Effect of Oxidation, Ph, and Ionic Strength on Calpastatin Inhibition of Μ- and M-Calpain K

Animal Science Publications Animal Science

4-2006 Effect of oxidation, pH, and ionic strength on calpastatin inhibition of μ- and m-calpain K. R. Maddock Carlin Iowa State University

Elisabeth J. Huff-Lonergan Iowa State University, [email protected]

L. J. Rowe Iowa State University

Steven M. Lonergan Iowa State University, [email protected]

Follow this and additional works at: http://lib.dr.iastate.edu/ans_pubs Part of the Agriculture Commons, and the Meat Science Commons The ompc lete bibliographic information for this item can be found at http://lib.dr.iastate.edu/ ans_pubs/27. For information on how to cite this item, please visit http://lib.dr.iastate.edu/ howtocite.html.

This Article is brought to you for free and open access by the Animal Science at Iowa State University Digital Repository. It has been accepted for inclusion in Animal Science Publications by an authorized administrator of Iowa State University Digital Repository. For more information, please contact [email protected]. Effect of oxidation, pH, and ionic strength on calpastatin inhibition of μ- and m-calpain

Abstract The bjo ective 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 er actions 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.

Keywords calpain, calpastatin, ionic strength, oxidation, proteolysis, pH

Disciplines Agriculture | Animal Sciences | Meat Science

Comments This article is from Journal of Animal Science 84 (2006): 925–937. Posted with permission.

This article is available at Iowa State University Digital Repository: http://lib.dr.iastate.edu/ans_pubs/27 Effect of oxidation, pH, and ionic strength on calpastatin inhibition of µ- and m-calpain K. R. Maddock Carlin, E. Huff-Lonergan, L. J. Rowe and S. M. Lonergan

J ANIM SCI 2006, 84:925-937.

The online version of this article, along with updated information and services, is located on the World Wide Web at: http://www.journalofanimalscience.org/content/84/4/925

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Downloaded from www.journalofanimalscience.org at Serials Acquisitions Dept on February 24, 2014 Effect of oxidation, pH, and ionic strength on calpastatin inhibition of ␮- and m-calpain

K. R. Maddock Carlin, E. Huff-Lonergan, L. J. Rowe, and S. M. Lonergan1

Department of Animal Science, Iowa State University, Ames 50011

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

Key words: calpain, calpastatin, ionic strength, oxidation, proteolysis, pH

INTRODUCTION et al., 1992; Koohmaraie, 1992). m-Calpain is also present in muscle, and its action in postmortem proteolysis The signiﬁcant changes in muscle cellular environ- and meat tenderization is highly debated (as reviewed ment that occur early postmortem are known to affect by Geesink et al., 2000). Calpains are regulated by meat quality (Huff-Lonergan et al., 1996). One of the several factors including calcium concentration, pH, most pronounced changes is the pH decline from near and the endogenous inhibitor calpastatin. An increase neutrality in living muscle to approximately 5.6 in in ionic strength has been shown to decrease ␮-calpain meat. Another change that occurs is the increase in activity by decreasing the stability of autolyzed ␮-cal- ionic strength from an approximate equivalent of 165 pain (Geesink and Koohmaraie, 2000; Li et al., 2004). mM NaCl in living muscle to approximately 295 mM Protein oxidation occurs in postmortem muscle NaCl in meat (Winger and Pope, 1981). ␮-Calpain is (Martinaud et al., 1997; Rowe et al., 2004b). Oxidation a main factor in postmortem proteolysis of cytoskeletal has been shown to decrease the ability of ␮-calpain to proteins and subsequent tenderization of meat (Goll 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 1Corresponding author: [email protected] Received April 6, 2005. under pH and ionic strength conditions observed in Accepted November 18, 2005. early postmortem muscle.

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Downloaded from www.journalofanimalscience.org at Serials Acquisitions Dept on February 24, 2014 926 Maddock Carlin et al. MATERIALS AND METHODS Calpain Activity Assays

To ensure that pH, ionic strength, and H O did not ␮ 2 2 Purification of Calpastatin, -Calpain, directly influence the peptide, assays were also con- and m-Calpain ducted with the enzyme carboxypeptidase Y (Calbio- chem, La Jolla, CA). Carboxypeptidase Y has a pH opti- Calpastatin, ␮-calpain, and m-calpain were purified mum of pH 5.5 to 6.5 and a stability of up to pH 8.0. from porcine skeletal muscle based on the procedures Suc-Leu-Leu-Val-Tyr-7-amino-4-methyl coumarin is a outlined by Thompson and Goll (2000) with minor modi- substrate for this enzyme (Stennicke et al., 1994). In fications as described by Maddock et al. (2005). Porcine these assays, carboxypeptidase Y was added in place semimembranosus sample (2 kg) was taken from a mar- of ␮- or m-calpain at 0.5 ␮L (0.0045 ␮M final concentra- ket barrow approximately 25 min after exsanguination, tion). Comparison of activity measured in the assays prepared as previously described, and loaded on a Q- at pH 7.5, 6.5, and 6.0; ionic strength of 165 and 295 Sepharose Fast Flow (Amersham Biosciences, Piscata- mM NaCl; and 0 or 16 ␮M H2O2 was done to determine way, NJ) anion-exchange column. Porcine calpastatin, whether these environmental conditions changed the ␮-calpain, and m-calpain were eluted in 3 separate susceptibility of the peptide to proteolysis. peaks off this column. The technique used in this experiment is a sensitive Purification of Calpastatin. Fractions containing activity assay using a calpain substrate, Suc-Leu-Leu- calpastatin activity were pooled and further purified Val-Tyr-7-amino-4-methyl coumarin (Bachem, Tor- using methods described by Thompson and Goll (2000) rence, CA; 10 mg/mL in dimethyl sulfoxide), which is with modifications taken from procedures of Geesink a known substrate of ␮- and m-calpain (Sasaki et al., and Koohmaraie (1998) as described by Maddock et 1984). The highly purified calpains (␮- or m-calpain; al. (2005). Purification was completed using successive 0.45 units of caseinolytic activity) were incubated with ␮ chromatography over Phenyl Sepharose 6 Fast Flow 170 M Suc-Leu-Leu-Val-Tyr-AMC in either the pres- (Amersham Biosciences), Blue Sepharose 6 Fast Flow ence of highly purified calpastatin (0.15 or 0.30 units (Amersham Biosciences), and EMD TMAE 650 S (EM measured against 0.45 units of m-calpain caseinolytic Science, Gibbstown, NJ). Purified calpastatin consisted activity) or 0 units of calpastatin under the following only of a 68-kDa band when analyzed by SDS-PAGE conditions: 1) pH 7.5, 165 mM NaCl; 2) pH 7.5, 295 mM and had a specific activity of 451 U/mg of protein. One NaCl; 3) pH 6.5, 165 mM NaCl; 4) pH 6.5, 295 mM unit of activity of calpastatin was defined as the ability NaCl; 5) pH 6.0, 165 mM NaCl; or 6) pH 6.0, 295 to inhibit 1 unit of m-calpain caseinolytic activity mM NaCl. The buffers were either 50 mM HEPES (pH (Koohmaraie et al., 1995). 7.5 and 6.5) or 50 mM 2-(4-morpholino)-ethane sulfonic acid (MES; pH 6.0). Oxidation treatments consisted of Purification of ␮- and m-Calpain. ␮- and m-Cal- the addition 16 ␮M H O immediately prior to addition pain were purified according to the methods of Thomp- 2 2 of calpain. The reactions were initiated with the addi- son and Goll (2000) with minor modifications as de- tion of 100 ␮M CaCl (␮-calpain) or 1 mM CaCl (m- scribed by Maddock et al. (2005). Fractions from the Q- 2 2 calpain). Calpain activity and percentage of inhibition sepharose column containing ␮-calpain activity were by calpastatin [percentage of inhibition = 1 − (calpain pooled, and m-calpain activity was pooled. The ␮-cal- activity without calpastatin ÷ calpain activity with calpain was purified using successive chromatography pastatin) × 100] were measured at 60 min in a TD-700 over a Phenyl Sepharose 6 Fast Flow, Butyl Sepharose fluorometer (Turner Designs, Sunnyvale, CA). A control 4 Fast Flow (Amersham Biosciences), EMD TMAE 650 with calcium (without the addition of ␮- or m-calpain) S, and DEAE-TSK Toyopearl (Supelco, Bellefonte, PA). for each pH and ionic strength condition was conducted ␮ The purified -calpain had a specific activity of 71.3 U/ to determine whether calcium affected the fluorescence mg of protein. One unit of calpain was defined as the of the peptide. An EDTA control (20 mM EDTA final amount of calpain required to increase the absorbance concentration added prior to addition of calpain and at 278 nM of the supernatant by 1 unit during1hof calcium) was included for each pH and ionic strength ° incubation at 25 C because of the release of trichloro- condition and was used for the baseline. acetic acid-soluble polypeptides resulting from the di- gestion of casein (Koohmaraie, 1990). Myofibril Isolation Fractions containing m-calpain activity were pooled and purified using successive chromatography over a Porcine semimembranosus (100 g; 45 min postmor- Phenyl Sepharose 6 Fast Flow, Reactive Red 120 tem) was homogenized in ice cold extraction buffer [10 (Sigma, St. Louis, MO), and DEAE-TSK Toyopearl. Pu- mM EDTA, 2 ␮M E-64, 100 mg of trypsin inhibitor/L, rified m-calpain had a specific activity of 147 U/mg of 2mM phenylmethylsulfonylfluoride (PMSF), and 100 protein. The purified ␮-calpain, m-calpain, and calpas- mM Tris-HCl; pH 8.3] using a polytron PT 3100 (Kin- tatin were stored in TEM (1 mM EDTA, 0.1% (vol/vol) metica AG, Littau, Switzerland) set at 10,000 rpm. β-mercaptoethanol, 40 mM Tris-HCl, pH 7.4) with the Samples were centrifuged (20,000 × g) for 30 min at addition of 1 mM sodium azide at 4°C. 4°C. Pellets were homogenized in 10 vol of standard

