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Effect of oxidation, pH, and ionic strength on calpastatin inhibition of µ- and m-calpain

Department of Animal Science, Iowa State University, Ames 50011

	Abstract

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The objective of this study was to evaluate the effect of oxidation on µ- and m-calpain activity at varying pH and ionic strength conditions in the presence of calpastatin. In 2 separate experiments, purified porcine skeletal muscle µ- or m-calpain (0.45 units of caseinolytic activity) was incubated in the presence of calpastatin (0, 0.15, or 0.30 units) at pH 7.5, 6.5, or 6.0 with either 165 or 295 mM NaCl. The reactions were initiated with the addition of CaCl₂ (100 µM for µ-calpain; 1 mM for m-calpain). In Experiment 1, µ- or m-calpain was incubated with the calpain substrate Suc-Leu-Leu-Val-Tyr-AMC (170 µM). Either 0 or 16 µ µM H₂O₂ was added to each assay. Activity was measured at 60 min. In Experiment 2, calpain was incubated with highly purified porcine myofibrils (4 mg/mL) under conditions described. Either 0 or 100 µM H₂O₂ was added immediately prior to the addition of calpain. Degradation of desmin was determined on samples collected at 2, 15, 60, and 120 min. Results from Experiment 1 indicated that oxidation decreased (P < 0.01) activity of µ-calpain. µ-Calpain had the greatest (P < 0.01) activity at pH 6.5, and m-calpain had the greatest (P < 0.01) activity at pH 7.5 at 60 min. m-Calpain activity was not detected at pH 6.0. µ- and m-calpain activity were lower (P < 0.01) at 295 mM NaCl than at 165 mM NaCl at all pH conditions. Oxidation lowered (P < 0.01) calpastatin inhibition of µ-and m-calpain at all pH and ionic strength combinations. In Experiment 2, oxidation decreased proteolytic activity of µ-calpain against desmin at pH 6.0 (P < 0.05 at 15, 60, and 120 min) and decreased m-calpain at all pH conditions. However, desmin degradation by µ-calpain was not as efficiently inhibited by calpastatin at pH 7.5 and as at pH 6.5 (P = 0.03 at 60 min) when oxidizing conditions were created. This is consistent with the results from Experiment 1, which indicated that oxidation decreased the ability of calpastatin to inhibit µ-calpain. These studies provide evidence that oxidation influences calpain activity and inhibition of calpains by calpastatin differently under varying environmental conditions. The results suggest that, at the higher pH conditions used, calpastatin may limit the possibility of oxidation-induced inactivation of µ-calpain.

Key Words: calpain • calpastatin • ionic strength • oxidation • proteolysis • pH

	INTRODUCTION

Top Abstract INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION IMPLICATIONS LITERATURE CITED

 
The significant changes in muscle cellular environment that occur early postmortem are known to affect meat quality (Huff-Lonergan et al., 1996). One of the most pronounced changes is the pH decline from near neutrality in living muscle to approximately 5.6 in meat. Another change that occurs is the increase in ionic strength from an approximate equivalent of 165 mM NaCl in living muscle to approximately 295 mM NaCl in meat (Winger and Pope, 1981). µ-Calpain is a main factor in postmortem proteolysis of cytoskeletal proteins and subsequent tenderization of meat (Goll et al., 1992; Koohmaraie, 1992). m-Calpain is also present in muscle, and its action in postmortem proteolysis and meat tenderization is highly debated (as reviewed by Geesink et al., 2000). Calpains are regulated by several factors including calcium concentration, pH, and the endogenous inhibitor calpastatin. An increase in ionic strength has been shown to decrease µ-calpain activity by decreasing the stability of autolyzed µ-calpain (Geesink and Koohmaraie, 2000; Li et al., 2004).

Protein oxidation occurs in postmortem muscle (Martinaud et al., 1997; Rowe et al., 2004b). Oxidation has been shown to decrease the ability of µ-calpain to degrade its substrates (Guttmann and Johnson, 1998; Rowe et al., 2004a). The objective of this study was to determine the extent to which oxidation influences calpain activity and calpain inhibition by calpastatin under pH and ionic strength conditions observed in early postmortem muscle.

