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The effect of recombinant caspase 3 on myofibrillar proteins in porcine skeletal muscle

Published online by Cambridge University Press:  01 August 2008

C. M. Kemp  and
T. Parr
C. M. Kemp
Affiliation:
Division of Nutritional Sciences, School of Biosciences, Sutton Bonington Campus, The University of Nottingham, Leicestershire, LE12 5RD, UK
T. Parr*
Affiliation:
Division of Nutritional Sciences, School of Biosciences, Sutton Bonington Campus, The University of Nottingham, Leicestershire, LE12 5RD, UK
*
E-mail: [email protected]
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Abstract

The objective of this study was to investigate the potential role of the caspase protease family in meat tenderisation by examining if caspase 3 was capable of causing myofibril protein degradation. Full-length human recombinant caspase 3 (rC3) was expressed in Escherichiacoli and purified. The rC3 was active in the presence of myofibrils isolated from porcine longissimus dorsi muscle (LD) and retained activity in a buffer system closely mimicking post mortem conditions. The effect of increasing concentrations of rC3, incubation temperature, as well as incubation time on the degradation of isolated myofibril proteins were all investigated in this study. Myofibril protein degradation was determined by SDS-PAGE and Western blotting. There was a visible increase in myofibril degradation with a decrease in proteins identified as desmin and troponin I and the detection of protein degradation products at approximately 32, 28 and 18 kDa with increasing concentrations of rC3. These degradation products were analysed using MALDI-TOF mass spectrometry and identified to occur from the proteolysis of actin, troponin T and myosin light chain, respectively. The production of these degradation products was not inhibited by 5 mM EDTA or semi-purified calpastatin but was inhibited by the caspase-specific inhibitor Ac-DEVD-CHO. The temperature at which isolated myofibrils were incubated with rC3 was also found to affect degradation, with increasing incubation temperatures causing increased desmin degradation and cleavage of pro-caspase 3 into its active isoform. Incubation of isolated myofibrils at 4°C for 5 days with rC3 resulted in the visible degradation of a number of myofibril proteins including desmin and troponin I. This study has shown that rC3 is capable of causing myofibril degradation, hydrolysing myofibril proteins under conditions that are similar to those found in muscle in the post mortem conditioning period.

Keywords

caspasemyofibrilsproteolysistenderization
Type
Full Paper
Information
animal , Volume 2 , Issue 8 , August 2008 , pp. 1254 - 1264
Copyright
Copyright © The Animal Consortium 2008

