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Changes in proteasome activity during postmortem aging of bovine muscle
Meat Science 61 (2002) 199–204 www.elsevier.com/locate/meatsci
Changes in proteasome activity during postmortem aging of bovine muscle Marie Lamarea, Richard G. Taylorb, Luc Farouta, Yves Brianda,*, Mariele Brianda a
Universite´ Blaise Pascal, Laboratoire de Biochimie Applique´e, associe´ INRA, 63174 Aubiere, France b INRA, Station de Recherche sur la Viande, Theix, 63122 St Genes Champanelle, France Received 23 April 2001; received in revised form 4 September 2001; accepted 7 September 2001
Abstract Changes in the chymotrypsin-like, trypsin-like, peptidylglutamylpeptide hydrolyzing and caseinolytic activities of proteasomes in bovine rectus abdominis muscle were measured during the first seven days of postmortem storage. Enzyme assays were performed in crude extracts under near-physiological conditions, since the activities are likely to be altered by purification. The different proteasome activities at cellular pH were stable at different times postmortem, and were 40, 76, 50 and 61% of their at-death value after 7 days of storage at 4 C. This considerable postmortem stability of proteasome activities, despite the marked decrease in pH, allows them to play a role in meat tenderization in synergy with other proteolytic systems. # 2002 Elsevier Science Ltd. All rights reserved. Keywords: Muscle proteolysis; Proteasome; Meat aging
1. Introduction The quality of meat most appreciated by consumers is tenderness, particularly in the case of beef (Boleman et al., 1997; Warkup, Marie, & Harrington, 1995). Tenderness is achieved through aging, during which structural changes occur in the muscle due to complex physicochemical mechanisms involving pH and ionic strength, synergistically combined with the action of cellular proteolytic enzymes (Ouali, 1990; Taylor, 1998). The proteolysis of cytoskeletal proteins is considered as the main enzymatic mechanism in the postmortem increase in tenderness (Koohmaraie, 1992b, 1994, 1996; Taylor, Geesink, Thompson, Koohmaraie, & Goll, 1995). Three proteolytic systems have been studied to varying degrees: the lysosomal proteases, calciumdependent proteases, and more recently proteasomes. While none of these systems can explain all the changes observed postmortem (Boehm, Kendall, Thompson, & Goll, 1998; Roncales et al., 1998), the role of cathepsins is probably limited to the first days after death (Kooh* Corresponding author. Tel.: +33-4-7340-7419; fax: +33-4-73407402. E-mail address: [email protected] (Y. Briand).
maraie, 1990; Koohmaraie, Babiker, Merker, & Dutson, 1988; Mestres Prates, Ribeire, & Dias Correia, 2001). Calpains have been extensively studied and their role in postmortem proteolysis and tenderization has been widely demonstrated (Boehm et al., 1998; Geesink, Ilian, Morton, & Bickerstaffe, 2000; Geesink & Koohmaraie, 1999; Huff-Lonergan, Mitsuhashi, Beekman, Parrish, Jr., Olson, & Robson, 1996; Koohmaraie et al., 1988; Thompson, Taylor, & Christiansen, 1992). The proteolysis of cytoskeletal proteins by calpains accounts for most proteolysis during meat tenderization under normal storage and pH conditions (Koohmaraie, 1996). Recently, Boehm et al. (1998), noted a marked drop in the activity of m-calpain from the first day postmortem onwards, while m-calpain activity was unchanged after 7 days. However, it is unlikely that m-calpain is fully active because the low calcium concentrations do not allow its autolysis and hence activation. Little work has been devoted to proteasomes. Koomharaie (1992a) has suggested that proteasomes are not involved in the early stages of the destabilization of myofibrils. Purified proteasomes from lobster (Mykles & Haire, 1995) and rabbit hydrolyze myofibrillar proteins, although in most cases they require activation by warming or addition of SDS (Matsuishi & Okitani, 1997; Otsuka, Homma, Shiga,
0309-1740/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved. PII: S0309-1740(01)00187-5
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2.3. Sample preparation Nomenclature SDS-PAGE Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis DTT Dithiotreitol BSA Bovine Serum Albumin T-L Trypsin-like ChT-L Chymotrypsin-like PGPH Peptidyl glutamyl peptide hydrolase TBS Tris-buffered saline.