	MATERIALS AND METHODS

Top Abstract INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION IMPLICATIONS LITERATURE CITED

 
Purification of Calpastatin, µ-Calpain, and m-Calpain

Calpastatin, µ-calpain, and m-calpain were purified from porcine skeletal muscle based on the procedures outlined by Thompson and Goll (2000) with minor modifications as described by Maddock et al. (2005). Porcine semimembranosus sample (2 kg) was taken from a market barrow approximately 25 min after exsanguination, prepared as previously described, and loaded on a Q-Sepharose Fast Flow (Amersham Biosciences, Piscataway, NJ) anion-exchange column. Porcine calpastatin, µ-calpain, and m-calpain were eluted in 3 separate peaks off this column.

Purification of Calpastatin. Fractions containing calpastatin activity were pooled and further purified using methods described by Thompson and Goll (2000) with modifications taken from procedures of Geesink and Koohmaraie (1998) as described by Maddock et al. (2005). Purification was completed using successive chromatography over Phenyl Sepharose 6 Fast Flow (Amersham Biosciences), Blue Sepharose 6 Fast Flow (Amersham Biosciences), and EMD TMAE 650 S (EM Science, Gibbstown, NJ). Purified calpastatin consisted only of a 68-kDa band when analyzed by SDS-PAGE and had a specific activity of 451 U/mg of protein. One unit of activity of calpastatin was defined as the ability to inhibit 1 unit of m-calpain caseinolytic activity (Koohmaraie et al., 1995).

Purification of µ- and m-Calpain. µ- and m-Calpain were purified according to the methods of Thompson and Goll (2000) with minor modifications as described by Maddock et al. (2005). Fractions from the Q-sepharose column containing µ-calpain activity were pooled, and m-calpain activity was pooled. The µ-calpain was purified using successive chromatography over a Phenyl Sepharose 6 Fast Flow, Butyl Sepharose 4 Fast Flow (Amersham Biosciences), EMD TMAE 650 S, and DEAE-TSK Toyopearl (Supelco, Bellefonte, PA). The purified µ-calpain had a specific activity of 71.3 U/mg of protein. One unit of calpain was defined as the amount of calpain required to increase the absorbance at 278 nM of the supernatant by 1 unit during 1 h of incubation at 25°C because of the release of trichloroacetic acid-soluble polypeptides resulting from the digestion of casein (Koohmaraie, 1990).

Fractions containing m-calpain activity were pooled and purified using successive chromatography over a Phenyl Sepharose 6 Fast Flow, Reactive Red 120 (Sigma, St. Louis, MO), and DEAE-TSK Toyopearl. Purified m-calpain had a specific activity of 147 U/mg of protein. The purified µ-calpain, m-calpain, and calpastatin were stored in TEM (1 mM EDTA, 0.1% (vol/vol) ß-mercaptoethanol, 40 mM Tris-HCl, pH 7.4) with the addition of 1 mM sodium azide at 4°C.

Calpain Activity Assays

To ensure that pH, ionic strength, and H₂O₂ did not directly influence the peptide, assays were also conducted with the enzyme carboxypeptidase Y (Calbiochem, La Jolla, CA). Carboxypeptidase Y has a pH optimum of pH 5.5 to 6.5 and a stability of up to pH 8.0. Suc-Leu-Leu-Val-Tyr-7-amino-4-methyl coumarin is a substrate for this enzyme (Stennicke et al., 1994). In these assays, carboxypeptidase Y was added in place of µ- or m-calpain at 0.5 µL (0.0045 µM final concentration). Comparison of activity measured in the assays at pH 7.5, 6.5, and 6.0; ionic strength of 165 and 295 mM NaCl; and 0 or 16 µM H₂O₂ was done to determine whether these environmental conditions changed the susceptibility of the peptide to proteolysis.