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References

Earnshaw, WC, Martins, LM, Kaufmann, SH 1999. Mammalian caspases: structure, activation, substrates, and functions during apoptosis. Annual Review of Biochemistry 68, 383–424.CrossRefGoogle ScholarPubMed
Fischer, U, Janicke, RU, Schulze-Osthoff, K 2003. Many cuts to ruin: a comprehensive update of caspase substrates. Cell Death and Differentiation 10, 76–100.CrossRefGoogle Scholar
Fuentes-Prior, P, Salvesen, GS 2004. The protein structures that shape caspase activity, specificity, activation and inhibition. Biochemical Journal 384, 201–232.CrossRefGoogle ScholarPubMed
Geesink, GH, Koohmaraie, M 1999. Effect of calpastatin on degradation of myofibrillar proteins by mu-calpain under postmortem conditions. Journal of Animal Science 77, 2685–2692.CrossRefGoogle ScholarPubMed
Geesink, GH, Kuchay, S, Chishti, AH, Koohmaraie, M 2006. Micro-calpain is essential for postmortem proteolysis of muscle proteins. Journal of Animal Science 84, 2834–2840.CrossRefGoogle ScholarPubMed
Goll DE, Young RB and Stromer MH 1974. Separation of subcellular organelles by differential and density gradient centrifugation. In Proceedings of the 27th Annual Reciprocal Meat Conference, pp. 250–267.Google Scholar
Green, DR 2005. Apoptotic pathways: ten minutes to dead. Cell 121, 671–674.CrossRefGoogle ScholarPubMed
Herrera-Mendez, CH, Becila, S, Boudjellal, A, Ouali, A 2006. Meat ageing: reconsideration of the current concept. Trends in Food Science and Technology 17, 394–405.CrossRefGoogle Scholar
Ho, CY, Stromer, MH, Robson, RM 1994. Identification of the 30 kDa polypeptide in post mortem skeletal muscle as a degradation product of troponin-T. Biochimie 76, 369–375.CrossRefGoogle ScholarPubMed
Holcik, M, Sonenberg, N 2005. Translational control in stress and apoptosis. Nature Reviews. Molecular Cell Biology 6, 318–327.CrossRefGoogle ScholarPubMed
Hopkins, DL, Taylor, RG 2004. Post-mortem muscle proteolysis and meat tenderisation. In Muscle development of livestock animals (ed. M te Pas, M Everts and H Haagsman), pp. 363–389. CAB International, Cambridge, MA, USA.Google Scholar
Hopkins, DL, Thompson, JM 2002. The degradation of myofibrillar proteins in beef and lamb using denaturing electrophoresis – an overview. Journal of Muscle Foods 13, 81–102.CrossRefGoogle Scholar
Huff-Lonergan, E, Mitsuhashi, T, Beekman, DD, Parrish, FC Jr, Olson, DG, Robson, RM 1996. Proteolysis of specific muscle structural proteins in mu-calapin at low pH and temperature is similar to degradation in postmortem bovine muscle. Journal of Animal Science 74, 993–1008.CrossRefGoogle Scholar
Hwang, IH, Park, BY, Kim, JH, Cho, SH, Lee, JM 2005. Assessment of postmortem proteolysis by gel-based proteome analysis and its relationship to meat quality traits in pig longissimus. Meat Science 69, 79–91.CrossRefGoogle ScholarPubMed
Kemp, CM, Bardsley, RG, Parr, T 2006a. Changes in caspase activity during the postmortem conditioning period and its relationship to shear force in porcine longissimus muscle. Journal of Animal Science 84, 2841–2846.CrossRefGoogle ScholarPubMed
Kemp, CM, Parr, T, Bardsley, RG, Buttery, PJ 2006b. Comparison of the relative expression of caspase isoforms in different porcine skeletal muscles. Meat Science 73, 426–431.CrossRefGoogle ScholarPubMed
Kent, MP, Veiseth, E, Therkildsen, M, Koohmaraie, M 2005. An assessment of extraction and assay techniques for quantification of calpain and calpastatin from small tissue samples. Journal of Animal Science 83, 2182–2188.CrossRefGoogle ScholarPubMed
Koohmaraie, M, Schollmeyer, JE, Dutson, TR 1986. Effect of low-calcium-requiring calcium activated factor on myofibrils under varying pH and temperature conditions. Journal of Food Science 51, 28–32.CrossRefGoogle Scholar
Koohmaraie, M, Shackelford, SD, Wheeler, TL, Lonergan, SM, Doumit, ME 1995. A muscle hypertrophy condition in lamb (callipyge): characterization of effects on muscle growth and meat quality traits. Journal of Animal Science 73, 3596–3607.CrossRefGoogle ScholarPubMed
