Isolation and characterization of mu-calpain, m-calpain, and calpastatin from postmortem muscle. I. Initial steps

Evidence has indicated that mu-calpain, m-calpain, and calpastatin have important roles in the proteolytic degradation that results in postmortem tenderization. Simple assays of these 3 proteins at different times postmortem, however, has shown that calpastatin and mu-calpain both rapidly lose their activity during postmortem storage, so that proteolytic activity of mu-calpain is nearly zero after 3 d postmortem, even when assayed at pH 7.5 and 25 degrees C, and ability of calpastatin to inhibit the calpains is 30% or less of its ability when assayed at death. m-Calpain, however, retains much of its proteolytic activity during postmortem storage, but the Ca(2+) requirement of m-calpain is much higher than that reported to exist in postmortem muscle. Consequently, it is unclear how the calpain system functions in postmortem muscle. To clarify this issue, we have initiated attempts to purify the 2 calpains and calpastatin from bovine semitendinosus muscle after 11-13 d postmortem. The known properties of the calpains and calpastatin in postmortem muscle have important effects on approaches that can be used to purify them. A hexyl-TSK hydrophobic interaction column is a critical first step in separating calpastatin from the 2 calpains in postmortem muscle. Dot-blot assays were used to detect proteolytically inactive mu-calpain. After 2 column chromatographic steps, 5 fractions can be identified: 1) calpastatin I that does not bind to an anion-exchange matrix, that does not completely inhibit the calpains, and that consists of small polypeptides <60 kDa; 2) calpastatin II that binds weakly to an anion-exchange matrix and that contains polypeptides <60 kDa; all these polypeptides are smaller than the native 115- to 125-kDa skeletal muscle calpastatin; 3) proteolytically active mu-calpain even though very little mu-calpain activity can be detected in zymogram assays of muscle extracts from 11- to 13-d postmortem muscle; this mu-calpain has an autolyzed 76-kDa large subunit but the small subunit consists of 24-, 26- and a small amount of unautolyzed 28-kDa polypeptides; 4) proteolytically active m-calpain that is not autolyzed; and 5) proteolytically inactive mu-calpain whose large subunit is autolyzed to a 76-kDa polypeptide and whose small subunit contains polypeptides similar to the proteolytically active mu-calpain. Hence, loss of calpastatin activity in postmortem muscle is due to its degradation, but the cause of the loss of mu-calpain activity remains unknown.     

Effect of postmortem storage on activity of mu- and m-calpain in five bovine muscles

An in situ system involving incubation of 60- to 80-g pieces of muscle at 4 degrees C under different conditions was used to determine the effects of time of postmortem storage, of pH, and of temperature on activities of mu- 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, mu-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 mu- 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 mu-calpain but not m-calpain is proteolytically active at pH 5.8, these studies have used calpains obtained from muscle at death. Both mu- 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 mu-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 mu-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 mu-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 mu-calpain molecule is completely active proteolytically, it is possible that the 76-/28-kDa mu-calpain molecule in postmortem muscle is proteolytically inactive and that this accounts for the loss of mu-calpain activity during postmortem storage. Because neither mu- 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.     

Ionic strength-induced inactivation of mu-calpain in postmortem muscle

The present study was conducted to study the stability of autolyzed mu-calpain activity and determine whether measurement of mu-calpain activity after anion exchange chromatography accurately reflects its activity in postmortem muscle. Ionic strength and pH affected the stability of partially autolyzed mu-calpain. Complete loss of activity was observed as a result of binding of autolyzed mu-calpain to DEAE-Sephacel when the large subunit of mu-calpain was autolyzed from 80 to 76 kDa. Therefore, determination of mu-calpain by standard anion exchange chromatography may underestimate mu-calpain activity in postmortem muscle. The activity of autolyzed mu-calpain was stabilized by inclusion of glycerol in the buffers, and this permitted us to investigate whether the apparent loss of mu-calpain activity in postmortem muscle is an artifact of the methodology. Despite the inclusion of glycerol in the buffers, a decrease in mu-calpain activity was observed during postmortem storage of muscle, even though the autolyzed enzyme was readily detectable by Western blotting in muscle extracts and column eluates. This result indicates that instability of autolyzed mu-calpain is a major cause for the decline in mu-calpain activity in postmortem muscle.     

Effect of calpastatin on degradation of myofibrillar proteins by mu-calpain under postmortem conditions.