Downloaded from www.journalofanimalscience.org at Serials Acquisitions Dept on February 24, 2014 Oxidation of calpain and calpastatin 927

salt solution (100 mM KCl, 2 mM MgCl2,1mM EGTA, Table 1. Carboxypeptidase Y control activity (%) based 1mM NaN3, and 20 mM K2HPO4; pH 7.0). Purification on measurements taken from reference (pH 7.5, 165 mM of myofibrils was completed by differential centrifuga- NaCl, control) after 60 min tion (Goll et al., 1974), and protein concentration was determined using the Biuret method as modified by 165 mM NaCl 295 mM NaCl

Robson et al. (1968). pH Control H2O2 Control H2O2

7.5 100 95.9 106 102 Incubation of Myofibrils with Calpain 6.5 99.3 102 92.1 92.7 6.0 96.4 95.6 93.4 98.1 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, lytic activity (Koohmaraie et al., 1995). A protein assay or 0.3 units measured against 0.45 units of m-calpain (Bradford, 1976) was conducted on each sample to de- caseinolytic activity/mL in the assay) under the follow- termine specific activity of each calpastatin sample. ing 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 Statistical Analysis 295 mM NaCl. The buffers were either 50 mM HEPES (pH 7.5 and 6.5) or 50 mM MES (pH 6.0). Oxidation Data were analyzed using a split-split plot design. treatment consisted of the addition of 0 or 100 ␮M H2O2. The whole plot was pH/ionic strength (7.5/165, 7.5/295, Concentrations of H2O2 were used based on concentra- 6.5/165, 6.5/295, 6.0/165, or 6.0/295). The split plot was tions used in previous studies (Guttmann et al., 1997). calpastatin (0, 0.15, or 0.30 units). The split-split plot ␮ ␮ The reactions were initiated with the addition of CaCl2 was H2O2 (0 or 16 M/100 M). Calpain activity at 60 at a final concentration of 100 ␮M (␮-calpain) or 1 mM min and densitometry of intact desmin were analyzed (m-calpain). Aliquots were removed at 2, 15, 60, and using PROC MIXED procedure in SAS (SAS Institute 120 min after the addition of CaCl2. The digests were Inc., Cary, NC). Least squares means were separated terminated by the addition 20 mM EDTA (final concen- using Tukey’s Honestly Significant Difference test. Sig- tration). The aliquots were centrifuged at 6,000 × g for nificance was defined as P < 0.05. 15 min at 4°C. A portion of the pellet was used for SDS- PAGE and immunoblotting to determine changes in RESULTS desmin. Experiments were replicated (n = 2) on different days. Carboxypeptidase Y Activity Control

SDS-PAGE and Western Blotting Ionic strength, pH, or H2O2 conditions (Table 1) used in these experiments did not alter susceptibility of the Sodium dodecyl sulfate-PAGE and membrane trans- peptide to carboxypeptidase Y activity. Fluorescence fer were conducted as described by Rowe et al. (2004a) measured at 60 min at pH 7.5,165 mM NaCl, and 0 using 10% polyacrylamide separating gels and a desmin ␮M H2O2 was defined as 100% of carboxypeptidase Y primary antibody (polyclonal rabbit antidesmin anti- activity. It was concluded that the synthetic peptide body, V2022, Biomedia, Foster City, CA) for desmin. A was not affected (P > 0.5) by the differing pH, ionic sensitive chemiluminescent (ECL Plus kit, Amersham strength, and oxidation conditions and was appropriate Biosciences) system was used to detect labeled protein to use for our study. bands using a charged coupled device camera (Fluro- Chem 8800, Alpha Innotech Corporation, San Leandro, Calpastatin Oxidation CA) and FluorChem IS-800 software (Alpha Innotech Corporation). Densitometry was completed using the Irreversibly blocking sulfhydryls with NEM did not AlphaEaseFC software (Alpha Innotech Corporation). alter the specific activity of calpastatin. The specific A reference sample was used on each blot to standardize activity of control calpastatin was 416 units of activity/ densitometry data to compare differences between mg of protein. The mean specific activity of the oxidized blots. calpastatin with NEM was 406.5, 410.5, and 398 units of activity/mg of protein for 4, 8, and 12 mM NEM, re- Oxidation of Calpastatin spectively.

Calpastatin (10 units) was incubated with an irre- Calpain Activity Assays versible sulfhydryl blocking agent N-ethylmaleimide (NEM;0,4,8,or12mM ﬁnal concentration; n = 2) for ␮-Calpain Activity. Fluorescence measurements at 1hat4°C. Samples were exhaustively dialyzed in 1 60 min indicated that the addition of H2O2 signiﬁcantly mM EDTA and 40 mM Tris-HCl (pH 7.5) to remove decreased (P < 0.05; Table 2) ␮-calpain activity regard- NEM. Activity of calpastatin after dialysis was deter- less of pH and ionic strength conditions. ␮-Calpain had mined by measuring inhibition of m-calpain caseino- greater activity at pH 6.5 (P < 0.01) than at pH 7.5 or

Downloaded from www.journalofanimalscience.org at Serials Acquisitions Dept on February 24, 2014 928 Maddock Carlin et al. ± ␮ Table 2. Least squares means ( SE) of -calpain activity at 60 min after addition of CaCl2