The technique used in this experiment is a sensitive activity assay using a calpain substrate, Suc-Leu-Leu-Val-Tyr-7-amino-4-methyl coumarin (Bachem, Torrence, CA; 10 mg/mL in dimethyl sulfoxide), which is a known substrate of µ- and m-calpain (Sasaki et al., 1984). The highly purified calpains (µ- or m-calpain; 0.45 units of caseinolytic activity) were incubated with 170 µM Suc-Leu-Leu-Val-Tyr-AMC in either the presence of highly purified calpastatin (0.15 or 0.30 units measured against 0.45 units of m-calpain caseinolytic activity) or 0 units of calpastatin under the following conditions: 1) pH 7.5, 165 mM NaCl; 2) pH 7.5, 295 mM NaCl; 3) pH 6.5, 165 mM NaCl; 4) pH 6.5, 295 mM NaCl; 5) pH 6.0, 165 mM NaCl; or 6) pH 6.0, 295 mM NaCl. The buffers were either 50 mM HEPES (pH 7.5 and 6.5) or 50 mM 2-(4-morpholino)-ethane sulfonic acid (MES; pH 6.0). Oxidation treatments consisted of the addition 16 µM H₂O₂ immediately prior to addition of calpain. The reactions were initiated with the addition of 100 µM CaCl₂ (µ-calpain) or 1 mM CaCl₂ (m-calpain). Calpain activity and percentage of inhibition by calpastatin [percentage of inhibition = 1 – (calpain activity without calpastatin ÷ calpain activity with calpastatin) x 100] were measured at 60 min in a TD-700 fluorometer (Turner Designs, Sunnyvale, CA). A control with calcium (without the addition of µ- or m-calpain) for each pH and ionic strength condition was conducted to determine whether calcium affected the fluorescence of the peptide. An EDTA control (20 mM EDTA final concentration added prior to addition of calpain and calcium) was included for each pH and ionic strength condition and was used for the baseline.

Myofibril Isolation

Porcine semimembranosus (100 g; 45 min postmortem) was homogenized in ice cold extraction buffer [10 mM EDTA, 2 µM E-64, 100 mg of trypsin inhibitor/L, 2 mM phenylmethylsulfonylfluoride (PMSF), and 100 mM Tris-HCl; pH 8.3] using a polytron PT 3100 (Kinmetica AG, Littau, Switzerland) set at 10,000 rpm. Samples were centrifuged (20,000 x g) for 30 min at 4°C. Pellets were homogenized in 10 vol of standard salt solution (100 mM KCl, 2 mM MgCl₂, 1 mM EGTA, 1 mM NaN₃, and 20 mM K₂HPO₄; pH 7.0). Purification of myofibrils was completed by differential centrifugation (Goll et al., 1974), and protein concentration was determined using the Biuret method as modified by Robson et al. (1968).

Incubation of Myofibrils with Calpain

The highly purified calpains (0.45 units of caseinolytic activity/mL in the assay) were incubated with highly purified porcine myofibrils in suspension (4 mg/mL of buffer) in the presence of calpastatin (0, 0.15, or 0.3 units measured against 0.45 units of m-calpain caseinolytic activity/mL in the assay) under the following conditions: pH 7.5 and 165 or 295 mM NaCl, pH 6.5 and 165 or 295 mM NaCl; and pH 6.0 and 165 or 295 mM NaCl. The buffers were either 50 mM HEPES (pH 7.5 and 6.5) or 50 mM MES (pH 6.0). Oxidation treatment consisted of the addition of 0 or 100 µM H₂O₂. Concentrations of H₂O₂ were used based on concentrations used in previous studies (Guttmann et al., 1997). The reactions were initiated with the addition of CaCl₂ at a final concentration of 100 µM (µ-calpain) or 1 mM (m-calpain). Aliquots were removed at 2, 15, 60, and 120 min after the addition of CaCl₂. The digests were terminated by the addition 20 mM EDTA (final concentration). The aliquots were centrifuged at 6,000 x g for 15 min at 4°C. A portion of the pellet was used for SDS-PAGE and immunoblotting to determine changes in desmin. Experiments were replicated (n = 2) on different days.

SDS-PAGE and Western Blotting

Sodium dodecyl sulfate-PAGE and membrane transfer were conducted as described by Rowe et al. (2004a) using 10% polyacrylamide separating gels and a desmin primary antibody (polyclonal rabbit antidesmin antibody, V2022, Biomedia, Foster City, CA) for desmin. A sensitive chemiluminescent (ECL Plus kit, Amersham Biosciences) system was used to detect labeled protein bands using a charged coupled device camera (Fluro-Chem 8800, Alpha Innotech Corporation, San Leandro, CA) and FluorChem IS-800 software (Alpha Innotech Corporation). Densitometry was completed using the AlphaEaseFC software (Alpha Innotech Corporation). A reference sample was used on each blot to standardize densitometry data to compare differences between blots.