To improve our understanding of the regulation of mu-calpain activity in situ during postmortem storage of muscle, the effect of different calpastatin levels on proteolysis of myofibrillar proteins by mu-calpain in a system closely mimicking postmortem conditions was studied. Increasing the amount of calpastatin in the incubations limited both the rate and extent of proteolysis of myofibrillar proteins and autolysis of mu-calpain. Excess calpastatin (i.e., a mu-calpain:calpastatin ratio of 1:4) did not inhibit proteolysis completely. Western blot analysis revealed that proteolysis of myofibrillar proteins virtually ceased after 7 d of incubation, despite the presence of partly autolyzed, therefore seemingly active, mu-calpain. A series of incubations of autolyzed mu-calpain revealed that the autolyzed form of this enzyme is unstable at an ionic strength observed in postmortem muscle. The possible significance of these results in terms of the regulation of mu-calpain activity in postmortem muscle is discussed.     

Effect of pH, temperature, and inhibitors on autolysis and catalytic activity of bovine skeletal muscle mu-calpain.

To improve our understanding of the regulation of calpain activity in situ during postmortem storage, the effects of pH, temperature, and inhibitors on the autolysis and subsequent proteolytic activity of mu-calpain were studied. Calpains (mu- and m-calpain) and calpastatin were purified from bovine skeletal muscle. All autolysis experiments were conducted in the absence of substrate at different pH (7.0, 6.2, and 5.8) and temperatures (25 and 5 degrees C). Autolysis of mu-calpain generated polypeptides with estimated masses of 61, 55, 40, 27, 23, and 18 kDa. The rate of autolysis was significantly increased with decreasing pH. The rate of degradation of the 80-kDa subunit was significantly decreased with decreasing temperature. However, degradation of the 30-kDa subunit was not affected by decreasing temperature. By conducting autolysis experiments at 5 degrees C and immunoblotting of autolytic fragments with anti-80 kDa, it was demonstrated that with the exception of 18 kDa, which originates from 30 kDa, all other fragments probably originate from degradation of the 80-kDa subunit. Calpastatin, leupeptin, and E-64 did not inhibit the initial step of autolysis, but they did inhibit further breakdown of these fragments. However, zinc, which also inhibits the proteolytic activity of calpain, only reduced the rate of autolysis, but did not inhibit it. The possible significance of these results in terms of the regulation of calpain in postmortem muscle is discussed.     

Effect of dietary protein on calpastatin in canine skeletal muscle

The cysteine proteinases, mu- and m-calpain, along with their inhibitor, calpastatin, have been hypothesized to play a role in skeletal muscle protein degradation. Because nutrition has previously been shown to influence the expression of calpastatin, the working hypothesis of this study was that the quantity and source of dietary protein could influence regulation of the calpain system in muscle. The objectives to support this hypothesis were to determine the effects of dietary protein (amount and source) on the expression of calpastatin in canine skeletal muscle. This study comprised eight diets with seven dogs per diet. A biopsy was taken from the biceps femoris of all 56 dogs before and after 10 wk on their respective diets. This experimental design allowed examination of change within individual dogs. Diets 1 to 4 contained 12% total protein derived from chicken and/or corn gluten meal in ratios of 100:0, 67:33, 33:67, and 0:100%, respectively. Diets 5 to 8 contained 28% total protein with protein sources and ratios identical to Diets 1 to 4. Differences in calpastatin were examined qualitatively using SDS-PAGE and immunoblotting, and semiquantitatively with densitometric analyses. The majority of the calpastatin blots showed three distinct calpastatin bands, the uppermost appearing at approximately 110 kDa. Diet 5 (28% CP, 100% chicken) resulted in an increase in the expression of the 110-kDa calpastatin band compared with the other two lower molecular weight bands in the same samples. Muscle from dogs fed Diet 5 showed greater increase in (P < 0.05) calpastatin intensity of the topmost band than those fed Diet 8 (0:100; chicken:corn gluten meal). Diet 5 (100:0; chicken:corn gluten meal) showed greater total calpastatin intensity than Diet 8 (0:100; chicken:corn gluten meal). These data suggest that dogs fed a diet containing a higher total percentage of chicken protein may have a greater potential to regulate calpain-mediated degradation of muscle protein than dogs fed diets containing corn gluten meal.     