165 mM NaCl 295 mM NaCl

pH and calpastatin Control H2O2 Control H2O2

7.5 0 units 63.99 ± 0.69d,w 36.73 ± 2.48c,y 40.77 ± 1.51b,x 13.81 ± 0.69c,z 0.15 units 50.16 ± 0.66e,x 32.15 ± 4.19c,y 33.18 ± 0.88c,y 12.98 ± 0.85c,z 0.30 units 35.22 ± 2.13f,x 22.98 ± 4.42d,y 20.67 ± 1.21e,y 9.14 ± 1.65de,z 6.5 0 units 117.72 ± 5.02a,w 50.07 ± 1.98a,y 58.99 ± 1.54a,x 25.44 ± 1.61a,z 0.15 units 102.68 ± 1.43b,w 43.72 ± 1.97b,x 39.86 ± 1.79b,y 19.24 ± 1.36b,z 0.30 units 78.12 ± 3.74c,w 36.23 ± 0.46c,x 26.41 ± 0.67d,y 13.83 ± 0.36c,z 6.0 0 units 11.85 ± 0.17g,y 6.45 ± 0.36e,z 18.25 ± 1.26e,x 11.48 ± 0.85c,y 0.15 units 8.94 ± 0.54h,y 4.54 ± 1.10f,z 12.31 ± 0.28f,x 8.59 ± 0.11d,y 0.30 units 7.97 ± 0.28i,x 4.59 ± 0.36f,y 8.48 ± 0.49g,x 7.66 ± 0.39e,x a–iWithin a column, least squares means lacking a common superscript differ (P < 0.05;n=3). w–zWithin a row, least squares means lacking a common superscript differ (P < 0.05).

6.0. ␮-Calpain activity was lower (P < 0.05) at 295 mM of H2O2 at pH 7.5 and 295 mM NaCl, 0.15 units of NaCl than at 165 mM NaCl. calpastatin only inhibited 5.99% of ␮-calpain activity. m-Calpain Activity. Similar to ␮-calpain, addition Higher ionic strength increased (P < 0.05) percentage of H2O2 signiﬁcantly decreased (P < 0.01; Table 3) activ- of inhibition of ␮-calpain activity by calpastatin at pH ity of m-calpain at all pH and ionic strength conditions. 6.5 and 6.0, but not at pH 7.5. The addition of H2O2 Furthermore, the presence of H2O2 in the assays com- not only caused a decrease in activity of ␮-calpain, as pletely prevented proteolytic activity of m-calpain at discussed previously, but also caused a decrease (P < pH 6.5 and 295 mM NaCl with or without the presence 0.05) in the percentage of inhibition of ␮-calpain activity of calpastatin. In contrast to ␮-calpain, m-calpain had by calpastatin at pH 7.5 and 6.5 at both ionic strengths greater activity at pH 7.5 (P < 0.01) than at pH 6.5. and at pH 6.0 and 295 mM NaCl. The effect (P > 0.05) Higher ionic strength decreased (P < 0.05) m-calpain of oxidation was not signiﬁcant at pH 6.0 and 165 activity. No measurable activity of m-calpain was de- mM NaCl. tected at pH 6.0, which was previously observed by m-Calpain Inhibition by Calpastatin. Calpastatin Maddock et al. (2005). inhibited (P < 0.05; Table 5) m-calpain activity at all ␮-Calpain Inhibition by Calpastatin. Calpastatin pH and ionic strength conditions. There was no main inhibited (P < 0.05; Table 4) ␮-calpain activity at all effect (P > 0.05) of pH on percentage of inhibition of m- pH and ionic strength conditions. Percentage of inhibi- calpain by calpastatin. Inhibition percentage was not tion of ␮-calpain activity by calpastatin was not affected reported at pH 6.5 and 295 mM NaCl because no m- by pH (P > 0.05), but it is noteworthy that at pH 6.5 calpain activity was detected, even when 0 units of and 165 mM NaCl, only 12.77% inhibition occurred calpastatin was added to the assay. An ionic strength when 0.15 units of calpastatin was added to the assay. effect (P < 0.05) was observed where, at 295 mM NaCl, This lower percentage of inhibition of ␮-calpain activity the percentage of inhibition of m-calpain by calpastatin by calpastatin was not observed when the higher ionic was greater than at 165 mM NaCl, with the exception of strength was used at pH 6.5. However, in the presence assays conducted at pH 7.5 and 0.3 units of calpastatin.

± Table 3. Least squares means ( SE) of m-calpain activity at 60 min after addition of CaCl2

165 mM NaCl 295 mM NaCl

pH and calpastatin Control H2O2 Control H2O2

7.5 0 units 93.14 ± 7.15a,x 23.59 ± 2.37a,z 53.83 ± 1.41a,y 26.44 ± 1.19a,z 0.15 units 46.34 ± 1.86b,x 15.06 ± 1.17b,z 21.52 ± 0.54b,y 16.21 ± 0.21b,z 0.30 units 21.38 ± 1.90c,x 7.92 ± 1.00c,z 11.83 ± 0.56c,y 6.92 ± 0.76c,z 6.5 0 units 18.00 ± 0.41d,x 7.82 ± 0.81c,y 6.34 ± 2.10d,y ND1 0.15 units 8.27 ± 0.33e,x 2.95 ± 1.54d,y 1.34 ± 0.47e,y ND 0.30 units 6.24 ± 0.40f,x 2.92 ± 0.17d,y 0.09 ± 0.19f,z ND

a–fWithin a column, least squares means lacking a common superscript differ (P < 0.05;n=3). w–zWithin a row, least squares means lacking a common superscript differ (P < 0.05). 1ND = not detected.

Downloaded from www.journalofanimalscience.org at Serials Acquisitions Dept on February 24, 2014 Oxidation of calpain and calpastatin 929 Table 4. Least squares means (±SE) of percentage of inhibition of ␮-calpain by 0.15 and 0.3 units of calpastatin1 calculated from ﬂuorescence measurements taken at 60 min after the addition of CaCl2

165 mM NaCl 295 mM NaCl

pH and calpastatin Control H2O2 Control H2O2

7.5 0.15 units 21.61 ± 2.16c,x 12.47 ± 6.19a,yz 18.62 ± 2.16c,xy 5.99 ± 6.18d,z 0.30 units 44.96 ± 2.95a,y 37.44 ± 6.95a,z 49.29 ± 2.95a,y 33.83 ± 6.95b,z 6.5 0.15 units 12.77 ± 1.21d,x 12.67 ± 3.93a,x 32.44 ± 3.04b,y 24.37 ± 5.38d,x 0.30 units 33.64 ± 3.18b,w 27.63 ± 0.93a,x 55.23 ± 1.15a,z 45.65 ± 1.42a,y 6.0 0.15 units 24.54 ± 4.62c,z 29.56 ± 15.70ab,yz 32.53 ± 1.51b,y 25.19 ± 0.94c,z 0.30 units 32.69 ± 2.39b,x 28.73 ± 5.55a,x 53.52 ± 2.66a,y 33.29 ± 3.35b,x

a–dWithin a column, least squares means lacking a common superscript differ (P < 0.05;n=3). x–zWithin a row, least squares means lacking a common superscript differ (P < 0.05). 1Inhibition was calculated as percentage of inhibition = [1− (calpain activity without calpastatin ÷ calpain activity with calpastatin)] × 100 (n = 3).

