Effect of postmortem storage on mu-calpain and m-calpain in ovine skeletal muscle.
Casein zymography was used to determine the effect of postmortem storage on the proteolytic activity of mu-calpain and m-calpain in lamb longissimus. Casein zymography assays were conducted on crude muscle extracts (only one centrifugation). Six market weight crossbred lambs were slaughtered and a portion of the longissimus lumborum was removed at death (within 15 min of exsanguination) and after 3, 6, 9, 12, 24, 72, and 360 h postmortem. Muscle samples were snap-frozen in liquid nitrogen and stored at -70 degrees C. Soluble muscle proteins were extracted from muscle samples and analyzed by in-gel casein assay to measure calpain proteolytic activity. There was a gradual decline in mu-calpain activity (P < 0.05) such that after 24 and 72 h postmortem, mu-calpain had lost 42 and 95% of its activity, respectively. After 360 h postmortem, no mu-calpain activity could be detected (under the conditions used in this study). Autolysis of mu-calpain could be detected as early as 3 h postmortem. It was demonstrated that the detectable level of mu-calpain activity is a function of the amount of muscle protein electrophoresed. Hence, the activity data reported are in relative terms, rather than absolute values. Furthermore, it was demonstrated that the activity data also are a function of the assay methods used. Different methods have different lower detection limits. Of the three assays examined, 14C-labeled casein was the most sensitive, then the in-gel casein assay, and the least-sensitive method was the standard casein assay. Unlike mu-calpain, postmortem storage had no effect on m-calpain (P > 0.05). When the calcium concentration of a muscle extract was increased to the level that induces m-calpain autolysis, m-calpain was autolyzed and its autolysis was readily detected by the in-gel casein assay. Collectively, these results demonstrate that calcium concentration in postmortem muscle is only high enough to activate mu-calpain. These results support the widely believed conclusion that mu-calpain-mediated proteolysis of key myofibrillar and cytoskeletal proteins is responsible for postmortem tenderization. Hence, understanding the regulation of mu-calpain in postmortem muscle should be the focus of future studies.
Veiseth
E
E
Department of Food Science, Agricultural University of Norway, 1432 Aas, Norway.
Shackelford
S D
SD
Wheeler
T L
TL
Koohmaraie
M
M
D000818:Animals; D001329:Autolysis; D015153:Blotting, Western / Q000662:veterinary; D002154:Calpain / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel / Q000662:veterinary; D004792:Enzyme Precursors / Q000378:metabolism*; D008460:Meat / Q000592:standards; D018482:Muscle, Skeletal / Q000378:metabolism*; D011180:Postmortem Changes; D012680:Sensitivity and Specificity; D012756:Sheep
D004792:Enzyme Precursors; D002154:Calpain; C098484:mu-calpain proenzyme