Effect of skeletal muscle decorin on collagen fibrillogenesis in vitro

The effect of skeletal muscle decorin on collagen fibrillogenesis was investigated, in order to provide background for understanding the functions of decorin in skeletal muscle. The self‐assembly of type I and III collagen with the addition of decorin or the core protein of decorin from bovine neonatal skeletal muscle was monitored using a spectrophotmeter. Time course changes in the absorbance of collagen solutions showed typical sigmoidal curves composed of three phases. The time of the initial phase was not different between the collagen solution with decorin and that without decorin. The increase rate of the absorbance in the second phase decreased with concentration of decorin added in collagen solutions. Similar effects on fibrillogenesis of type I and III collagens were observed when the core protein of decorin was added in collagen solutions. These results suggest that regulation of collagen fibrillogenesis by decorin depends on its core protein. The networks of reconstructed collagen fibrils with decorin were looser than those without decorin. Bovine skeletal muscle decorin could participate in the regulation of collagen fibrillogenesis and in the arrangement of collagen fibrils in the intramuscular connective tissue.     

Simultaneous histochemical determination of three fiber types in single sections of ovine, bovine and porcine skeletal muscle

Classification of muscle fiber types involves tedious comparisons of serial muscle tissue sections. Procedures that would incorporate pertinent histochemical properties into one simplified assay for the successful differentiation and determination of fiber types were evaluated using muscle samples from three different species (ovine, bovine and porcine). For ovine and porcine muscle tissue, the best staining procedure involved initial preincubation of the tissue section at 4.35 (for ovine) and 4.30 (for porcine) followed by incubating for succinate dehydrogenase (SDH) activity, then staining for myofibrillar adenosine triphosphatase (ATPase) activity after an acid preincubation and, finally, counterstaining with hematoxylin. For bovine muscle tissue, the procedure involves first incubating for SDH activity, after which the section is incubated at pH 4.15 and then stained for myofibrillar ATPase activity after an acid preincubation, with a concluding counterstaining using hematoxylin. These procedures permit successful differentiation and identification of three fiber types (beta R, alpha R and alpha W) from a single section of muscle tissue with approximately a 65% saving in time and materials. The results of histochemically typing muscle fibers in a single tissue section stained with these techniques were similar to those obtained with the traditional methods comparing serial sections.     

Indicators of tenderization are detectable by 12 h postmortem in ovine longissimus

Postmortem changes in osmotic pressure; ionic strength; pH; temperature; mu- and m-calpain; calpastatin; desmin degradation; and myofibril fragmentation index (MFI) were determined in ovine longissimus muscle. Our objectives were to characterize changes in these variables and to identify postmortem time points at which significant proteolysis and tenderization (as measured by change in MFI) could be detected. Seven crossbred (Dorset x Romanov) lambs were slaughtered, and samples of the longissimus muscle were removed at 0, 3, 6, 9, 12, 24, 72, and 360 h postmortem. Osmotic pressure increased (P < 0.05) from 379 to 528 mOsm during the postmortem storage period, with two-thirds of the increase occurring within the first 24 h. By measuring conductivity, we showed that ionic strength increased (P < 0.05) from 8.13 to 9.78 mS/cm during the storage period, which is equivalent to 79 and 97 mM NaCl solutions, respectively. In accordance with pH and temperature, conductivity reached ultimate levels at 24 h postmortem. Within 9 h postmortem, mu-calpain activity had decreased (P < 0.05) from at-death values and continued to decrease until 72 h, at which time it was undetectable. It was still possible to detect the 76-kDa isoforms (a product of the autolysis of the 80-kDa subunit of mu-calpain) immunologically, which implies that the loss of activity was not caused by extensive autolysis. In contrast, m-calpain activity remained constant throughout the aging period, whereas calpastatin activity was stable until 24 h postmortem, after which it gradually decreased. Autolysis products of mu-calpain were detected at 3 h postmortem, indicating that mu-calpain was activated some time between 0 and 3 h postmortem. Moreover, the effect of mu-calpain activity on myofibrillar substrates was first observed at 9 h postmortem, when a 23% loss of native desmin was detected. This degradation translated into an increase in MFI at 12 h. Collectively, these results imply that mu-calpain is active in postmortem muscle in the presence of calpastatin, and that effects of mu-calpain activity as determined by increased MFI are detectable during the first 12 h postmortem.     