Interestingly, the same effect [H2O2 causing a decrease The combination of calpastatin and H2O2 appears to (P < 0.05) in percentage of inhibition of m-calpain by work synergistically to limit the activity of ␮-calpain calpastatin], which was observed with m-calpain, was at pH 6.0. The inhibition of ␮-calpain by calpastatin also observed for ␮-calpain. This effect was observed at was very different at pH 6.5 and 7.5. The addition of pH 7.5 and 165 mM NaCl at both calpastatin levels H2O2 appears to decrease the degradation of desmin and pH 7.5 and 295 mM NaCl when 0.15 units of calpas- under the conditions when 0 units of calpastatin was tatin was present. added to the digests. This is apparent at both pH and ionic strength conditions and is consistent with assays Digest of Myoﬁbrils and Western Blots with the calpain substrate. Unexpected results oc- of Desmin Degradation curred when calpastatin was added to the digests. These results are best evaluated when observing the ␮-Calpain Myoﬁbril Digests. Differences in desmin blots from the digests at pH 6.5 and 165 mM NaCl. It degradation caused by oxidation and calpastatin were appears that the addition of H2O2 in the presence of observed (Figure 1; Table 6). At pH 6.0, calpastatin calpastatin results in greater degradation of intact des- decreased degradation of desmin as indicated by detec- min. When H2O2 is added to the digests in the presence tion of intact desmin at 60 and 120 min of incubation of 0 units of calpastatin, intact desmin is detected as with increasing levels of calpastatin at both 165 and late as 120 min of incubation. When 0.15 units of calpas- 295 mM NaCl. An effect of oxidation was observed tatin was added, the degradation of intact desmin is where the addition of H2O2 to the digest decreased the almost complete by 60 min as indicated by the very degradation of desmin; this was particularly obvious at light band. Finally, when 0.3 units of calpastatin was 295 mM NaCl when 0 units of calpastatin was added added to the assay, degradation of intact desmin ap- to the digests, and intact desmin was detected at all pears to be almost complete by 15 min. A similar pattern time points. is observed at 295 mM NaCl and pH 7.5 at both ionic

Table 5. Least squares means (±SE) of percentageof inhibition of m-calpain by 0.15 and 0.3 units of calpastatin1 calculated from ﬂuorescence measurements taken at 60 min after the addition of CaCl2

165 mM NaCl 295 mM NaCl

pH and calpastatin Control H2O2 Control H2O2

7.5 0.15 units 50.24 ± 2.01c,x 36.15 ± 4.96b,z 60.01 ± 1.01c,w 38.68 ± 0.81b,z 0.30 units 77.05 ± 2.04a,x 66.41 ± 4.23a,y 78.03 ± 3.04b,x 73.84 ± 2.13a,x 6.5 0.15 units 54.07 ± 1.84c,x 62.23 ± 19.71a,y 78.94 ± 7.44b,z — 0.30 units 65.35 ± 2.25b,y 62.74 ± 2.22a,y 98.53 ± 3.02a,z —

a–cWithin a column, least squares means lacking a common superscript differ (P < 0.05;n=3). x–zWithin a row, least squares means lacking a common superscript differ (P < 0.05). 1Inhibition was calculated as percentage of inhibition = [1− (calpain activity without calpastatin ÷ calpain activity with calpastatin)] × 100 (n = 3).

Downloaded from www.journalofanimalscience.org at Serials Acquisitions Dept on February 24, 2014 930 Maddock Carlin et al.

Figure 1. Western blots of desmin degraded by ␮-calpain. Each lane was loaded with 20 ␮g 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 ␮-calpain.

strength conditions. These effects are similar to the Densitometry data describing intact desmin degrada- results observed in the calpain assays where percentage tion (n = 2) are shown in Table 6, and statistical main of inhibition of ␮-calpain was decreased in the presence effects and interactions are shown in Table 7. After of H2O2. 2 min of incubation, no signiﬁcant main effects were

Downloaded from www.journalofanimalscience.org at Serials Acquisitions Dept on February 24, 2014 Oxidation of calpain and calpastatin 931 ± ± ± ± ± ± ± ± xyz wxyz wxyz yz yz yz yz z 0 0.38 0.21 0.27 0.04 0.27 0.14 0.01 0.24 0.38 0.27 0.14 0.01 0.21 0.04 0.45 0.27 ± ± ± ± ± ± ± ± ± ab,xyz ab,yz ab,yz ab,yz ab,yz b,yz ab,xyz a,wx ab 2 0.43 0.26 0.23 0.18 0.16 0.36 0.29 0.32 0.09 0.05 0.05 0.17 0.17 0.11 0.28 0.16 0.58 0.39 O 2 H ± ± ± ± ± ± ± ± ± ab,x b,y b,xyz ab,yz a,x b,wx ab,wxyz ab,wx ab 0.64 0.53 0.71 0.83 0.44 0.65 0.13 0.02 0.13 0.57 0.03 0.04 0.04 0.09 0.38 0.72 0.53 ± ± ± ± ± ± ± ± ± x y xyz x wxyz wx wx yz NaCl 0.03 0.17 0.44 0.18 0.23 0.11 0.12 0.77 0.70 0.94 0.52 1.16 0.59 0.92 0.15 0.21 1.11 0.72 0.63 ± ± ± a,yz a,xy ab 295 m M b,z b,z b,z b,z b,z b,z 0 0.21 0 0 0 0.41 0 0 0.02 0.73 0.02 0.41 ± ± ± ± ± a,xy b,xyz ab b,z bz b,z b,z b,z b,z 0.09 0.07 0.35 0 0 0 0 0.09 0.09 0.84 0.03 0.03 0.07 0.35 Control ± ± ± ± ± ± ± ± ± ab,wxy ab,xyz ab,y a,xy ab,yz ab,wxyz b,xyz b,wyz ab 0.19 0.50 0.20 0.40 0.12 1.02 0.12 0.63 0.56 0.53 0.49 0.19 0.11 0.28 0.11 0.63 0.39 ± ± ± ± ± ± ± ± ± x y x wxyz wx wx yz w 0.05 0.07 0.06 0.57 0.12 0.14 0.75 0.67 0.90 1.11 0.65 0.99 0.70 0.05 0.71 0.78 0.72 0.03 0.28 ± ± ± ± ± ± ± ± ab,wxy ab,z ab,yz ab,x ab,yz ab,wxyz a,y ab b,z 0.51 0.07 0.07 0.15 0.15 0.12 0.12 0.76 0.65 0.30 0.30 0.15 0.03 0 0.68 0.41 0.28 ␮ -calpain (n = 2) 0.05). < 0.05). ± ± ± ± ± ± ± ± P ab,xy bcd,z bcd,yz abc,yz abc,yz abc,wxyz a,wx ab < 2 O P c,z 2 0.10 0.10 0.10 0.16 0.06 0.27 0.19 0.89 0.36 0.59 0.32 0.16 0 0.75 0.10 0.55 H ± ± ± ± ± ± ± ± ± xyz y xy x wxyz wxy yz w 0.30 0.10 0.28 0.02 0.50 0.13 0.48 0.70 0.68 1.25 0.51 0.78 1.03 0.41 0.83 0.01 0.78 ± ± ± ± ± ± ± ± ± xyz y xy wxyz wx wx yz yz NaCl 0.25 0.36 0.24 0.56 0.09 0.11 1.33 0.89 0.66 0.38 0.57 0.91 0.73 1.34 0.91 0.60 0.80 ± ± 165 m M yz z z z z z z z 0 0 0 0 0 0 0 0.05 0.05 0.38 ± ± ± ± yz yz yz z z z z z 0 0 0 0 0 0.06 0.02 0.04 0.45 0.06 0.02 0.04 Control ± ± ± ± ± ± ± ± ± y y wxyz wxyz wx wx yz yz 0.09 0.05 0.23 0.46 0.06 0.09 0.21 0.41 0.61 0.45 1.11 0.54 0.60 0.72 0.10 0.38 0.43 ± ± ± ± ± ± ± ± ± xy xy y wxy wx yz w w 0.20 0.45 0.47 0.07 0.08 0.04 0.06 0.12 0.45 0.38 0.07 0.02 0.10 0.63 0.87 Relative intensity ( ± SE) of intact desmin after incubation with Within a row, least squares means lacking a common superscript differ ( Within a column, least squares means lacking a common superscript differ ( a–c w–z 0 units0.15 units 0.73 0.72 0 units0.15 units 1.27 1.13 0.3 units 1.36 0 units0.15 units 0.79 0.71 0.3 units 0.90 0.3 units 0.17 Table 6. pH and calpastatin 2 min 15 min 60 min 120 min 2 min 15 min 60 min 120 min 2 min 15 min 60 min 120 min 2 min 15 min 60 min 120 min 7.5 6.5 6.0

