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 mucalpain-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. ABSTRACT:Postmortem (PM) and m-calpaininduced degradation of specific skeletal muscle proteins was monitored by SDS-PAGE and Western blotting. Samples were removed from bovine longissimus thoracis ( L T ) 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°C. Samples were removed for WarnerBratzler shear force analysis and biochemical analysis at 1, 3, 7, 14, 28, and 56 d PM. The protease m-calpain was purified from bovine skeletal muscle and used to digest at-death MF at pH 5.6, 4°C, 100 mM CaCl 2 . Degradation of the proteins titin, nebulin, filamin, desmin, and troponin-T was monitored in the PM and m-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 ( T 1 ) 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, ...

Purified myofibril (MF) and homogenized whole muscle (WM) samples were prepared from A maturity market steers. Samples were removed at 0, 1, 3, 7, 14, and 28 d postmortem. The MF and WM samples from all steers were analyzed by SDS-PAGE (5% gels) and by Western blot analysis using monoclonal antibodies to titin and nebulin. The rates of degradation of the intact forms of titin and nebulin, with regard to differences dependent on sample type (MF vs WM), were examined. The results showed that there was very little difference in the rate of postmortem degradation of the intact form of titin or of intact nebulin with respect to the two types of samples examined. Analysis of MF and WM preparations revealed that titin and nebulin were progressively degraded, each at its own rate, with nebulin degrading faster, as postmortem storage time increased. Examination of MF and WM samples showed that the intact form of titin (T1) was absent at the same time postmortem in both sample types. Intact nebulin was not detected in MF and WM preparations at the same time postmortem with respect to sample type examined. Our results indicate that either purified MF or WM samples can be used satisfactorily to analyze the rate of degradation of the intact forms of both titin and nebulin.

An improved method was investigated for sodium dodecyl sulfate polyacrylamide slab gel electrophoresis (SDS-PAGE) to facilitate the analysis of the giant myofibrillar proteins, connectin and nebulin, in fish meat by using jack mackerel (Trachurus japonicus) as the sample fish. It was established that separation of the alpha-connectin band from the beta-connectin band by SDS-PAGE could be achieved by using 3-5% gradient gels with glycerol to facilitate the formation of a gradient with polymerization at 35 degrees C. SDS-PAGE samples of white dorsal muscle from the jack mackerel were homogenized with a 2% SDS solution containing an inhibitor mixture (1 microg/mL of phenylmethanesulfonyl fluoride, 1 microg/mL of leupeptin, and 1 microg/mL of E-64) and heated at 50 degrees C for 20 min. Heating these samples at 100 degrees C for 2 min resulted in the disintegration of connectin but did not affect nebulin. A purified myofibril sample and a whole muscle sample showed similar changes in the overall rate of degradation of whole connectin and nebulin during the postmortem storage period, but it was clear that beta-connectin was cleaved from alpha-connectin during the preparation of myofibrils at the early stage postmortem. Storage of the SDS-PAGE samples at -85 degrees C was preferable to storage at -18 degrees C for a long period.

The present study examined the changes in texture and protein components during cold storage of different squid varieties. Raw oval squid, Japanese common squid and arrow squid were sliced fresh and the muscles were stored at 4°C for 0, 4, 8, 12, 18, 24, 48 and 120 h. The rheological measurements, protein components and amounts of collagen were examined. The adhesiveness of each squid increased significantly in the early stage of cold storage. In all varieties, penetration decreased at 4 h, which is considered to be rigor mortis, then increased. The amounts of total collagen, 20°C water‐soluble collagen and 70°C water‐soluble collagen did not change significantly in each variety during cold storage. Sodium dodecylsulfate–polyacrylamide gel electrophoresis (SDS‐PAGE) pattern showed that the 580 kDa component gradually disappeared up to 48 h. The correlations between the amounts of 580 kDa component and adhesiveness or firmness were high. Models of fit based on chemical kinetics accurately expressed the behavior of adhesiveness, firmness and penetration showing that 63.2% of adhesiveness changes occurred in 13–19 h and that 63.2% of firmness changes occurred in 18–24 h.