Immunoblot analysis of calpastatin degradation: evidence for cleavage by calpain in postmortem muscle.
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. To accomplish this, purified calpastatin 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 recombinant bovine skeletal muscle calpastatin were used to monitor calpastatin degradation. 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 discrete pattern of calpastatin degradation products that included immunoreactive bands at approximately 100, 80, 65, 54, 32, and 29 kDa. Undegraded calpastatin (130 kDa) was barely detectable after 72 h of postmortem storage at 4 degrees 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, mu-calpain, cathepsin B, proteasome, trypsin, or chymotrypsin. Each of these enzymes degraded calpastatin. Immunoreactive fragments resulting 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 that observed in longissimus. Thus, m- and(or) mu-calpain seem to be responsible for calpastatin degradation during postmortem storage of meat.
Doumit
M E
ME
Roman L. Hruska U.S. Meat Animal Research Center, ARS, USDA, Clay Center, NE 68933-0166, USA.
Koohmaraie
M
M
D000818:Animals; D015153:Blotting, Western / Q000662:veterinary; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D002154:Calpain / Q000037:antagonists & inhibitors / Q000378:metabolism*; D002417:Cattle / Q000378:metabolism*; D015853:Cysteine Proteinase Inhibitors / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel / Q000662:veterinary; D008460:Meat / Q000592:standards; D051379:Mice; D008970:Molecular Weight; D009132:Muscles / Q000201:enzymology*; D011180:Postmortem Changes
D002135:Calcium-Binding Proteins; D015853:Cysteine Proteinase Inhibitors; C033668:calpastatin; D002154:Calpain