Downloaded from www.journalofanimalscience.org at Serials Acquisitions Dept on February 24, 2014 932 Maddock Carlin et al. Table 7. P-values of main effects and interactions of environmental conditions on ␮- calpain degradation of desmin

Treatment effect 2 min 15 min 60 min 120 min

pH 0.08 0.45 0.78 0.74 Ionic strength 0.44 0.48 0.97 0.92 Calpastatin 0.94 0.48 0.41 0.58 Oxidation 0.77 0.04 0.001 0.04 pH × ionic strength 0.18 0.46 0.80 0.89 pH × calpastatin 0.36 0.61 0.23 0.33 pH × oxidation 0.35 0.22 0.02 0.21 Ionic strength × calpastatin 0.72 0.52 0.87 0.88 Ionic strength × oxidation 0.79 0.15 0.008 0.13 Calpastatin × oxidation 0.99 0.24 0.007 0.05 pH × ionic strength × calpastatin 0.29 0.32 0.66 0.71 pH × ionic strength × oxidation 0.66 0.24 0.07 0.48 pH × calpastatin × oxidation 0.91 0.56 0.03 0.12 Ionic strength × calpastatin × oxidation 0.98 0.42 0.06 0.17 pH × ionic strength × calpastatin × oxidation 0.89 0.31 0.19 0.36

observed. There was a trend toward an effect of pH (P = calpastatin (P = 0.002) appeared, indicating that cal- 0.08) where, at pH 6.5, degradation of desmin was less pastatin was inhibiting degradation of intact desmin than at pH 7.5 and 6.0 at all 3 levels of calpastatin. by m-calpain. An interaction of pH × calpastatin (P < After 15 min of incubation, an effect of oxidation (P = 0.001) occurs because adding more calpastatin de- 0.04) was observed. The addition of H2O2, in general, creased desmin degradation at pH 7.5 but not at pH caused a decrease in the degradation of desmin, as indi- 6.5. A main effect of oxidation does not appear in the cated by higher densitometry measurements. At 60 min analysis of densitometry measurements, but there was of incubation, many effects (P < 0.05) were observed. an interaction of pH × oxidation (P < 0.001) and calpas- Oxidation decreased (P < 0.001) desmin degradation as tatin × oxidation (P = 0.005), resulting in the pH × indicated by more intact desmin. The interactions of calpastatin × oxidation interaction (P = 0.002), where, pH × oxidation, calpastatin × oxidation, and pH × cal- at pH 7.5, the addition of H2O2 inhibited desmin degra- pastatin × oxidation were significant. At pH 6.0, the dation when 0 units of calpastatin was added at both combination of H2O2 and calpastatin appeared to be ionic strength conditions and at pH 6.5; there was not additive in its inhibitory effects on ␮-calpain activity a significant effect of oxidation. The ionic strength × vs. pH 7.5 and 6.5, where the combination appeared to calpastatin interaction (P = 0.013) indicated that cal- decrease inhibition. After 120 min of incubation, many pastatin was a better inhibitor of desmin degradation of the significant effects observed at 60 min were not by m-calpain at 295 mM NaCl than at 165 mM NaCl, apparent. This was due to the almost complete degrada- which is consistent with the results observed in the tion of intact desmin that occured by 60 min under the fluorescent assays (Table 5). By 120 min of incubation, different environmental conditions. An effect of oxida- there is a reduction in the significance of the particular tion (P = 0.04) was still observed where, in general, the main effects and interactions observed at 60 min. presence of H2O2 decreased degradation of desmin by ␮-calpain. DISCUSSION m-Calpain Myofibril Digests. Visual analysis of Western blots for desmin from the myofibril digests During the conversion of muscle to meat, many with m-calpain showed greater proteolysis of intact des- changes occur within the environment of the muscle min at pH 7.5 than at pH 6.5 and less proteolysis at (Winger and Pope, 1981). Loss of homeostasis and de- 295 mM NaCl than at 165 mM NaCl (Figure 2). More pleted oxygen ultimately allow for these changes, which intact desmin was observed when calpastatin and H2O2 include a pH decline from near neutrality to approxi- were present. In these samples, the addition of calpas- mately 5.6 and a continued increase in ionic strength. tatin and H2O2 appeared to be additive in the inhibitory These changes, when combined with the temperature effect on m-calpain at pH 6.5 and 7.5. decrease that occurs during harvest, can affect other Densitometry data describing intact desmin degrada- postmortem processes that affect meat quality. These tion are shown in Table 8, and main effects and interac- specific environmental changes have been shown to af- tions are shown in Table 9. After 15 min of incubation, fect activity of calpains (Kendall et al., 1993; Huff-Lon- the pH × calpastatin interaction (P = 0.013) indicated ergan et al., 1996). Another change in environment that that there was more degradation of desmin at pH 7.5 occurs with the loss of homeostasis is an increase in and 0 units of calpastatin than at 0.15 and 0.3 units of oxidative conditions (Martinaud et al., 1997; Harris et calpastatin at pH 7.5 and at all levels of calpastatin al., 2001). Oxidation can also affect calpain activity. A at pH 6.5. By 60 min of incubation, a main effect of study by Guttmann et al. (1997) evaluated ␮-calpain

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Figure 2. Western blots of desmin degraded by m-calpain. Each lane was loaded with 20 ␮g 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.

activity when exposed to H2O2 and observed that pro- early postmortem muscle. Two different methods were teolytic activity of ␮-calpain was strongly inhibited. used to evaluate the sum of the effects of pH, ionic Rowe et al. (2004b) used irradiation to demonstrate strength, and oxidation of ␮- and m-calpain activity in that inactivation of ␮-calpain resulted in a decrease in the presence or absence of calpastatin. The first method postmortem proteolysis and a decrease in tenderiza- used a calpain substrate to precisely measure activity tion. Based on these observations, it is important to of ␮- and m-calpain and their inhibition by calpastatin. understand how environmental conditions of pH, ionic The second method used purified myofibrils as a sub- strength, and oxidation interact to affect calpain activ- strate and evaluation of degradation of the protein des- ity and additionally how the presence of calpastatin min. This allowed definition of the effects of environ- adds to these effects. ment on the activities of ␮- and m-calpain and their In the current study, the objective was to determine inhibition by calpastatin using a substrate found in the extent to which oxidation influences ␮- and m-cal- meat. It was concluded that both the calpain activity pain activity and calpain inhibition by calpastatin un- and inhibition of calpain by calpastatin can be dramati- der the pH and ionic strength conditions observed in cally affected by pH, ionic strength, and oxidation. The

Downloaded from www.journalofanimalscience.org at Serials Acquisitions Dept on February 24, 2014 934 Maddock Carlin et al. effects of environment were profoundly different be- ± ± ± ± ± ab,yz ab a,y ab

ab tween ␮- and m-calpain. b,z 0 0.62 0.05 1.08 1.13 0.68 0.61 0.42 pH and Ionic Strength ± ± ± ± ± ± y xy yz A previous study was conducted using the calpain 2 0.03 0.10 1.14 0.55 1.10 0.73 0.63 0.39 0.47