Lametsch, R, Roepstorff, P, Moller, HS, Bendixen, E 2004. Identification of myofibrillar substrates for μ-calpain. Meat Science 68, 515–521.CrossRefGoogle ScholarPubMed
Liu, H, Chang, DW, Yang, X 2005. Interdimer processing and linearity of procaspase-3 activation. A unifying mechanism for the activation of initiator and effector caspases. Journal of Biological Chemistry 280, 11578–11582.CrossRefGoogle ScholarPubMed
McGinnis, KM, Gnegy, ME, Park, YH, Mukerjee, N, Wang, KK 1999. Procaspase-3 and poly(ADP)ribose polymerase (PARP) are calpain substrates. Biochemical and Biophysical Research Communications 263, 94–99.CrossRefGoogle ScholarPubMed
Neumar, RW, Xu, YA, Gada, H, Guttmann, RP, Siman, R 2003. Cross-talk between calpain and caspase proteolytic systems during neuronal apoptosis. Journal of Biological Chemistry 278, 14162–14167.CrossRefGoogle ScholarPubMed
Ouali, A, Herrera-Mendez, CH, Coulis, G, Becila, S, Boudjellal, A, Aubry, L, Sentandreu, MA 2006. Revisiting the conversion of muscle into meat and the underlying mechanisms. Meat Science 74, 44–58.CrossRefGoogle ScholarPubMed
Porzio, MA, Pearson, AM 1977. Improved resolution of myofibrillar proteins with sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Biochimica et Biophysica Acta 490, 27–34.CrossRefGoogle ScholarPubMed
Savell, JW, Mueller, SL, Baird, BE 2005. The chilling of carcasses. Meat Science 70, 449–459.CrossRefGoogle ScholarPubMed
Sensky, PL, Parr, T, Bardsley, RG, Buttery, PJ 1996. The relationship between plasma epinephrine concentration and the activity of the calpain enzyme-system in porcine longissimus muscle. Journal of Animal Science 74, 380–387.CrossRefGoogle ScholarPubMed
Sentandreu, MA, Coulis, G, Ouali, A 2002. Role of muscle endopeptidases and their inhibitors in meat tenderness. Trends in Food Science and Technology 13, 400–421.CrossRefGoogle Scholar
Smulders, F, Toldra, F, Flores, J, Prieto, M 1992. New technologies for meat and meat science. Audet Tijdschriften, Utrecht, The Netherlands.Google Scholar
Stennicke, HR, Salvesen, GS 1999. Caspases: preparation and characterization. Methods 17, 313–319.CrossRefGoogle ScholarPubMed
Taylor, RG, Geesink, GH, Thompson, VF, Koohmaraie, M, Goll, DE 1995. Is Z-disk degradation responsible for postmortem tenderization? Journal of Animal Science 73, 1351–1367.CrossRefGoogle ScholarPubMed
Tews, DS 2005. Muscle-fiber apoptosis in neuromuscular diseases. Muscle and Nerve 32, 443–458.CrossRefGoogle ScholarPubMed
Thornberry, NA, Lazebnik, Y 1998. Caspases: enemies within. Science 281, 1312–1316.CrossRefGoogle Scholar
Towbin, H, Staehelin, T, Gordon, J 1979. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proceedings of the National Academy of Sciences of the United States of America 76, 4350–4354.CrossRefGoogle ScholarPubMed
Veiseth, E, Shackelford, SD, Wheeler, TL, Koohmaraie, M 2004. Indicators of tenderization are detectable by 12 h postmortem in ovine longissimus. Journal of Animal Science 82, 1428–1436.CrossRefGoogle ScholarPubMed
Wang, KK 2000. Calpain and caspase: can you tell the difference? Trends in Neurosciences 23, 20–26.CrossRefGoogle ScholarPubMed
Wang, KK, Posmantur, R, Nadimpalli, R, Nath, R, Mohan, P, Nixon, RA, Talanian, RV, Keegan, M, Herzog, L, Allen, H 1998. Caspase-mediated fragmentation of calpain inhibitor protein calpastatin during apoptosis. Archives of Biochemistry and Biophysics 356, 187–196.CrossRefGoogle ScholarPubMed
Warriss, PD 2000. Meat science: an introductory text. CAB International, Wallingford, UK.CrossRefGoogle Scholar
Whipple, G, Koohmaraie, M, Dikeman, ME, Crouse, JD, Hunt, MC, Klemm, RD 1990. Evaluation of attributes that affect longissimus muscle tenderness in Bos taurus and Bos indicus cattle. Journal of Animal Science 68, 2716–2728.CrossRefGoogle ScholarPubMed
Winger, RJ, Pope, CG 1981. Osmotic properties of post-rigor beef muscle. Meat Science 5, 355–369.CrossRefGoogle ScholarPubMed