Rectus abdominis muscle kept at 4 C was sampled at different times postmortem from 1 h to 7 days. Slices 1 cm thick were cut at 90 to the line of the muscle fibre. The top slice, which had been in contact with air, was discarded, and the second underlying slice was used for the study. Slices were immediately frozen in liquid nitrogen, and kept at 80 C until used. 2.4. pH measurement
Ushiki, Ikeuchi, & Suzuki, 1998; Yamaguchi et al., 1996). We recently showed that purified bovine proteasomes damage myofibrillar structures without prior activation (Taylor, Tassy, Briand, Robert, Briand, & Ouali, 1995). We therefore suggested that the combined action of calpains and physicochemical changes in the muscle destabilized the myofibrillar structures, leading to slow denaturation of proteins, which consequently become accessible to proteasome 20S (Robert, Briand, Taylor, & Briand, 1999) and are thus hydrolyzed (Davies, 2001). The aim of this work was to show that the proteasome remains active during postmortem aging. We developed a method which measures proteasome activity without prior purification, which is likely to alter the activity, and which mimics intracellular conditions (Farout, Lamare, Cardozo, Harrisson, Briand, & Briand, 2000). Our results show that the proteasomes are still substantially active 7 days after death and at a pH below 6, thus supporting the hypothesis of their putative action in the tenderization process.
Two grams of rectus abdominis muscle were thawed in 10 ml of iodoacetate solution (150 mM potassium chloride, 5 mM sodium iodoacetate) at 4 C, then homogenized using a Polytron device. The postmortem pH of the muscle homogenate was measured directly by means of a pH meter, while stirring gently at 4 C. 2.5. Preparation of crude extracts Extracts were prepared according to Farout et al. (2000). Muscle (500 mg) was suspended (1:10 w:v) in 50 mM Tris/HCl buffer pH 8.0 containing 10% glycerol, 1 mM EDTA, 1 mM EGTA, 50 nM E64, 2.5 mM pepstatin A and homogenized with a Polytron device. Crude extracts were prepared by centrifuging the homogenates at 100,000 g for 1 h and were studied directly. Protein concentration was determined by the Bradford method using BioRad reagent and BSA as standard. 2.6. Peptidase and caseinolytic assays
2. Materials and methods 2.1. Materials N-t-Boc-LSTR-AMC and Z-LLE-NA (Z, benzyloxycarbonyl-one letter amino acid codes; AMC, 4 methylcoumaryl-7 amide; NA, 2-naphthylamide) were from Sigma, and N-Suc-LLVY-AMC (N-Suc, N-Succinyl) was from Bachem. The protease inhibitors L transepoxisuccinyl L leucylamido-(4 guanido)-butane (E64) and pepstatin A were from Sigma. The [14C]-methylated casein was from NEN and the enhanced chemiluminescent kit (ECL) from Amersham Pharmacia Biotech. The polyvinyl difluoride (PVDF) blotting membrane was from Millipore. The anti-iota (a1) antibody was from Cappel. 2.2. Muscle Seven 7-year-old Charolais cows were used. Rectus abdominis muscle (300–400 g) was removed from the carcasses immediately after slaughter and kept at 4 C.
All assays were done in triplicate. Hydrolysis of fluorogenic substrates was determined in reaction mixtures (200 ml) containing 50 mM Tris/HCl buffer pH 7.5, 1 mM DTT, 30 to 100 mg of protein and 40 mM NSuc-LLVY-AMC, 40 mM LSTR-AMC or 100 mM ZLLE-NA. After incubation for 30 min at 37 C, the reaction was stopped by adding 800 ml of 100 mM monochloroacetate–30 mM sodium acetate. Fluorescence was monitored on a Hitachi E2000 fluorimeter (excitation 370 nm, emission 420 nm for AMC substrates and excitation 333 nm, emission 410 nm for NA substrate). To assay the degradation of exogenous protein, breakdown of [14C]-methylcasein in the crude extracts was measured by monitoring the release of trichloroacetic acid-soluble radioactivity using a b liquid scintillation counter (Beckman LS 6500). Samples of about 20,000 dpm of [14C]-methylcasein (3.4 mCi/mg) were incubated at 37 C for 2 h in a total volume of 200 ml of a reaction mixture containing 50 mM Tris/HCl pH 8.0, 1 mM DTT and 100 mg of protein.
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2.7. Immunological analysis Immuno-electrophoretic blot analysis was carried out by the method of Towbin, Staehlin, and Gordon (1979). Samples (10 mg protein) were separated by SDS-PAGE according to Laemmli (1970) and transferred electrophoretically to PVDF sheets. The membranes were saturated with 10% skimmed milk, 0.1% Tween in TBS buffer for 2 h at room temperature before overnight incubation at 4 C with the mouse monoclonal antiiota (a1) antibody. After washing in TBS+0.1% Tween, the second antibody coupled to horseradish peroxidase was added for 1 h at room temperature. Membranes were revealed by chemiluminescence using the ECL kit.