O substrate to evaluate ␮- and m-calpain activity in the 2

H presence of reducing agents (Maddock et al., 2005). ± ± ± ± ± ± x xy yz Ionic strength and pH effects similar to calpain activity 0.10 0.02 1.12 1.11 0.77 0.71 0.87 0.23 0.56 0.21 0.33 0.37 assay results in the current study were observed on the activities of m- and ␮-calpain and their inhibition by ± ± ± ± ± ± x ab yz w calpastatin. As observed in this study, m-calpain activity was also greatest at pH 7.5, and activity was least NaCl 0.17 0.23 1.15 1.17 0.88 0.78 0.92 0.40 0.230.85 0.270.02 0.410.33 0.47 0.38 0.38at pH 0.4 6.0. Additionally, ␮-calpain activity was also greater at pH 6.5 vs. pH 7.5 or 6.0. Western blotting of ± ± ± ± a,y ab ab ab ␮ 295 m M -calpain autolysis revealed that autolysis occurred at b,z b,z 0 0.07 0.42 0 0.29 0.43 0.80 1.14 0.75 0.65 a slower rate at pH 6.5 than at pH 7.5. It was hypothesized that the observed activity differences were not ± ± ± ± ± ␮

a,y a a,y a a due to a faster activation of -calpain, but rather to a ␮ b,z slower autolytic inactivation of -calpain at pH 6.5. It 0 0.23 0.44 0.16 1.09 1.10 0.78 0.81 0.86 was also determined that the rate of hydrolysis in the Control ± ± ± ± ± ± ﬁrst 5 min of the assay was greater at pH 6.5 than at xyz a wy yz pH 7.5. The ionic strength effects observed in Maddock 0.46 0.51 0.03 1.15 1.15 0.79 0.86 0.87 0.95 0.48 et al. (2005) and the current study indicate that both ␮- and m-calpain activities are decreased with increasing ± ± ± ± ± ± x a wx yz ionic strength. Geesink and Koohmaraie (2000) observed that ␮-calpain activity decreased with increas- 0.10 0.36 0.20 0.47 0.060.23 0.18 0.15 0.20 0.37 1.06 1.19 0.89 0.88 1.01 1.02 ing ionic strengths and indicated that the decrease in ␮ ± ± ± ± ± ± activity of -calpain was due to a decrease in the stabil- a,y a ab,yz ab a b,z ity of the molecule. Li et al. (2004) hypothesized that an 0.06 0.04 0.27 0.38 0.17 0.14 1.04 1.05 0.50 0.65 0.14 0.44 elevated ionic strength (equal to 100 mM KCl) caused disassociation of the 2 calpain subunits, allowing for ± ± ± ± ± ± 0.05). ab,xy ab,y ab b,yz abc a formation of dimers and trimers of the large subunit, < 0.05). P 2 1.27 1.03 0.60 0.64 0.59 0.09 0.37 0.45 0.02 which irreversibly inactivated the proteinase. These re- < O 2 P sults indicate that pH and ionic strength have the po- H ± ± ± ± ± ± tential to affect activity of ␮- and m-calpain through x y yz autolytic inactivation and dissociation. 0.30 0.22 1.29 1.09 0.73 0.94 0.95 0.41 0.80 In the current study, inhibition of ␮-calpain and m- calpain by calpastatin were not affected by pH. This is ± ± ± ± ± ± x wx yz consistent with reports by Geesink and Koohmaraie NaCl 0.22 0.33 1.33 0.43 1.25 0.92 0.98 0.91 1.13 (1999) and Otsuka and Goll (1987). Calpastatin is the endogenous inhibitor of ␮- and m-calpain and does not ± ± ± ±

z inhibit any other protease that it has been tested

165 m M 2+

z z against (Goll et al., 2003). The presence of Ca is re- 0 0 0.06 0.65 0.520.38 0.550.33 0.23 0.54 0.14 0.12 0.10 0.26 0.05 0.17 0.66 0.38 0.40 quired for calpastatin to bind to the calpains to inhibit

± ± ± ± ± their activity (Cottin et al., 1981). In porcine muscle, ab,y abc b,y abc a the ratio of calpastatin activity to ␮-calpain activity is c,z 0.21 0.02 0 0.84 0.99 0.24 0.48 0.66 approximately 1.5:1 (Ouali and Talmant, 1990). The ratio of calpastatin to calpain used in this study was 1:3 Control ± ± ± ± ± ±

xyz y wx and 2:3 to evaluate the effect of calpastatin on calpain without the occurrence of complete inhibition. Maddock 0.24 0.09 0.22 0.55 0.93 1.14 1.07 0.67 0.93 et al. (2005) reported an effect of pH on inhibition of

± ± ± ± ± ± m-calpain, where inhibition was greater at pH 6.5 than x y w at pH 7.5. The discrepancy between this study and the 0.13 0.09 0.19 0.250.03 0.44 0.12 0.26 0.14 0.33 0.04 0.03 previous study is likely due to the presence of a reducing

Relative intensity ( ± SE) of intact desmin after incubation with m-calpain (n = 2) agent in Maddock et al. (2005) when the activity of m-calpain was maximized; in this study, no reducing and Within a row, least squares means lacking a common superscript differ ( Within a column, least squares means lacking a common superscript differ ( × agents were used to maximize the effect of oxidation a–c w–z 0 units0.15 units 0.93 0.94 0.3 units 1.12 0 units0.15 units 0.93 0.83 0.3 units 0.95 Table 8. pH calpastatin 2 min 15 min 60 min 120 min 2 min 15 min 60 min 120 min 2 min 15 min 60 min 120 min 2 min 15 min 60 min 120 min 7.5 6.5 on activity and inhibition.

Downloaded from www.journalofanimalscience.org at Serials Acquisitions Dept on February 24, 2014 Oxidation of calpain and calpastatin 935 Table 9. P-values of main effects and interactions of environmental conditions on m- calpain degradation of desmin

Treatment effect 2 min 15 min 60 min 120 min

pH 0.08 0.08 0.20 0.55 Ionic strength 0.99 0.99 0.49 0.24 Calpastatin 0.46 0.11 0.002 0.003 Oxidation 0.38 0.54 0.12 0.15 pH × ionic strength 0.32 0.37 0.55 0.41 pH × calpastatin 0.10 0.01 0.0006 0.002 pH × oxidation 0.19 0.21 0.047 0.09 Ionic strength × calpastatin 0.60 0.21 0.012 0.001 Ionic strength × oxidation 0.71 0.95 0.36 0.08 Calpastatin × oxidation 0.72 0.36 0.005 0.005 pH × ionic strength × calpastatin 0.29 0.07 0.011 0.003 pH × ionic strength × oxidation 0.57 0.71 0.24 0.29 pH × calpastatin × oxidation 0.32 0.11 0.002 0.001 Ionic strength × calpastatin × oxidation 0.87 0.64 0.047 0.006 pH × ionic strength × calpastatin × oxidation 0.76 0.48 0.05 0.006