Oxidation of Calpastatin

Calpastatin (10 units) was incubated with an irreversible sulfhydryl blocking agent N-ethylmaleimide (NEM; 0, 4, 8, or 12 mM final concentration; n = 2) for 1 h at 4°C. Samples were exhaustively dialyzed in 1 mM EDTA and 40 mM Tris-HCl (pH 7.5) to remove NEM. Activity of calpastatin after dialysis was determined by measuring inhibition of m-calpain caseinolytic activity (Koohmaraie et al., 1995). A protein assay (Bradford, 1976) was conducted on each sample to determine specific activity of each calpastatin sample.

Statistical Analysis

Data were analyzed using a split-split plot design. The whole plot was pH/ionic strength (7.5/165, 7.5/295, 6.5/165, 6.5/295, 6.0/165, or 6.0/295). The split plot was calpastatin (0, 0.15, or 0.30 units). The split-split plot was H₂O₂ (0 or 16 µM/100 µM). Calpain activity at 60 min and densitometry of intact desmin were analyzed using PROC MIXED procedure in SAS (SAS Institute Inc., Cary, NC). Least squares means were separated using Tukey’s Honestly Significant Difference test. Significance was defined as P < 0.05.

	RESULTS

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Carboxypeptidase Y Activity Control

Ionic strength, pH, or H₂O₂ conditions (Table 1) used in these experiments did not alter susceptibility of the peptide to carboxypeptidase Y activity. Fluorescence measured at 60 min at pH 7.5,165 mM NaCl, and 0 µM H₂O₂ was defined as 100% of carboxypeptidase Y activity. It was concluded that the synthetic peptide was not affected (P > 0.5) by the differing pH, ionic strength, and oxidation conditions and was appropriate to use for our study.

Irreversibly blocking sulfhydryls with NEM did not alter the specific activity of calpastatin. The specific activity of control calpastatin was 416 units of activity/mg of protein. The mean specific activity of the oxidized calpastatin with NEM was 406.5, 410.5, and 398 units of activity/mg of protein for 4, 8, and 12 mM NEM, respectively.

Calpain Activity Assays

µ-Calpain Activity. Fluorescence measurements at 60 min indicated that the addition of H₂O₂ significantly decreased (P < 0.05; Table 2) µ-calpain activity regardless of pH and ionic strength conditions. µ-Calpain had greater activity at pH 6.5 (P < 0.01) than at pH 7.5 or 6.0. µ-Calpain activity was lower (P < 0.05) at 295 mM NaCl than at 165 mM NaCl.

µ-Calpain Myofibril Digests. Differences in desmin degradation caused by oxidation and calpastatin were observed (Figure 1; Table 6). At pH 6.0, calpastatin decreased degradation of desmin as indicated by detection of intact desmin at 60 and 120 min of incubation with increasing levels of calpastatin at both 165 and 295 mM NaCl. An effect of oxidation was observed where the addition of H₂O₂ to the digest decreased the degradation of desmin; this was particularly obvious at 295 mM NaCl when 0 units of calpastatin was added to the digests, and intact desmin was detected at all time points.

View larger version (35K): [in this window] [in a new window]   Figure 1. Western blots of desmin degraded by µ-calpain. Each lane was loaded with 20 µg of protein. Lane 1 (labeled S) is a standard sample used for densitometry standardization. Lanes 2 through 5 show the degradation profile of desmin from samples without H2O2 taken at 2, 15, 60, and 120 min after initiation of the digest. Lanes 6 through 10 depict the degradation profile of desmin from samples with H2O2 taken at 2, 15, 60, and 120 min after initiation of the digest. Each grouping of pH and ionic strength depicts 3 Western blots, each with differing levels (0, 0.15, and 0.3 units) of calpastatin added to the digest. Disappearance of bands over time indicate degradation of intact desmin by µ-calpain.

Densitometry data describing intact desmin degradation (n = 2) are shown in Table 6, and statistical main effects and interactions are shown in Table 7. After 2 min of incubation, no significant main effects were observed. There was a trend toward an effect of pH (P = 0.08) where, at pH 6.5, degradation of desmin was less than at pH 7.5 and 6.0 at all 3 levels of calpastatin. After 15 min of incubation, an effect of oxidation (P = 0.04) was observed. The addition of H₂O₂, in general, caused a decrease in the degradation of desmin, as indicated by higher densitometry measurements. At 60 min of incubation, many effects (P < 0.05) were observed. Oxidation decreased (P < 0.001) desmin degradation as indicated by more intact desmin. The interactions of pH x oxidation, calpastatin x oxidation, and pH x calpastatin x oxidation were significant. At pH 6.0, the combination of H₂O₂ and calpastatin appeared to be additive in its inhibitory effects on µ-calpain activity vs. pH 7.5 and 6.5, where the combination appeared to decrease inhibition. After 120 min of incubation, many of the significant effects observed at 60 min were not apparent. This was due to the almost complete degradation of intact desmin that occured by 60 min under the different environmental conditions. An effect of oxidation (P = 0.04) was still observed where, in general, the presence of H₂O₂ decreased degradation of desmin by µ-calpain.

