Immunoblot analysis of calpastatin degradation: Evidence for cleavage by calpain in postmortem muscle

A negative correlation exists between calpastatin activity and meat tendern ess. Therefore, it is important to determine the mechanism of calpastatin i nactivation in postmortem skeletal muscle. Western immunoblot analysis was performed to determine the protease(s) responsible for degradation of muscl e calpastatin during postmortem storage. To accomplish this, purified calpa statin was digested with different proteases in vitro, and their pattern of calpastatin degradation was compared with that of calpastatin degradation in postmortem muscle. Polyclonal antibodies raised in mice against recombin ant, bovine skeletal muscle calpastatin were used to monitor calpastatin de gradation. Lamb longissimus was stored at 4 degrees C and sampled at 0, 6, 12, 24, 72, 168, and 336 h postmortem Postmortem storage produced a discret e pattern of calpastatin degradation products that included immunoreactive bands at approximately 100, 80, 65, 54, 32, and 29 kDa. Undegraded calpasta tin (130 kDa) was barely detectable after 72 h of postmortem storage at 4 d egrees C, and no immunoreactive calpastatin was observed by 336 h postmorte m. For in vitro proteolysis, lamb longissimus calpastatin (0 h postmortem) was purified using Affi-Gel Blue chromatography. Calpastatin was digested w ith m-calpain, mu-calpain, cathepsin B, proteasome, trypsin, or chymotrypsi n. Each of these enzymes degraded calpastatin. Immunoreactive fragments res ulting from digestion of calpastatin with m- and mu-calpain were similar to each other and closely resembled those observed during postmortem aging of lamb longissimus at 4 degrees C. Digestion of calpastatin with mu-calpain reduced calpastatin activity. Degradation of calpastatin by other proteases resulted in unique patterns of immunoreactive fragments, distinct from tha t observed in longissimus. Thus, m- and(or) mu-calpain seem to be responsib le for calpastatin degradation during postmortem storage of meat.