Oxidation This important observation provides evidence that oxidation of calpastatin does not affect its action on inhibi- ␮ Because - and m-calpain are cysteine proteases, tion of calpains. their activity requires the exchange of electrons be- Many of the singular effects of environment did not tween the active site cysteine and histidine residues appear to be significant on ␮- or m-calpain activity. (Medhi, 1991). The cysteine residue site is highly sus- However, the specific objectives of these experiments ceptible to oxidation by H2O2 (Neumann, 1972). Oxida- were to evaluate the interactions between environmen- tion of calpain with H2O2 can allow for inactivation of tal conditions. The interaction of oxidation and calpas- calpain (Guttmann et al., 1997), indicating that the tatin indicates that these environmental combinations active site cysteine residue may be oxidized. Oxidation do have an effect on the proteolytic activity of ␮- and of proteins does occur during postmortem aging (Mar- m-calpain; therefore, they also have implications in tinaud et al., 1997; Rowe et al., 2004a), as antioxidants postmortem muscle and in living muscle tissue. are expended, and is indicated by increased protein The presence of H2O2 decreased the percentage of carbonyl content and decreased sulfhydryl concentra- inhibition of both ␮- and m-calpain by calpastatin when tion. An oxidative environment decreases degradation using the calpain substrate. This corresponds to the of substrates by calpain (Guttmann et al., 1997; Gutt- effects observed in the ␮-calpain-degraded myofibrils, mann and Johnson, 1998), and this oxidation is shown where the addition of H2O2 in the presence of calpas- to be reversible (Rowe et al., 2004b). Oxidation or more tatin allowed for greater degradation of desmin at the specifically, thiolation, is a common reaction of H2O2 higher pH of 7.5 and 6.5. These observations were with sulfhydryl groups that occurs quickly and is re- unique to ␮-calpain in the digests of myofibrils. versible with the introduction of a reducing environ- The role of calpastatin in binding to and inhibiting ment (Saurin et al., 2004). Hydrogen peroxide is widely calpain is an area of research that is currently getting produced in cells and allows for the reversal of oxida- some attention, as the exact mechanism is not under- tion; thereby, it is appropriate for the study of oxidation stood (Goll et al., 2003). As previously discussed, Ca2+ in muscle and meat. is required for binding of calpastatin to calpains (Cottin Oxidation decreased the proteolytic activity of ␮-and et al., 1981). The Ca2+ concentration required for the m-calpain (Tables 2 and 3) in the activity assays. When binding of calpastatin to ␮- and m-calpain is lower than using myofibrils as a substrate, the main effect of oxida- the Ca2+ concentration required for proteolytic activity tion (Table 7) was specifically noted on ␮-calpain. The at half-maximal rate. This indicates that, if present, effect of oxidation on m-calpain (Table 9) in this system calpastatin will bind and inhibit calpain before it can was detected only when also considering interactions become proteolytically active (Kapprell and Goll, 1989). with pH and ionic strength in combination with calpas- Calpastatin is considered a competitive inhibitor of cal- tatin. Previous studies observed similar effects of oxida- pains based on kinetic evidence (Croall and McGrody, tion on ␮-calpain activity in meat (Rowe et al., 2004b) 1994). However, evidence indicates that calpastatin and in vitro (Guttmann et al., 1997; Guttmann and does not bind at the active site of calpain (Nishimura Johnson, 1998). and Goll, 1991; Croall and McGrody, 1994), but rather Evidence that cysteine residues of calpastatin are near the active site in a way that blocks access of sub- not affected by oxidation is provided in this study by strates as hypothesized by Todd et al. (2003). In the incubations with NEM, which covalently blocks sulfhy- current study, when a calpastatin/␮-calpain complex dryl groups, resulting in effects similar to oxidation. was formed and then exposed to H2O2, greater degrada-

Downloaded from www.journalofanimalscience.org at Serials Acquisitions Dept on February 24, 2014 936 Maddock Carlin et al. tion of desmin was observed at pH 6.5 and 7.5. Oxida- ment of calpastatin regulates oxidation of ␮-calpain and tion of the active site cysteine residue decreases calpain allows activation to occur. activity (Guttmann et al., 1997). It is possible that the In summary, activity differences were observed be- interaction of calpastatin with ␮-calpain occurs in a cause of pH, where ␮-calpain had greater activity at way that blocks H2O2 from accessing and oxidizing the pH 6.5 than at pH 7.5; the opposite was true for m- active site cysteine residue, thus preventing oxidative calpain. Oxidation of m-calpain with H2O2 resulted in inactivation of ␮-calpain. a decrease in proteolytic activity, and the addition of The results of this study indicate that ␮-calpain and calpastatin decreased proteolytic activity even further m-calpain react differently to different environments. as demonstrated by the decrease in desmin degradation ␮ Interestingly, the environmental conditions that are (Figure 2). Oxidation of -calpain with H2O2 did de- observed in postmortem muscle are the conditions that crease proteolytic activity, but oxidation of the calpas- ␮ appear to be most favorable for activation of ␮-calpain. tatin/ -calpain complex with H2O2 resulted in in- This is consistent with previous hypotheses (as re- creased proteolysis of desmin (Figure 1) and, therefore, viewed by Geesink et al., 2000) that indicate ␮-calpain appeared to increase activity of ␮-calpain in the pres- and not m-calpain is responsible for the proteolysis that ence of H2O2. This novel observation is important be- ␮ occurs in postmortem muscle and, therefore, subse- cause it demonstrates that inhibition of -calpain by quent tenderization. calpastatin is diminished by oxidation. The effects of oxidation observed in this study have implications for meat quality. Rowe at al. (2004b) evalu- IMPLICATIONS ated how oxidative conditions initiated by irradiation early postmortem caused inhibition of ␮-calpain activ- Oxidative conditions in postmortem muscle could ity, slowing the rate of proteolysis occurring during have an effect on proteolytic activity that occurs during aging and, thereby, decreasing tenderization. Antioxi- the aging process. The differences observed between ␮ dants are currently becoming more prevalently used in oxidation of -calpain vs. m-calpain provide further evi- ␮ the meat industry to increase shelf-life by preventing dence of the role of -calpain in postmortem proteolysis. oxidation of lipids that cause rancidity. Previous re- The results may also explain some of the variation ob- search has also evaluated the use of antioxidants as a served in tenderness, as the interactions of pH, ionic feed supplement and how their use can affect meat strength, and oxidation in addition to the presence of ␮ tenderization. Harris et al. (2001) evaluated increased calpastatin cause -calpain to act differently than if levels of α-tocopherol in beef through supplemental vi- examined under each environmental factor alone. tamin E in the diet in combination with injecting steaks These results also indicate a need for greater under- with CaCl . They found that beef with increased levels standing of the interaction of calpastatin with the cal- 2 pains to understand how these proteins function in the of α-tocopherol exhibited a faster rate of decline of War- muscle, not only postmortem, but also living muscle. ner-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 LITERATURE CITED degradation in beef from calves fed a diet supplemented Bradford, M. 1976. A rapid and sensitive method for the quantitation with vitamin E. of microgram quantities of protein-dye binding. Anal. Biochem. Oxidation has been shown to decrease activity of cal- 72:248–254. pains, but in contrast, calpains have also been shown Cottin, P., P. L. Vidalenc, and A. Duscastaing. 1981. Ca2+-dependent 2+ to play a role in protein degradation when oxidative association between a Ca -activated neutral proteinase (CaANP) and its specific inhibitor. FEBS Lett. 136:221–224. conditions are higher than normal. A relationship be- Croall, D. E., and K. S. McGrody. 1994. Domain structure of calpain: tween protein oxidation and proteolysis has been Mapping the binding site for calpastatin. Biochemistry clearly established (as reviewed by Mehlhase and 33:13223–13230. Grune, 2002), where it has been established that oxida- Geesink, G. H., M. A. Ilian, J. D. Morton, and R. Bickerstaffe. 2000. tion of proteins can enhance susceptibility to proteolytic Involvement of calpains in postmortem tenderisation: A review of recent research. Proc. N.Z. Soc. Anim. Prod. 60:99–102. degradation. However, at the same time, the excessive Geesink, G. H., and M. Koohmaraie. 1998. An improved purification oxidation may cause a decrease in susceptibility to deg- protocol for heart and skeletal muscle calpastatin reveals two radation. Oxidative stress in combination with ob- isoforms resulting from alternative splicing. Arch. Biochem. Bio- served intracellular Ca2+ concentration in a mouse em- phys. 356:19–24. bryonal carcinoma cell line (Ray et al., 2000) and a rat Geesink, G. H., and M. Koohmaraie. 1999. Effect of calpastatin on degradation of myofibrillar proteins by ␮-calpain under postmor- adrenal gland cell line (Ishihara et al., 2000) has been tem conditions. J. Anim. Sci. 77:2685–2692. shown to increase activation of calpains. Pronzato et Geesink, G. H., and M. Koohmaraie. 2000. Ionic strength-induced al. (1993) observed that the level of oxidative stress inactivation of ␮-calpain in postmortem muscle. J. Anim. Sci. can affect calpain activity, where moderate levels of 78:2336–2343. Goll, D. E., R. G. Taylor, J. A. Christiansen, and V. F. Thompson. oxidation enhance activity, but increased oxidative ex- 1992. Role of proteinases and protein turnover in muscle growth posure can cause inactivation of calpains. These obser- and meat quality. Pages 25–36 inProc. 44th Annu. Recip. Meat vations could provide an explanation of how the involve- Conf. Natl. Livest. Meat Board, Chicago, IL.

