suggests that the calpains are involved. Injection of synthetic or fungal inhibitors that inhibit the calpains but do not inhibit various cathepsins into muscle in situ inhibited degradation of titin and troponin T, whereas injection of an inhibitor of cathepsin B and L did not affect rate of postmortem tenderization or degradation of titin and troponin T (Uytterhaegen et al., 1994). Very little degradation of myosin or actin occurs during postmortem storage at 2 to 4°C for periods up to 7 to 10 d (Bandman and Zdanis, 1988). The cathepsins degrade both actin and myosin rapidly, but the calpains are unique in that they do not degrade undenatured actin and degrade myosin only very slowly and to a limited degree (S. W. Mares, V. F. Thompson, G. Beinbrech, and D. E. Goll, University of Arizona, unpublished results). Hence, the cathepsins are not responsible to any significant extent for postmortem tenderization. The proteasome requires ATP, which is absent in postmortem muscle, for ubiquitination of substrates, so it is unlikely that the proteasome is active in postmortem muscle.
Although the in situ assays used in the experiments described in this paper do not mimic exactly the conditions that occur in situ during postmortem storage, the loss of µ-calpain activity during the first 48 to 72 h postmortem is similar to that observed in previous studies in which µ-calpain activity was assayed after extraction from muscle at different times postmortem (Vidalenc et al., 1983; Ducastaing et al., 1985; Koohmaraie et al., 1987; Boehm et al., 1998; Kanawa et al., 2002). That prevention of µ-calpain autolysis in the current study also prevented loss of its proteolytic activity may suggest that autolysis of µ-calpain so weakens the interaction between the 2 calpain subunits that they dissociate even at the low ionic strengths to which they were exposed before zymography assay. The evidence accumulated thus far indicates that, depending on the conditions of postmortem storage, µ-calpain autolysis begins as soon as 24 to 48 h postmortem and that µ-calpain in bovine muscle may be nearly completely in the autolyzed state after 6 to 7 d postmortem (Boehm et al., 1998). Although it could be suggested that the autolyzed µ-calpain is so unstable that its 2 subunits dissociate, and proteolytic activity is lost even when exposed to ionic strengths of 150 mM or less in vitro, the results of the Western analyses presented in this manuscript show that the small 28-kDa subunit remains associated with the 80-kDa subunit even after storage of muscle samples in 120 mM KCl, followed by homogenization and DEAE ion-exchange chromatography. Hence, the 2 subunits of µ-calpain remain together during postmortem storage and salt-induced dissociation of the calpain subunits does not explain why µ-calpain loses its proteolytic activity during postmortem storage. The small subunit of µ-calpain is not autolyzed during postmortem storage, and the N-terminal 91 AA in the small subunit that are not removed because this subunit is not autolyzed may provide additional contacts between the small and large subunits and prevent their dissociation in postmortem muscle. That the small 28-kDa subunit is not autolyzed also raises the possibility that the 76-/28-kDa calpain molecule is proteolytically inactive and differs from the 76-/18-kDa calpain molecule that retains complete activity. Purification or partial purification of proteolytically inactive µ-calpain from postmortem muscle and characterization of its properties will be needed to answer this question definitively.
On the other hand, m-calpain in bovine skeletal muscle does not seem to be autolyzed after 7 d postmortem (Boehm et al., 1998; and this paper), which may explain why m-calpain retains its proteolytic activity for some time during postmortem storage. However, whether m-calpain is or can be proteolytically active in postmortem muscle depends on the Ca2+ concentration in this muscle, a property that has not been carefully determined, or on information showing that m-calpain in postmortem muscle is activated by some mechanism that reduces its Ca2+ requirement below 100 µM. The results of the current study showing that m-calpain has almost no proteolytic activity at pH 5.8, the pH of mammalian muscle after 24 h postmortem, also argues strongly against an important role of m-calpain in postmortem tenderization.