3. Results 3.1. Proteasome activity during postmortem aging The proteasome’s three main peptidase and caseinolytic activities were measured in crude muscle extracts obtained at different times postmortem, as indicated in Section 2. Under our experimental conditions, only proteasome proteolytic activities are measured (Farout et al., 2000), under widely used standard conditions, notably in terms of a pH of 7.5, a value allowing measurement of the different activities under identical conditions. We also measured the effect of
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the pH on the proteasome activities, and thus calculated the true activity in the muscle at different times postmortem. 3.1.1. Proteasome activities as a function of pH Fig. 1 shows the proteasome activities measured in crude extracts as a function of pH. The optimum pH was 6.5–7 for chymotrypsin-like (ChT-L) and peptidylglutamylpeptide hydrolyzing (PGPH) activities, and more alkaline for trypsin-like (T-L) and caseinolytic activities. These optimum pHs measured in crude extracts were slightly lower (0.5 of a unit, data not shown) than those measured using pure proteasomes. Using purified bovine proteasomes, Arbona and Koohmaraie (1993) measured an optimum pH of 8 for the substrate Gly-Gly-Leu. Matsuishi and Okitani (1997), noted an optimum pH of 8 for purified rabbit proteasomes using the substrate LLVY. 3.1.2. Postmortem proteasome activities The four proteasome activities were measured at pH 7.5 at different times postmortem (Fig. 2). In the first hours, the activities were stable, with increases in PGPH and caseinolytic activities, possibly due to activation of the proteasome by the change in physicochemical conditions, notably the increase in ionic strength and the drop in pH strength (Zamora, Debiton, Lepetit, Lebert, Dransfield, & Ouali, 1996). The activities were close to 50% of the at-death values 72 h postmortem, and above 30% after 7 days.
Fig. 1. pH dependence of proteasome activities. Relative activity was expressed as a percentage of the maximum. Results are averages SE of 7 differents experiments.
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Fig. 3. Western blot analysis of crude extract using anti alpha 1 subunit antibody.
Fig. 4. pH decline of bovine rectus abdominis muscle during meat aging.
Fig. 2. Change in activities of proteasome during postmortem aging. Relative activity was expressed as a percentage of the time 1 h. Results are averagesSE of 7 differents experiments. Relative activity measured in standard condition (white bars). Relative activity calculated at actual postmortem pH (black bars).
Western blot analysis of the alpha 1 subunit of the proteasome (Fig. 3) showed that proteasome levels remained constant during this same period, and indicated that the decrease in activity resulted from denaturation of the proteasome and probably from a structural modification due to the intracellular physicochemical conditions. These activities measured under standard conditions indicate the proteasome’s proteolytic potential, but it is interesting to measure the actual activity of the proteasome at the pH corresponding to the time considered. As shown in Fig. 4, the pH decreased rapidly over the first 24 h postmortem to a minimum of 5.7, which was unchanged 7 days postmortem. The activities at postmortem pH (Fig. 2, white bars) were calculated from the activities measured under
standard conditions (Fig. 2, black bars) corrected for the effect of the pH as shown in Fig. 1. The PGPH and ChT-L activities were above the standard activities measured at pH 7.5, irrespective of the storage time of the muscle, whereas the T-L and caseinolytic activities were lower. Given the trend in pH, these activities were relatively stable postmortem and corresponded 168 h postmortem to 40, 76, 50 and 61%, respectively of the at-death ChT-L, PGPH, T-L and caseinolytic activities. The caseinolytic activity after 7 days (at pH 5.7) was 36% of the activity measured under optimum conditions (pH 7.5) 1 h postmortem.