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Goll, D. E., V. F. Thompson, H. Li, W. Wei, and J. Cong. 2003. The Neumann, N. P. 1972. Oxidation with hydrogen peroxide. Methods calpain system. Physiol. Rev. 83:731–801. Enzymol. 25:393–400. Goll, D. E., R. B. Young, and M. H. Stromer. 1974. Separation of Nishimura, T., and D. E. Goll. 1991. Binding of calpain fragments subcellular organelles in differential and density gradient cen- to calpastatin. J. Biol. Chem. 266:11842–11850. trifugation. Proc. Recip. Meat Conf. 37:250–254. Otsuka, Y., and D. E. Goll. 1987. Purification of the Ca2+-dependent Guttmann, R. P., J. S. Elce, P. D. Bell, J. C. Isbell, and G. V. W. proteinases inhibitor from bovine cardiac muscle and its interac- Johnson. 1997. Oxidation inhibits substrate proteolysis but not tion with the millimolar Ca2+-dependent proteinases. J. Biol. autolysis. J. Biol. Chem. 272:2005–2012. Chem. 262:5839–5851. Guttmann, R. P., and G. V. W. Johnson. 1998. Oxidative stress inhib- Ouali, A., and A. Talmant. 1990. Calpains and calpastatin distribu- its calpain activity in situ. J. Biol. Chem. 273:13331–13338. tion in bovine, porcine, and ovine skeletal muscle. Meat Sci. Harris, S. E., E. Huff-Lonergan, S. M. Lonergan, W. R. Jones, and 28:331–348. D. Rankins. 2001. Antioxidant status affects color stability and Pronzato, M. A., C. Domenicotti, E. Russo, A. Bellochio, M. Patrone, tenderness of calcium chloride-injected beef. J. Anim. Sci. U. M. Marinari, E. Melloni, and G. Poli. 1993. Modulation of rat 79:666–677. protein kinase C during in vitro CCl4-induced oxidative stress. Huff-Lonergan, E., T. Mitsuhashi, D. D. Beekman, F. C. Parrish, Jr., Biochem. Biophys. Res. Commun. 194:635–641. D. G. Olson, and R. M. Robson. 1996. Proteolysis of specific Ray, S. K., M. Fidan, M. W. Noak, G. G. Wilford, E. L. Hogan, and ␮ muscle structural proteins by -calpain at low pH and tempera- N. L. Banik. 2000. Oxidative stress and Ca2+ influx upregulate ture is similar to degradation in postmortem bovine muscle. J. and induce apoptosis in PC12 cells. Brain Res. 852:326–334. Anim. Sci. 74:993–1008. Robson, R. M., D. E. Goll, and M. J. Temple. 1968. Determination of Ishihara, I., Y. Minamai, T. Nishizaki, T. Matsuoka, and H. Yama- protein in “tris” buffer by the biuret reaction. Anal. Biochem. mura. 2000. Activation of calpain precedes morphological alter- 24:339–341. ations during hydrogen peroxide-induced apoptosis in neu- Rowe, L. J., K. R. Maddock, S. M. Lonergan, and E. Huff-Lonergan. ronally differentiated mouse embryonal carcinoma P19 cell line. 2004a. Influence of early postmortem oxidation on beef quality. Neurosci. Lett. 279:97–100. J. Anim. Sci. 82:785–793. Kapprell, K., and D. E. Goll. 1989. Effect of Ca2+ on binding of the Rowe, L. J., K. R. Maddock, S. M. Lonergan, and E. Huff-Lonergan. calpains to calpastatin. J. Biol. Chem. 264:17888–17896. 2004b. Oxidative environments decrease tenderization of beef Kendall, T. L., M. Koohmaraie, J. R. Arbona, S. E. Williams, and L. steaks through inactivation of ␮-calpain. J. Anim. Sci. L. Young. 1993. Effect of pH and ionic strength on bovine m- 82:3254–3266. calpain and calpastatin activity. J. Anim. Sci. 71:96–104. Sasaki, T., T. Kikuchi, N. Yumoto, N. Yoshimura, and T. Murachi. Koohmaraie, M. 1990. Quantification of Ca2+-dependent protease ac- 1984. Comparative specificity and kinetic studies on porcine tivities by hydrophobic and ion-exchange chromatography. J. calpain I and calpain II with naturally occurring peptides and Anim. Sci. 68:659–665. synthetic fluorogenic substrates. J. Biol. Chem. 259:12489– Koohmaraie, M. 1992. The role of Ca2+-dependent proteases (calpains) in postmortem proteolysis and meat tenderness. Biochimie 12494. 74:239–245. Saurin, A. T., H. Neubert, J. P. Brennan, and P. Eaton. 2004. Wide- Koohmaraie, M., S. D. Shackelford, T. L. Wheeler, S. M. Lonergan, spread sulfenic acid formation in tissues in response to hydrogen and M. E. Doumit. 1995. A muscle hypertrophy condition in peroxide. Proc. Natl. Acad. Sci. USA 101:17982–17987. lamb (callipyge): Characterization of effects on muscle growth Stennicke, H. R., U. H. Mortensen, U. Christensen, S. J. Remington, and meat quality traits. J. Anim. Sci. 73:3596–3607. and K. Breddam. 1994. Effects of introduced aspartic and glu- Li, H., V. F. Thompson, and D. E. Goll. 2004. Effects of autolysis on tamic acid residues on the Pi substrate specificity, pH depen- properties of ␮- and m-calpain. Biochim. Biophys. Acta dence, and stability of carboxypeptidase Y. Protein Eng. 1691:91–103. 7:911–916. Maddock, K. R., E. Huff-Lonergan, L. J. Rowe, and S. M. Lonergan. Thompson, V. F., and D. E. Goll. 2000. Purification of ␮-calpain, m- 2005. Effect of pH and ionic strength on calpastatin inhibition calpain, and calpastatin from animal tissues. Methods Mol. Biol. of ␮- and m-calpain. J. Anim. Sci. 83:1370–1376. 144:3–16. Martinaud, A., Y. Mercier, P. Marinova, C. Tassy, P. Gatellier, and M. Todd, B., D. Moore, C. C. S. Deivananyagum, G. Lin, D. Chattopad- Renerre. 1997. Comparison of oxidative processes on myofibrillar hyay, M. Maki, K. K. W. Wang, and S. V. L. Narayana. 2003. proteins from beef during maturation and by different model A structural model for the inhibition of calpain by calpastatin: oxidation systems. J. Agric. Food Chem. 45:2481–2487. Crystal structures of the native domain IV of calpain and its Medhi, S. 1991. Cell-penetrating inhibitors of calpain. Trends Bio- complexes with calpastatin peptide and a small molecule inhibi- chem. Sci. 16:150–153. tor. J. Mol. Biol. 328:131–146. Mehlhase, J., and T. Grune. 2002. Proteolytic response to oxidative Winger, R. J., and C. G. Pope. 1981. Osmotic properties of post-rigor stress in mammalian cells. Biol. Chem. 383:559–567. beef muscle. Meat Sci. 5:355–369.

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