View larger version (31K): [in this window] [in a new window]   Figure 2. Western blots of desmin degraded by m-calpain. Each lane was loaded with 20 µg of protein. Lane 1 (labeled S) is a standard sample used for densitometry standardization. Lanes 2 through 5 show the degradation profile of desmin from samples without H2O2 taken at 2, 15, 60, and 120 min after initiation of the digest. Lanes 6 through 10 depict the degradation profile of desmin from samples with H2O2 taken at 2, 15, 60, and 120 min after initiation of the digest. Each grouping of pH and ionic strength depicts 3 Western blots, each with differing levels (0, 0.15, and 0.3 units) of calpastatin added to the digest. Disappearance of bands over time indicates degradation of desmin by m-calpain.

	DISCUSSION

Top Abstract INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION IMPLICATIONS LITERATURE CITED

In the current study, the objective was to determine the extent to which oxidation influences µ- and m-calpain activity and calpain inhibition by calpastatin under the pH and ionic strength conditions observed in early postmortem muscle. Two different methods were used to evaluate the sum of the effects of pH, ionic strength, and oxidation of µ- and m-calpain activity in the presence or absence of calpastatin. The first method used a calpain substrate to precisely measure activity of µ- and m-calpain and their inhibition by calpastatin. The second method used purified myofibrils as a substrate and evaluation of degradation of the protein desmin. This allowed definition of the effects of environment on the activities of µ- and m-calpain and their inhibition by calpastatin using a substrate found in meat. It was concluded that both the calpain activity and inhibition of calpain by calpastatin can be dramatically affected by pH, ionic strength, and oxidation. The effects of environment were profoundly different between µ- and m-calpain.

pH and Ionic Strength

A previous study was conducted using the calpain substrate to evaluate µ- and m-calpain activity in the presence of reducing agents (Maddock et al., 2005). Ionic strength and pH effects similar to calpain activity assay results in the current study were observed on the activities of m- and µ-calpain and their inhibition by calpastatin. As observed in this study, m-calpain activity was also greatest at pH 7.5, and activity was least at pH 6.0. Additionally, µ-calpain activity was also greater at pH 6.5 vs. pH 7.5 or 6.0. Western blotting of µ-calpain autolysis revealed that autolysis occurred at a slower rate at pH 6.5 than at pH 7.5. It was hypothesized that the observed activity differences were not due to a faster activation of µ-calpain, but rather to a slower autolytic inactivation of µ-calpain at pH 6.5. It was also determined that the rate of hydrolysis in the first 5 min of the assay was greater at pH 6.5 than at pH 7.5. The ionic strength effects observed in Maddock et al. (2005) and the current study indicate that both µ-and m-calpain activities are decreased with increasing ionic strength. Geesink and Koohmaraie (2000) observed that µ-calpain activity decreased with increasing ionic strengths and indicated that the decrease in activity of µ-calpain was due to a decrease in the stability of the molecule. Li et al. (2004) hypothesized that an elevated ionic strength (equal to 100 mM KCl) caused disassociation of the 2 calpain subunits, allowing for formation of dimers and trimers of the large subunit, which irreversibly inactivated the proteinase. These results indicate that pH and ionic strength have the potential to affect activity of µ- and m-calpain through autolytic inactivation and dissociation.