In conclusion, the results from this study suggest that additional information on the properties of the calpains in postmortem muscle is needed before their role in postmortem proteolysis can be understood. It is also possible that alternatives to µ- and m-calpain should be sought as an explanation for some of the proteolytic degradation that occurs in postmortem muscle. A recent report has described proteolysis of the calpain-sensitive proteins, nebulin, metavinculin, vinculin, dystrophin, desmin, and troponin T, in the hind-limb muscle of mice that have had the µ-calpain gene disrupted, so the animal contained no or very little µ-calpain (Geesink et al., 2006). After 3 d of postmortem storage, only 52.6% of the at-death dystrophin, 79.2% of the at-death vinculin, and 87.4% of the at-death desmin remained in the hind-limb muscle from the knockout mice. Clearly, postmortem degradation of these 3 calpain-sensitive proteins could not have been caused by µ-calpain. On the other hand, 109.2 and 99.9% of at-death metavinculin and troponin T, respectively, remained in the hind-limb muscles from the knockout mice after 3 d of postmortem storage, whereas only 0.8 and 83.8% of these 2 proteins remained after 3 d postmortem in the hind-limb muscles from the control mice. Interpreted in the simplest manner, these results suggest that µ-calpain has a crucial role in postmortem degradation of some but not all cytoskeletal proteins in muscle during postmortem storage, and as suggested by the results of the present manuscript, attention should be directed toward the possible roles of other proteolytic enzymes in postmortem degradation of cytoskeletal proteins. Although several studies have reported that the skeletal-muscle calpain, calpain-3, has a role in proteolytic degradation in postmortem muscle (Ilian et al., 2001a,b), careful studies by several investigators have found no evidence that calpain-3 has any role in postmortem proteolysis (Parr et al., 1999; Geesink et al., 2005). Given the known properties of cal-pain-3, it seems very unlikely that it is active in postmortem muscle. Recent studies have indicated that the caspases, which have been thought to be activated only during apoptosis, may also be active in living (and postmortem?) cells (Belizário et al., 2001; Schwerk and Schultze-Osthoff, 2003). Moreover, a recent report found a highly significant relationship (r = –0.75) between amount of a caspase degradation product of spectrin (indicative of caspase activity) and shear force of porcine LM (Kemp et al., 2006). It would be interesting to learn whether the caspases degrade cytoskeletal proteins and, if so, whether their degradation of cytoskeletal proteins resembles the degradation observed in postmortem muscle. It should be stressed that the evidence reported in this manuscript does not indicate that the calpains are not involved in postmortem proteolysis and tenderization, but they suggest that the postmortem proteolysis that contributes to tenderization may be a more complex process than has been believed and that proteolytic systems in addition to the calpain system may be involved.
Finally, this work is one of few studies that have compared the changes in calpain activity during postmortem storage among different muscles. Of the 5 muscles included in this study, postmortem changes in cal-pain activity were very similar among 4 of them, the dorsal and ventral areas of the longissimus dorsi, the semimembranosus, and the psoas major, but postmortem changes in both µ-and m-calpain activities occurred much more slowly in the triceps brachii than in any of the other 4 muscles studied. The reason for this difference is unclear at present; in the bovine, the triceps brachii does not differ markedly from the other 5 muscles in fiber type composition (Totland and Kryvi, 1991).
In summary, although considerable evidence indicates that the 2 calpains, µ-calpain and m-calpain, have an important role in the postmortem proteolysis that increases meat tenderness, it remains unclear how the calpains function in postmortem muscle. When measured in in vitro assays, proteolytic activity of µ-calpain decreases rapidly during postmortem aging and µ-calpain activity typically is less than 5% of its at-death activity after 72 h postmortem in bovine skeletal muscle. The other calpain, m-calpain, requires high Ca2+ concentrations for proteolytic activity, and it is unclear that Ca2+ concentrations ever become high enough to initiate m-calpain activity. The work described in this manuscript shows that, after 48 h postmortem, the cal-pains may not be proteolytically active at pH 5.8, the pH of postmortem muscle, and suggests that, in addition to the calpains, other proteolytic systems such as the caspases may contribute to postmortem proteolysis.
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Footnotes
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1 This work was supported by grants from the USDA National Research Initiative Competitive Grants Program, 9801191, 2002-35206-11630, and 2005-35206-15268; the National Institutes of Health, AR052108-02; the Muscular Dystrophy Association; and the Arizona Agriculture Experiment Station, Project 28, a contributing project to USDA Regional Research Project NC-131. We thank Janet Christner for help in preparing the manuscript and Hamdi Ahmad and his associates at the University of Arizona Livestock and Meats Complex for their help in obtaining the muscle samples used in this research. 
2 Present address: Centro de Investigación en Alimentación y Desarrollo AC, Hermosillo, Sonora, México.
3 Corresponding author: darrel.goll{at}arizona.edu
Received for publication March 13, 2007.
Accepted for publication May 14, 2007.
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Abstract
INTRODUCTION
