Changes in the calpains and calpastatin during postmortem storage of bovine muscle.
Changes in activity and protein status of micro-calpain, m-calpain, and calpastatin in bovine semimembranosus muscle during the first 7d of postmortem storage were monitored by using assays of proteolytic activity, SDS-polyacrylamide gel electrophoresis, and Western blot analysis. Extractable m-calpain activity changed slightly during the first 7d after death (decreased to 63% of at-death activity after 7d), whereas extractable calpastatin activity decreased substantially (to 60% of at-death activity after 1d and to 30% of at-death activity after 7d of postmortem storage) during this period. Extractable micro-calpain activity also decreased rapidly (to 20% of at-death activity at 1d and to less than 4% of its at-death activity at 7d after death) during postmortem storage. Western blot analysis showed that the 80-kDa subunit of m-calpain remained undegraded during the first 7d after death but that the 125- to 130-kDa calpastatin polypeptide was gone entirely at 7d after death. Hence, the calpastatin activity remaining at 7d originates from calpastatin polypeptides that are 42 kDa or smaller. The 80-kDa micro-calpain subunit was almost entirely in the 76-kDa autolyzed form at 7d after death; this form is proteolytically active in in vitro systems, and it is unclear why the postmortem, autolyzed micro-calpain is not active. Over 50% of total muscle micro-calpain is tightly bound to myofibrils 7d after death; this micro-calpain is also nearly inactive proteolytically. Unless postmortem muscle contains some factor that enables micro-calpain in this muscle to be proteolytically active, it is not clear whether micro-calpain could be responsible for any appreciable postmortem myofibrillar proteolysis.
Muscle Biology Group, University of Arizona, Tucson 85721, USA.
M L
ML
Boehm
T L
TL
Kendall
V F
VF
Thompson
D E
DE
Goll
D000818:Animals; D015153:Blotting, Western / Q000662:veterinary; D002135:Calcium-Binding Proteins / Q000032:analysis / Q000378:metabolism*; D002154:Calpain / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D002364:Caseins / Q000378:metabolism; D002417:Cattle / Q000378:metabolism*; D015853:Cysteine Proteinase Inhibitors / Q000032:analysis / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel / Q000662:veterinary; D005260:Female; D005519:Food Preservation; D006863:Hydrogen-Ion Concentration; D008297:Male; D008460:Meat / Q000032:analysis / Q000592:standards; D009124:Muscle Proteins / Q000378:metabolism; D018482:Muscle, Skeletal / Q000737:chemistry / Q000201:enzymology / Q000378:metabolism*; D009210:Myofibrils / Q000737:chemistry / Q000378:metabolism; D011180:Postmortem Changes
D002135:Calcium-Binding Proteins; D002364:Caseins; D015853:Cysteine Proteinase Inhibitors; D009124:Muscle Proteins; C033668:calpastatin; D002154:Calpain