4. Discussion The proteasome is considered by numerous authors as a neutral protease, and its role in postmortem tenderization of meat has probably been minimized. We have shown in bovine muscle that proteasomes can damage myofibrillar structures and hydrolyze myofibrillar proteins (Robert, Briand, Taylor, & Briand, 1999). A few years ago, Mykles and Haire (1995) showed that lobster 20S proteasome can hydrolyze myofibrillar proteins. More recently, similar data were recorded in rabbits (Matsuishi & Okitani, 1997; Otsuka, Homma, Shiga, Ushiki, Ikeuchi, & Suzuki, 1998). Yet these experiments in the lobster and rabbit were done on proteasomes purified by a procedure requiring treatment with SDS or heat. Postmortem conditions alter the capacity of different proteolytic systems and we felt it important to study the time-dependent changes in proteasome activity
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and the actual proteasome activities in situ during muscle aging, bearing in mind changes in pH. We therefore studied changes in proteasome activity in crude extracts under conditions in which the other proteases were inactive. These conditions were closer to the natural conditions, and the use of crude extracts obviated any perturbations associated with purification. Our results show that the proteasome activities measured under standard conditions decreased slowly postmortem, but remained substantial after seven days: 28, 54, 32 and 154% of the at-death ChT-L, T-L, PGPH and caseinolytic activities. These decreased activities probably result from structural changes, since the quantity of proteasome measured by Western blot with anti-subunit alpha1 antibody does not vary with time. The increase in caseinolytic activity may be due to proteasome activation, which facilitates substrate access (Davies, 2001). The activities at the actual postmortem cellular pH were, respectively 40, 76, 50 and 61% of the at-death activities. These residual activities are still high and probably account for some of the postmortem changes in myofibrillar structures. These results agree with those of Arbona and Koohmaraie (1993) who measured activity after proteasome purification. There is abundant evidence that calpains, and more particularly m-calpain, are involved in the degradation of costameres and the loss of cytoskeletal integrity during the first 48 h postmortem (Boehm et al., 1998; Geesink & Koohmaraie, 1999; Koohmaraie, 1992b; Taylor et al., 1995; Zamora, Debiton, Lepetit, Lebert, Dransfield, & Ouali, 1996). However, proteolysis is low during this period, and increases between days 3 and 14 (Boehm et al., 1998). It has also been shown that the activity of autolyzed m-calpain (i.e. the active calpain) drops substantially postmortem (Boehm et al., 1998; Zamora, Debiton, Lepetit, Lebert, Dransfield, & Ouali, 1996) to 20% after 24 h (Boehm et al., 1998). Other proteolytic systems must therefore intervene after this stage. This role has been attributed to m-calpain (Boehm et al., 1998) and more recently to calpain p94 (Ilian et al., 2001). We show here that the proteasomes can also participate in this second wave of proteolysis, and that their activity remains high after death. The 20S proteasome proteolyzes damaged or structurally altered proteins (Davies, 2001). The primary postmortem action of m-calpain, in synergy with the large increase in ionic strength (Zamora et al., 1996) and the drop in pH, results in substantial denaturation of myofibrillar proteins, which therefore become easy targets for other proteases, including the 20S proteasome. The 20S proteasome could also account for the proteolysis of autolyzed m-calpain, whose stability is poor postmortem (Geesink & Koohmaraie, 2000) because the structural changes induced by autolysis make it more susceptible to proteolysis, and for the degradation and resulting loss of activity of calpastatin (Boehm et al., 1998;
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Ducastaing, Valin, Schollmeyer & Cross, 1985; Koohmaraie, Seideman, Schollmeyer, Dutson, & Crouse, 1987). Although m-calpain’s role seems clear, the participation of other proteases in the mechanism of postmortem meat tenderization should be studied, particularly under special conditions. At high pH, the meat is more tender (Jeremiah, Tong, & Gibson, 1991) and the structural changes are different, especially with regards to loss of density of the Z-line (Yu & Lee, 1986) such as occur when myofibers are incubated with proteasomes (Robert, Briand, Taylor & Briand, 1999; Taylor et al., 1995). Proteasomes may also be involved in other modifications, such as the change in Z-line density in type I fibres (Gann & Merkel, 1978). Under these special conditions, there is hydrolysis of actin and myosin, two proteins which are potential substrates of proteasomes or of cathepsins.
Acknowledgements This work was supported by the Ministry de l’Enseignement et de la Recherche and the Ministry de l’Agriculture (AIP- INRA, 178).