A risk-assessed evaluation of postmortem meat inspection procedures for ovine thysanosomiasis

A risk assessment model was used to evaluate 4 meat inspection procedures for routine detection of Thysanosoma actinioides, a tapeworm that commonly infests the bile duct system of adult sheep in the United States. The overall sensitivities of different procedures ranged from 11.96 to 58.70%, and the nondetection rates per 1,000 viscera sets that passed inspection ranged from 38 to 77. On the basis of performance attributes for each procedure produced by the risk assessment model, the consumer was exposed to an aesthetic defect that was in 9 to 17 of every 1,000 livers passed for human consumption. The amount of wastage was also identified (3 to 39 livers/1,000 passed). Viewing of the incision site of the common hepatic duct was the most preferable routine inspection procedure, and matched pair analysis revealed that there was no additional benefit in combining this procedure with routine expression of a portion of bile from the gallbladder.     

Effect of storage on measurement of ionized calcium and acid-base variables in equine, bovine, ovine, and canine venous blood.

The stability of blood ionized calcium (Ca2+) and acid-base variables in equine, bovine, ovine, and canine venous blood samples (n = 15, in each group) stored at 4 C for 3, 6, 9, 24, or 48 hours was studied. Variables included blood Ca2+ and standard ionized calcium (Ca2+ corrected to pH 7.4) concentrations, pH, blood carbon dioxide and oxygen tensions, base excess, bicarbonate concentration, and total carbon dioxide content. Results indicate that storage of blood samples at 4 C for up to 48 hours, despite appreciable acid-base changes, is associated with less than 1.5% change in equine, bovine, and ovine blood Ca2+ concentrations. Similar changes were observed in canine blood during the first 9 hours' storage. After 24 and 48 hours' storage, clinically relevant decrease (10.5 and 15.5%) in canine blood Ca2+ concentration was measured. Therefore, Ca2+ concentration in equine, bovine, and ovine venous blood samples stored up to 48 hours, and in canine blood samples stored up to 9 hours at 4 C is of diagnostic use.     

Effect of pH and ionic strength on mu- and m-calpain inhibition by calpastatin

The objectives of this study were to determine the extent to which pH and ionic strength influence mu- and m-calpain activity and the inhibition of calpains by calpastatin. Calpastatin, mu-calpain, and m-calpain were purified from at-death porcine semimembranosus. Mu-calpain or m-calpain (0.45 U) were incubated with the calpain substrate Suc-Leu-Leu-Val-Tyr-7-amino-4-methyl coumarin in the presence of calpastatin (0, 0.15, or 0.30 U of calpain inhibitory activity) under the following pH and ionic strength conditions: pH 7.5 and 165 mM NaCl or 295 mM NaCl; pH 6.5 and 165 mM NaCl or 295 mM NaCl; and pH 6.0 and 165 mM NaCl or 295 mM NaCl. The reactions were initiated with addition of 100 microM (mu-calpain) or 1 mM CaCl2 (m-calpain), and calpain activity was recorded at 30 and 60 min. Mu-calpain had the greatest (P < 0.01) activity at pH 6.5 at each ionic strength. Higher ionic strength decreased mu-calpain activity (P < 0.01) at all pH conditions. Inhibition percent of mu-calpain by calpastatin was not affected by pH; however, it was influenced by ionic strength. Inhibition of mu-calpain by calpastatin was higher (P < 0.01) at 295 mM NaCl than at 165 mM NaCl when 0.3 units of calpastatin were included in the assay. Activity of m-calpain was greater (P < 0.01) at pH 7.5 than at pH 6.5. m-Calpain activity was not detected at pH 6.0. Inhibition of m-calpain was greater (P < 0.01) when 0.15 and 0.3 U calpastatin were added at pH 6.5 than 7.5 at 165 mM NaCl, whereas percentage inhibition of m-calpain was greater (P < 0.01) at 295 mM than 165 mM NaCl at pH 7.5 and 6.5. These observations provide new evidence that defines further the influence of pH decline and increased ionic strength on mu-calpain, m-calpain, and calpastatin activity, thereby helping to more accurately define a role for these enzymes in the process of postmortem tenderization.     

Effect of days fed, carcass grade traits, and subcutaneous fat removal on postmortem muscle characteristics and beef palatability.

