Author
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DOUMIT, MATTHEW - MI STATE UNIV, E. LANSING |
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Koohmaraie, Mohammad |
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Submitted to: Journal of Animal Science
Publication Type: Peer Reviewed Journal Publication Acceptance Date: 9/23/1998 Publication Date: N/A Citation: N/A Interpretive Summary: Consumers consider tenderness to be the single most important component of meat quality. This fact is easily confirmed by the positive relationship between the price of a cut of meat and its relative tenderness. Inconsistency in meat tenderness has been identified as one of the major problems facing the beef industry. Difference in the rate and extent of postmortem tenderization is responsible for the variation in meat tenderness at the consumer level. Breakdown of key muscle proteins is responsible for meat tenderization that occurs during postmortem storage of meat. Calpastatin activity at 24 h postmortem is inversely proportional to postmortem tenderization and accounts for a greater proportion of the variation in beef tenderness (approximately 40%) than any other single. In light of the relationship between postmortem calpastatin activity and meat tenderness, it is important to determine the mechanism of calpastatin inactivation in postmortem muscle. Understanding calpastatin degradation may provide insight for development of improved methods for meat tenderization. The objective of the experiments described here was to determine the protease(s) responsible for degradation of muscle calpastatin during postmortem storage. This was accomplished using Western immunoblot analysis to compare muscle calpastatin degradation products resulting from postmortem storage and in vitro digestion with purified proteases. Results indicated that only m- and u-calpain were able to reproduce postmortem changes in calpastatin. Technical Abstract: Calpastatin is an endogenous calpain inhibitor that regulates postmortem proteolysis and, thus, meat tenderization. A negative correlation exists between calpastatin activity and meat tenderness, therefore, it is important to determine the mechanism of calpastatin inactivation in postmortem skeletal muscle. Western immunoblot analysis was performed to determine the protease(s) responsible for degradation of muscle calpastatin during postmortem storage. Polyclonal antibodies raised in mice against recombinant bovine skeletal muscle calpastatin were used to monitor calpastatin degradation. Lamb longissimus was stored at 4 deg C and sampled at 0, 6, 12, 24, 72, 168, and 336 h postmortem. Postmortem storage produced a discrete pattern of calpastatin degradation products which included immunoreactive bands at approximately 100, 80, 65, 54, 32, and 29 kDa. Undegraded calpastatin (130 kDa) was barely detectable after 72 h postmortem storage at 4 deg C and no immunoreactive calpastatin was observed by 336 h postmortem. For in vitro proteolysis, lamb longissimus calpastatin (0 h postmortem) was purified using Affi-Gel Blue chromatography. Calpastatin was digested with m-calpain, u-calpain, cathepsin B, proteosome, trypsin, or chymotrypsin. Each of these enzymes degraded calpastatin. Immunoreactive fragments resulting from digestion of calpastatin with m- and u-calpain were similar to each other, and closely resembled those observed during postmortem aging of lamb longissimus at 4 deg C. Degradation of calpastatin by other proteases resulted in unique patterns of immunoreactive fragments, distinct from that observed in longissimus. Thus, m- and/or u-calpain appear to be responsible for calpastatin degradation. |