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Mykles, D. L., & Haire, M. F. (1995). Branched-chain-amino-acidpreferring peptidase activity of the lobster multicatalytic proteinase (proteasome) and the degradation of myofibrillar proteins. Biochemical Journal, 306, 285–291. Otsuka, Y., Homma, N., Shiga, K., Ushiki, J., Ikeuchi, Y., & Suzuki, A. (1998). Purification and properties of rabbit muscle proteasome, and its effect on myofibrillar structure. Meat Science, 49, 365–378. Ouali, A. (1990). Sensory quality of meat as affected by muscle biochemistry and modern technologies. In L. O. Fiems, B. G. Cottyn, & D. Demeyer (pp. 85–105). Amsterdam: Elsevier Science Publishers. Robert, N., Briand, M., Taylor, R. G., & Briand, Y. (1999). The effect of proteasome on myofibrillar structures in bovine skeletal muscle. Meat Science, 51, 149–153. Roncales, P., Geesink, G. H., Van Laack, R L., Jaime, I. Beltran, J. A., Barnier V., & Smulders, F. J. M. (1995). Meat tenderisation: enzymatic mechanisms. In: A. Ouali, D. Demeyer, & F. J. M. Smulders (Eds.), Expression of tissue proteinases and regulation of protein degradation as related to meat quality. Utrecht, The Netherlands: ECCEAMST (pp 311–332). Taylor, R. G., Geesink, G. H., Thompson, V. F., Koohmaraie, M., & Goll, D. E. (1995). Is Z-Disk degradation responsible for postmortem tenderization? Journal of Animal Science, 73, 1351–1367. Taylor, R. G., Tassy, C., Briand, M., Robert, N., Briand, Y., & Ouali, A. (1995). Proteolytic activity of proteasomes on myofibrillar structures. Molecular Biology Reports, 21, 71–73. Taylor, R. G. (1998). Structural basis for meat toughness and tenderness. Polish Journal of Food and Nutrition Sciences, 7, 37–52. Towbin, H., Staehlin, T., & Gordon, J. (1979). Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and application. Proceeding of National Academy of Sciences, USA, 76, 4350–4357. Warkup, C., Marie, S., & Harrington, G. (1995). Consumer perceptions of texture: the most important quality attribute of meat? In: A. Ouali, D. Demeyer, & F. J. M. Smulders (Eds.), Expression of tissue proteinases and regulation of protein degradation as related to meat quality. Utrecht, The Netherlands: ECCEAMST (pp 225–297). Yamaguchi, M., Muguruma, M., Sako, T., Nakayama, T., Yamamoto, S., Tangkawattana, P., Oba, T., Takehana, K., Muto, M., Nakade, T., & Yamano, S. (1996). Is there a protease that preferentially cleaves the M-line in partially dehydrated muscle? Meat Science, 42, 225–233. Yu, L. P., & Lee, Y. B. (1986). Effects of postmortem pH and temperature on bovine muscle structure and meat tenderness. Journal of Food Science, 51, 774–780. Zamora, F., Debiton, E. J., Lepetit, J., Lebert, A., Dransfield, E., & Ouali, A. (1996). Predicting variability of ageing and toughness in beef M. Longissimus lumborum et thoracis. Meat Science, 43, 321–333.
Changes in proteasome activity during postmortem aging of bovine muscle Marie Lamarea, Richard G. Taylorb, Luc Farouta, Yves Brianda,*, Mariele Brianda a
Universite´ Blaise Pascal, Laboratoire de Biochimie Applique´e, associe´ INRA, 63174 Aubiere, France b INRA, Station de Recherche sur la Viande, Theix, 63122 St Genes Champanelle, France Received 23 April 2001; received in revised form 4 September 2001; accepted 7 September 2001
Abstract Changes in the chymotrypsin-like, trypsin-like, peptidylglutamylpeptide hydrolyzing and caseinolytic activities of proteasomes in bovine rectus abdominis muscle were measured during the first seven days of postmortem storage. Enzyme assays were performed in crude extracts under near-physiological conditions, since the activities are likely to be altered by purification. The different proteasome activities at cellular pH were stable at different times postmortem, and were 40, 76, 50 and 61% of their at-death value after 7 days of storage at 4 C. This considerable postmortem stability of proteasome activities, despite the marked decrease in pH, allows them to play a role in meat tenderization in synergy with other proteolytic systems. # 2002 Elsevier Science Ltd. All rights reserved. Keywords: Muscle proteolysis; Proteasome; Meat aging
1. Introduction The quality of meat most appreciated by consumers is tenderness, particularly in the case of beef (Boleman et al., 1997; Warkup, Marie, & Harrington, 1995). Tenderness is achieved through aging, during which structural changes occur in the muscle due to complex physicochemical mechanisms involving pH and ionic strength, synergistically combined with the action of cellular proteolytic enzymes (Ouali, 1990; Taylor, 1998). The proteolysis of cytoskeletal proteins is considered as the main enzymatic mechanism in the postmortem increase in tenderness (Koohmaraie, 1992b, 1994, 1996; Taylor, Geesink, Thompson, Koohmaraie, & Goll, 1995). Three proteolytic systems have been studied to varying degrees: the lysosomal proteases, calciumdependent proteases, and more recently proteasomes. While none of these systems can explain all the changes observed postmortem (Boehm, Kendall, Thompson, & Goll, 1998; Roncales et al., 1998), the role of cathepsins is probably limited to the first days after death (Kooh* Corresponding author. Tel.: +33-4-7340-7419; fax: +33-4-73407402. E-mail address: [email protected] (Y. Briand).