In the current study, inhibition of µ-calpain and m-calpain by calpastatin were not affected by pH. This is consistent with reports by Geesink and Koohmaraie (1999) and Otsuka and Goll (1987). Calpastatin is the endogenous inhibitor of µ- and m-calpain and does not inhibit any other protease that it has been tested against (Goll et al., 2003). The presence of Ca²⁺ is required for calpastatin to bind to the calpains to inhibit their activity (Cottin et al., 1981). In porcine muscle, the ratio of calpastatin activity to µ-calpain activity is approximately 1.5:1 (Ouali and Talmant, 1990). The ratio of calpastatin to calpain used in this study was 1:3 and 2:3 to evaluate the effect of calpastatin on calpain without the occurrence of complete inhibition. Maddock et al. (2005) reported an effect of pH on inhibition of m-calpain, where inhibition was greater at pH 6.5 than at pH 7.5. The discrepancy between this study and the previous study is likely due to the presence of a reducing agent in Maddock et al. (2005) when the activity of m-calpain was maximized; in this study, no reducing agents were used to maximize the effect of oxidation on activity and inhibition.

Oxidation

Because µ- and m-calpain are cysteine proteases, their activity requires the exchange of electrons between the active site cysteine and histidine residues (Medhi, 1991). The cysteine residue site is highly susceptible to oxidation by H₂O₂ (Neumann, 1972). Oxidation of calpain with H₂O₂ can allow for inactivation of calpain (Guttmann et al., 1997), indicating that the active site cysteine residue may be oxidized. Oxidation of proteins does occur during postmortem aging (Martinaud et al., 1997; Rowe et al., 2004a), as antioxidants are expended, and is indicated by increased protein carbonyl content and decreased sulfhydryl concentration. An oxidative environment decreases degradation of substrates by calpain (Guttmann et al., 1997; Guttmann and Johnson, 1998), and this oxidation is shown to be reversible (Rowe et al., 2004b). Oxidation or more specifically, thiolation, is a common reaction of H₂O₂ with sulfhydryl groups that occurs quickly and is reversible with the introduction of a reducing environment (Saurin et al., 2004). Hydrogen peroxide is widely produced in cells and allows for the reversal of oxidation; thereby, it is appropriate for the study of oxidation in muscle and meat.

Oxidation decreased the proteolytic activity of µ-and m-calpain (Tables 2 and 3) in the activity assays. When using myofibrils as a substrate, the main effect of oxidation (Table 7) was specifically noted on µ-calpain. The effect of oxidation on m-calpain (Table 9) in this system was detected only when also considering interactions with pH and ionic strength in combination with calpastatin. Previous studies observed similar effects of oxidation on µ-calpain activity in meat (Rowe et al., 2004b) and in vitro (Guttmann et al., 1997; Guttmann and Johnson, 1998).

Evidence that cysteine residues of calpastatin are not affected by oxidation is provided in this study by incubations with NEM, which covalently blocks sulfhydryl groups, resulting in effects similar to oxidation. This important observation provides evidence that oxidation of calpastatin does not affect its action on inhibition of calpains.

Many of the singular effects of environment did not appear to be significant on µ- or m-calpain activity. However, the specific objectives of these experiments were to evaluate the interactions between environmental conditions. The interaction of oxidation and calpastatin indicates that these environmental combinations do have an effect on the proteolytic activity of µ- and m-calpain; therefore, they also have implications in postmortem muscle and in living muscle tissue.

The presence of H₂O₂ decreased the percentage of inhibition of both µ- and m-calpain by calpastatin when using the calpain substrate. This corresponds to the effects observed in the µ-calpain-degraded myofibrils, where the addition of H₂O₂ in the presence of calpastatin allowed for greater degradation of desmin at the higher pH of 7.5 and 6.5. These observations were unique to µ-calpain in the digests of myofibrils.

The role of calpastatin in binding to and inhibiting calpain is an area of research that is currently getting some attention, as the exact mechanism is not understood (Goll et al., 2003). As previously discussed, Ca²⁺ is required for binding of calpastatin to calpains (Cottin et al., 1981). The Ca²⁺ concentration required for the binding of calpastatin to µ- and m-calpain is lower than the Ca²⁺ concentration required for proteolytic activity at half-maximal rate. This indicates that, if present, calpastatin will bind and inhibit calpain before it can become proteolytically active (Kapprell and Goll, 1989). Calpastatin is considered a competitive inhibitor of calpains based on kinetic evidence (Croall and McGrody, 1994). However, evidence indicates that calpastatin does not bind at the active site of calpain (Nishimura and Goll, 1991; Croall and McGrody, 1994), but rather near the active site in a way that blocks access of substrates as hypothesized by Todd et al. (2003). In the current study, when a calpastatin/µ-calpain complex was formed and then exposed to H₂O₂, greater degradation of desmin was observed at pH 6.5 and 7.5. Oxidation of the active site cysteine residue decreases calpain activity (Guttmann et al., 1997). It is possible that the interaction of calpastatin with µ-calpain occurs in a way that blocks H₂O₂ from accessing and oxidizing the active site cysteine residue, thus preventing oxidative inactivation of µ-calpain.

