Proteolysis of specific muscle structural proteins by mu-calpain at low pH and temperature is similar to degradation in postmortem bovine muscle.
Postmortem (PM) and mu-calpain-induced degradation of specific skeletal muscle proteins was monitored by SDS-PAGE and Western blotting. Samples were removed from bovine longissimus thoracis (LT) at approximately 45 min PM for the preparation of at-death (0-d) myofibrils (MF). The LT was excised at 1 d PM, vacuum-packaged, and stored at 2 degrees C. Samples were removed for Warner-Bratzler shear force analysis and biochemical analysis at 1, 3, 7, 14, 28, and 56 d PM. The protease mu-calpain was purified from bovine skeletal muscle and used to digest at-death MF at pH 5.6, 4 degrees C, 100 microM CaCl2. Degradation of the proteins titin, nebulin, filamin, desmin, and troponin-T was monitored in the PM and mu-calpain-digested samples by using SDS-PAGE and Western blotting. The PM samples that had significantly lower shear force (LSF) values (P < .05) at 1 d PM exhibited faster degradation of these five proteins than the higher shear force (HSF) samples. In LSF samples, the intact titin band (T1) was absent by 7 d PM and nebulin was absent by 3 d PM. In LSF samples, some filamin was degraded by 3 d PM, but in HSF samples degradation was not apparent until 14 d PM. In LSF samples, desmin was degraded more rapidly PM than in HSF samples. Troponin-T was broken down PM to yield two major polypeptides of approximately 28 and 30 kDa; these polypeptides appeared earlier PM in LSF samples. Degradation products, similar to those observed PM, for all five proteins also were detected in Western blots of mu-calpain-digested MF, suggesting the calpain system plays a key role in PM protein degradation.
Huff-Lonergan
E
E
Department of Animal Science, Iowa State University, Ames 50011, USA.
Mitsuhashi
T
T
Beekman
D D
DD
Parrish
F C
FC
Olson
D G
DG
Robson
R M
RM
D000818:Animals; D015153:Blotting, Western / Q000662:veterinary; D002154:Calpain / Q000494:pharmacology*; D002417:Cattle / Q000378:metabolism*; D064211:Connectin; D003285:Contractile Proteins / Q000032:analysis / Q000378:metabolism; D003893:Desmin / Q000032:analysis / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel / Q000662:veterinary; D064448:Filamins; D006863:Hydrogen-Ion Concentration; D008297:Male; D008840:Microfilament Proteins / Q000032:analysis / Q000378:metabolism; D009124:Muscle Proteins / Q000032:analysis / Q000378:metabolism*; D018482:Muscle, Skeletal / Q000378:metabolism* / Q000473:pathology; D011180:Postmortem Changes; D011494:Protein Kinases / Q000032:analysis / Q000378:metabolism; D013696:Temperature; D014336:Troponin / Q000032:analysis / Q000378:metabolism
D064211:Connectin; D003285:Contractile Proteins; D003893:Desmin; D064448:Filamins; D008840:Microfilament Proteins; D009124:Muscle Proteins; D014336:Troponin; C035620:nebulin; D011494:Protein Kinases; D002154:Calpain