maraie, 1990; Koohmaraie, Babiker, Merker, & Dutson, 1988; Mestres Prates, Ribeire, & Dias Correia, 2001). Calpains have been extensively studied and their role in postmortem proteolysis and tenderization has been widely demonstrated (Boehm et al., 1998; Geesink, Ilian, Morton, & Bickerstaffe, 2000; Geesink & Koohmaraie, 1999; Huff-Lonergan, Mitsuhashi, Beekman, Parrish, Jr., Olson, & Robson, 1996; Koohmaraie et al., 1988; Thompson, Taylor, & Christiansen, 1992). The proteolysis of cytoskeletal proteins by calpains accounts for most proteolysis during meat tenderization under normal storage and pH conditions (Koohmaraie, 1996). Recently, Boehm et al. (1998), noted a marked drop in the activity of m-calpain from the first day postmortem onwards, while m-calpain activity was unchanged after 7 days. However, it is unlikely that m-calpain is fully active because the low calcium concentrations do not allow its autolysis and hence activation. Little work has been devoted to proteasomes. Koomharaie (1992a) has suggested that proteasomes are not involved in the early stages of the destabilization of myofibrils. Purified proteasomes from lobster (Mykles & Haire, 1995) and rabbit hydrolyze myofibrillar proteins, although in most cases they require activation by warming or addition of SDS (Matsuishi & Okitani, 1997; Otsuka, Homma, Shiga,
0309-1740/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved. PII: S0309-1740(01)00187-5
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2.3. Sample preparation Nomenclature SDS-PAGE Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis DTT Dithiotreitol BSA Bovine Serum Albumin T-L Trypsin-like ChT-L Chymotrypsin-like PGPH Peptidyl glutamyl peptide hydrolase TBS Tris-buffered saline.
Rectus abdominis muscle kept at 4 C was sampled at different times postmortem from 1 h to 7 days. Slices 1 cm thick were cut at 90 to the line of the muscle fibre. The top slice, which had been in contact with air, was discarded, and the second underlying slice was used for the study. Slices were immediately frozen in liquid nitrogen, and kept at 80 C until used. 2.4. pH measurement
Ushiki, Ikeuchi, & Suzuki, 1998; Yamaguchi et al., 1996). We recently showed that purified bovine proteasomes damage myofibrillar structures without prior activation (Taylor, Tassy, Briand, Robert, Briand, & Ouali, 1995). We therefore suggested that the combined action of calpains and physicochemical changes in the muscle destabilized the myofibrillar structures, leading to slow denaturation of proteins, which consequently become accessible to proteasome 20S (Robert, Briand, Taylor, & Briand, 1999) and are thus hydrolyzed (Davies, 2001). The aim of this work was to show that the proteasome remains active during postmortem aging. We developed a method which measures proteasome activity without prior purification, which is likely to alter the activity, and which mimics intracellular conditions (Farout, Lamare, Cardozo, Harrisson, Briand, & Briand, 2000). Our results show that the proteasomes are still substantially active 7 days after death and at a pH below 6, thus supporting the hypothesis of their putative action in the tenderization process.
Two grams of rectus abdominis muscle were thawed in 10 ml of iodoacetate solution (150 mM potassium chloride, 5 mM sodium iodoacetate) at 4 C, then homogenized using a Polytron device. The postmortem pH of the muscle homogenate was measured directly by means of a pH meter, while stirring gently at 4 C. 2.5. Preparation of crude extracts Extracts were prepared according to Farout et al. (2000). Muscle (500 mg) was suspended (1:10 w:v) in 50 mM Tris/HCl buffer pH 8.0 containing 10% glycerol, 1 mM EDTA, 1 mM EGTA, 50 nM E64, 2.5 mM pepstatin A and homogenized with a Polytron device. Crude extracts were prepared by centrifuging the homogenates at 100,000 g for 1 h and were studied directly. Protein concentration was determined by the Bradford method using BioRad reagent and BSA as standard. 2.6. Peptidase and caseinolytic assays
2. Materials and methods 2.1. Materials N-t-Boc-LSTR-AMC and Z-LLE-NA (Z, benzyloxycarbonyl-one letter amino acid codes; AMC, 4 methylcoumaryl-7 amide; NA, 2-naphthylamide) were from Sigma, and N-Suc-LLVY-AMC (N-Suc, N-Succinyl) was from Bachem. The protease inhibitors L transepoxisuccinyl L leucylamido-(4 guanido)-butane (E64) and pepstatin A were from Sigma. The [14C]-methylated casein was from NEN and the enhanced chemiluminescent kit (ECL) from Amersham Pharmacia Biotech. The polyvinyl difluoride (PVDF) blotting membrane was from Millipore. The anti-iota (a1) antibody was from Cappel. 2.2. Muscle Seven 7-year-old Charolais cows were used. Rectus abdominis muscle (300–400 g) was removed from the carcasses immediately after slaughter and kept at 4 C.