Angus x Hereford steers (n = 48) similar in frame size and in muscle thickness were allotted to eight groups (n = 6) of similar mean live weight for serial slaughter at 28-d intervals (0 to 196 d). Except for d-0 steers, which served as grass-fed controls, all steers were fed a high-concentrate diet during the finishing period. Upon slaughter, one side of each carcass was trimmed of subcutaneous fat in the wholesale rib region. Postmortem longissimus muscle (LM) temperature was monitored for each side during the 24-h chilling period. After quality and yield grade data were collected, rib steaks were removed and aged (7 d) and sensory traits of the steaks were evaluated. Most carcass grade traits increased linearly (P less than .01) with days on feed, whereas most sensory panel variables and marbling increased curvilinearly (P less than .05). Generally, after 56 d on feed, carcasses chilled at slower rates (P less than .05) with increased days fed. Taste panel tenderness, amount of perceived connective tissue, and shear force values peaked at 112 d and were slightly less desirable for cattle fed longer than 112 d (quadratic term, P less than .01). Postmortem muscle temperature at 2.5 h was the chilling time most highly correlated with tenderness values among untrimmed sides. Correlations for shear force with 2.5-h LM temperature, marbling score, days fed, fat thickness, and carcass weight were -.63, -.61, -.56, -.55, and -.53, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of clenbuterol on growth,carcase and skeletal muscle characteristics in broiler chickens

1. Male and female broiler chickens (144 in total) were given diets supplemented with clenbuterol (CB) at 0 (control) and at 1 mg/kg between 28 and 49 d of age to study the effect of CB on growth, carcase and skeletal muscle.

2. CB improved growth in males by increasing daily weight gain and final live weight and by lowering food conversion ratio. In females it changed the carcase composition by reducing abdominal fat pad and by increasing the proportion of protein. Consequently, carcase protein gain was increased in both sexes (11% and 16%, respectively).

3. Skeletal muscle weights were enhanced by between 6% and 22%. Muscle fibre diameters were increased in extensor hallucis longus (EHL) but not in gastrocnemius (GAS) muscle. This increase was more pronounced in females. EHL total muscle fibre number remained unchanged. The proportion of fast‐twitch glycolytic fibres was increased at the expense of fast‐twitch oxidative fibres in males only. Nuclear/cytoplasm and DNA/protein ratios tended to be decreased by CB.

4. From the elevated EHL muscle RNA/DNA, unchanged protein/RNA and translation activity it is suggested that CB stimulated protein synthesis at the pretranslational level. Reduced protein degradation is deduced from decreased neutral calcium‐dependent proteolytic activity.

5. It is concluded that broiler chickens respond to long‐term CB treatment as has been shown in various mammals. However, the sex‐specific response in growth, carcase composition and skeletal muscle cellularity is more clearly apparent in broiler chickens.     

Effect of the beta-adrenergic agonist L644,969 on muscle growth, endogenous proteinase activities, and postmortem proteolysis in wether lambs.

To examine the effect of a beta-adrenergic agonist (BAA) on muscle growth, proteinase activities, and postmortem proteolysis, 16 wether lambs were randomly assigned to receive 0 or 4 ppm of L644,969 in a completely mixed high-concentrate diet for 6 wk. Weight of the biceps femoris was 18.6% heavier in treated lambs. At 0 h after slaughter, treated lambs had higher cathepsin B (35.6%), cathepsins B + L (19.1%), calpastatin (62.8%), and m-calpain (24.6%) than control lambs, but both groups had similar mu-calpain activities. In both longissimus and biceps femoris muscles, treated lambs had higher protein and RNA and lower DNA concentrations. However, total DNA was not affected, indicating that the increase in muscle mass was probably due to muscle hypertrophy rather than to hyperplasia. The pattern of postmortem proteolysis was significantly altered by BAA feeding. In treated lambs, postmortem storage had no effect on the myofibril fragmentation index and degradation of desmin and troponin-T. These results indicate that the ability of the muscle to undergo postmortem proteolysis has been dramatically reduced with BAA feeding. Similar proteolytic systems are thought to be involved in antemortem and postmortem degradation of myofibrillar proteins, so BAA-mediated protein accretion is probably due, at least in part, to reduced protein degradation. To examine whether protein synthesis was altered with BAA feeding, the level of skeletal muscle alpha-actin mRNA was quantified. Longissimus muscle alpha-actin mRNA abundance was 30% greater in BAA-fed lambs. Collectively, these results indicate that dietary administration of BAA increases muscle mass through hypertrophy and that the increase in muscle protein accretion is due to reduced degradation and possibly to increased synthesis of muscle proteins.