The results of this study indicate that µ-calpain and m-calpain react differently to different environments. Interestingly, the environmental conditions that are observed in postmortem muscle are the conditions that appear to be most favorable for activation of µ-calpain. This is consistent with previous hypotheses (as reviewed by Geesink et al., 2000) that indicate µ-calpain and not m-calpain is responsible for the proteolysis that occurs in postmortem muscle and, therefore, subsequent tenderization.

The effects of oxidation observed in this study have implications for meat quality. Rowe at al. (2004b) evaluated how oxidative conditions initiated by irradiation early postmortem caused inhibition of µ-calpain activity, slowing the rate of proteolysis occurring during aging and, thereby, decreasing tenderization. Antioxidants are currently becoming more prevalently used in the meat industry to increase shelf-life by preventing oxidation of lipids that cause rancidity. Previous research has also evaluated the use of antioxidants as a feed supplement and how their use can affect meat tenderization. Harris et al. (2001) evaluated increased levels of -tocopherol in beef through supplemental vitamin E in the diet in combination with injecting steaks with CaCl₂. They found that beef with increased levels of -tocopherol exhibited a faster rate of decline of Warner-Bratzler shear force values and a faster rate of troponin-T degradation. Additionally, Rowe at al. (2004b) observed an increase in the rate of troponin-T degradation in beef from calves fed a diet supplemented with vitamin E.

Oxidation has been shown to decrease activity of calpains, but in contrast, calpains have also been shown to play a role in protein degradation when oxidative conditions are higher than normal. A relationship between protein oxidation and proteolysis has been clearly established (as reviewed by Mehlhase and Grune, 2002), where it has been established that oxidation of proteins can enhance susceptibility to proteolytic degradation. However, at the same time, the excessive oxidation may cause a decrease in susceptibility to degradation. Oxidative stress in combination with observed intracellular Ca²⁺ concentration in a mouse embryonal carcinoma cell line (Ray et al., 2000) and a rat adrenal gland cell line (Ishihara et al., 2000) has been shown to increase activation of calpains. Pronzato et al. (1993) observed that the level of oxidative stress can affect calpain activity, where moderate levels of oxidation enhance activity, but increased oxidative exposure can cause inactivation of calpains. These observations could provide an explanation of how the involvement of calpastatin regulates oxidation of µ-calpain and allows activation to occur.

In summary, activity differences were observed because of pH, where µ-calpain had greater activity at pH 6.5 than at pH 7.5; the opposite was true for m-calpain. Oxidation of m-calpain with H₂O₂ resulted in a decrease in proteolytic activity, and the addition of calpastatin decreased proteolytic activity even further as demonstrated by the decrease in desmin degradation (Figure 2). Oxidation of µ-calpain with H₂O₂ did decrease proteolytic activity, but oxidation of the calpastatin/µ-calpain complex with H₂O₂ resulted in increased proteolysis of desmin (Figure 1) and, therefore, appeared to increase activity of µ-calpain in the presence of H₂O₂. This novel observation is important because it demonstrates that inhibition of µ-calpain by calpastatin is diminished by oxidation.

	IMPLICATIONS

Top Abstract INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION IMPLICATIONS LITERATURE CITED

 
Oxidative conditions in postmortem muscle could have an effect on proteolytic activity that occurs during the aging process. The differences observed between oxidation of µ-calpain vs. m-calpain provide further evidence of the role of µ-calpain in postmortem proteolysis. The results may also explain some of the variation observed in tenderness, as the interactions of pH, ionic strength, and oxidation in addition to the presence of calpastatin cause µ-calpain to act differently than if examined under each environmental factor alone. These results also indicate a need for greater understanding of the interaction of calpastatin with the calpains to understand how these proteins function in the muscle, not only postmortem, but also living muscle.

¹ Corresponding author: slonerga{at}iastate.edu

Received for publication April 6, 2005. Accepted for publication November 18, 2005.

	LITERATURE CITED

Top Abstract INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION IMPLICATIONS LITERATURE CITED

 


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