All assays were done in triplicate. Hydrolysis of fluorogenic substrates was determined in reaction mixtures (200 ml) containing 50 mM Tris/HCl buffer pH 7.5, 1 mM DTT, 30 to 100 mg of protein and 40 mM NSuc-LLVY-AMC, 40 mM LSTR-AMC or 100 mM ZLLE-NA. After incubation for 30 min at 37 C, the reaction was stopped by adding 800 ml of 100 mM monochloroacetate–30 mM sodium acetate. Fluorescence was monitored on a Hitachi E2000 fluorimeter (excitation 370 nm, emission 420 nm for AMC substrates and excitation 333 nm, emission 410 nm for NA substrate). To assay the degradation of exogenous protein, breakdown of [14C]-methylcasein in the crude extracts was measured by monitoring the release of trichloroacetic acid-soluble radioactivity using a b liquid scintillation counter (Beckman LS 6500). Samples of about 20,000 dpm of [14C]-methylcasein (3.4 mCi/mg) were incubated at 37 C for 2 h in a total volume of 200 ml of a reaction mixture containing 50 mM Tris/HCl pH 8.0, 1 mM DTT and 100 mg of protein.
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2.7. Immunological analysis Immuno-electrophoretic blot analysis was carried out by the method of Towbin, Staehlin, and Gordon (1979). Samples (10 mg protein) were separated by SDS-PAGE according to Laemmli (1970) and transferred electrophoretically to PVDF sheets. The membranes were saturated with 10% skimmed milk, 0.1% Tween in TBS buffer for 2 h at room temperature before overnight incubation at 4 C with the mouse monoclonal antiiota (a1) antibody. After washing in TBS+0.1% Tween, the second antibody coupled to horseradish peroxidase was added for 1 h at room temperature. Membranes were revealed by chemiluminescence using the ECL kit.
3. Results 3.1. Proteasome activity during postmortem aging The proteasome’s three main peptidase and caseinolytic activities were measured in crude muscle extracts obtained at different times postmortem, as indicated in Section 2. Under our experimental conditions, only proteasome proteolytic activities are measured (Farout et al., 2000), under widely used standard conditions, notably in terms of a pH of 7.5, a value allowing measurement of the different activities under identical conditions. We also measured the effect of
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the pH on the proteasome activities, and thus calculated the true activity in the muscle at different times postmortem. 3.1.1. Proteasome activities as a function of pH Fig. 1 shows the proteasome activities measured in crude extracts as a function of pH. The optimum pH was 6.5–7 for chymotrypsin-like (ChT-L) and peptidylglutamylpeptide hydrolyzing (PGPH) activities, and more alkaline for trypsin-like (T-L) and caseinolytic activities. These optimum pHs measured in crude extracts were slightly lower (0.5 of a unit, data not shown) than those measured using pure proteasomes. Using purified bovine proteasomes, Arbona and Koohmaraie (1993) measured an optimum pH of 8 for the substrate Gly-Gly-Leu. Matsuishi and Okitani (1997), noted an optimum pH of 8 for purified rabbit proteasomes using the substrate LLVY. 3.1.2. Postmortem proteasome activities The four proteasome activities were measured at pH 7.5 at different times postmortem (Fig. 2). In the first hours, the activities were stable, with increases in PGPH and caseinolytic activities, possibly due to activation of the proteasome by the change in physicochemical conditions, notably the increase in ionic strength and the drop in pH strength (Zamora, Debiton, Lepetit, Lebert, Dransfield, & Ouali, 1996). The activities were close to 50% of the at-death values 72 h postmortem, and above 30% after 7 days.
Fig. 1. pH dependence of proteasome activities. Relative activity was expressed as a percentage of the maximum. Results are averages SE of 7 differents experiments.
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Fig. 3. Western blot analysis of crude extract using anti alpha 1 subunit antibody.
Fig. 4. pH decline of bovine rectus abdominis muscle during meat aging.
Fig. 2. Change in activities of proteasome during postmortem aging. Relative activity was expressed as a percentage of the time 1 h. Results are averagesSE of 7 differents experiments. Relative activity measured in standard condition (white bars). Relative activity calculated at actual postmortem pH (black bars).
Western blot analysis of the alpha 1 subunit of the proteasome (Fig. 3) showed that proteasome levels remained constant during this same period, and indicated that the decrease in activity resulted from denaturation of the proteasome and probably from a structural modification due to the intracellular physicochemical conditions. These activities measured under standard conditions indicate the proteasome’s proteolytic potential, but it is interesting to measure the actual activity of the proteasome at the pH corresponding to the time considered. As shown in Fig. 4, the pH decreased rapidly over the first 24 h postmortem to a minimum of 5.7, which was unchanged 7 days postmortem. The activities at postmortem pH (Fig. 2, white bars) were calculated from the activities measured under
standard conditions (Fig. 2, black bars) corrected for the effect of the pH as shown in Fig. 1. The PGPH and ChT-L activities were above the standard activities measured at pH 7.5, irrespective of the storage time of the muscle, whereas the T-L and caseinolytic activities were lower. Given the trend in pH, these activities were relatively stable postmortem and corresponded 168 h postmortem to 40, 76, 50 and 61%, respectively of the at-death ChT-L, PGPH, T-L and caseinolytic activities. The caseinolytic activity after 7 days (at pH 5.7) was 36% of the activity measured under optimum conditions (pH 7.5) 1 h postmortem.
4. Discussion The proteasome is considered by numerous authors as a neutral protease, and its role in postmortem tenderization of meat has probably been minimized. We have shown in bovine muscle that proteasomes can damage myofibrillar structures and hydrolyze myofibrillar proteins (Robert, Briand, Taylor, & Briand, 1999). A few years ago, Mykles and Haire (1995) showed that lobster 20S proteasome can hydrolyze myofibrillar proteins. More recently, similar data were recorded in rabbits (Matsuishi & Okitani, 1997; Otsuka, Homma, Shiga, Ushiki, Ikeuchi, & Suzuki, 1998). Yet these experiments in the lobster and rabbit were done on proteasomes purified by a procedure requiring treatment with SDS or heat. Postmortem conditions alter the capacity of different proteolytic systems and we felt it important to study the time-dependent changes in proteasome activity
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and the actual proteasome activities in situ during muscle aging, bearing in mind changes in pH. We therefore studied changes in proteasome activity in crude extracts under conditions in which the other proteases were inactive. These conditions were closer to the natural conditions, and the use of crude extracts obviated any perturbations associated with purification. Our results show that the proteasome activities measured under standard conditions decreased slowly postmortem, but remained substantial after seven days: 28, 54, 32 and 154% of the at-death ChT-L, T-L, PGPH and caseinolytic activities. These decreased activities probably result from structural changes, since the quantity of proteasome measured by Western blot with anti-subunit alpha1 antibody does not vary with time. The increase in caseinolytic activity may be due to proteasome activation, which facilitates substrate access (Davies, 2001). The activities at the actual postmortem cellular pH were, respectively 40, 76, 50 and 61% of the at-death activities. These residual activities are still high and probably account for some of the postmortem changes in myofibrillar structures. These results agree with those of Arbona and Koohmaraie (1993) who measured activity after proteasome purification. There is abundant evidence that calpains, and more particularly m-calpain, are involved in the degradation of costameres and the loss of cytoskeletal integrity during the first 48 h postmortem (Boehm et al., 1998; Geesink & Koohmaraie, 1999; Koohmaraie, 1992b; Taylor et al., 1995; Zamora, Debiton, Lepetit, Lebert, Dransfield, & Ouali, 1996). However, proteolysis is low during this period, and increases between days 3 and 14 (Boehm et al., 1998). It has also been shown that the activity of autolyzed m-calpain (i.e. the active calpain) drops substantially postmortem (Boehm et al., 1998; Zamora, Debiton, Lepetit, Lebert, Dransfield, & Ouali, 1996) to 20% after 24 h (Boehm et al., 1998). Other proteolytic systems must therefore intervene after this stage. This role has been attributed to m-calpain (Boehm et al., 1998) and more recently to calpain p94 (Ilian et al., 2001). We show here that the proteasomes can also participate in this second wave of proteolysis, and that their activity remains high after death. The 20S proteasome proteolyzes damaged or structurally altered proteins (Davies, 2001). The primary postmortem action of m-calpain, in synergy with the large increase in ionic strength (Zamora et al., 1996) and the drop in pH, results in substantial denaturation of myofibrillar proteins, which therefore become easy targets for other proteases, including the 20S proteasome. The 20S proteasome could also account for the proteolysis of autolyzed m-calpain, whose stability is poor postmortem (Geesink & Koohmaraie, 2000) because the structural changes induced by autolysis make it more susceptible to proteolysis, and for the degradation and resulting loss of activity of calpastatin (Boehm et al., 1998;
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Ducastaing, Valin, Schollmeyer & Cross, 1985; Koohmaraie, Seideman, Schollmeyer, Dutson, & Crouse, 1987). Although m-calpain’s role seems clear, the participation of other proteases in the mechanism of postmortem meat tenderization should be studied, particularly under special conditions. At high pH, the meat is more tender (Jeremiah, Tong, & Gibson, 1991) and the structural changes are different, especially with regards to loss of density of the Z-line (Yu & Lee, 1986) such as occur when myofibers are incubated with proteasomes (Robert, Briand, Taylor & Briand, 1999; Taylor et al., 1995). Proteasomes may also be involved in other modifications, such as the change in Z-line density in type I fibres (Gann & Merkel, 1978). Under these special conditions, there is hydrolysis of actin and myosin, two proteins which are potential substrates of proteasomes or of cathepsins.
Acknowledgements This work was supported by the Ministry de l’Enseignement et de la Recherche and the Ministry de l’Agriculture (AIP- INRA, 178).
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