Effect of postmortem storage on mu-calpain and m-calpain in ovine skeletal muscle.

Abstract

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.

Postmortem degradation of skeletal muscle proteins: a novel approach to determine the time since death

“…Note that at 4 μM CaCl 2 , the degraded calpain 1 is also active The native and the autolyzed forms have been shown to be active and bind tightly to intracellular structures such as myofibrils, where they proteolyze titin, nebulin, and desmin [31,36]. Native calpain 1 activity decreased faster than calpain 2 activity which is in agreement with the results in pig and lamb [32,45]. This is supported by Western blot analysis in bovine and ovine muscle, which demonstrated that the 80-kDa subunit of calpain 1 almost entirely degraded into a 76-kDa subunit within the first 7 days postmortem, while the 80-kDa subunit of calpain 2 remained undegraded in this period [42,46].…”

Effects of High Oxygen Packaging on Tenderness and Water Holding Capacity of Pork Through Protein Oxidation

“…The myofibrillar samples were frozen at −80°C until used for assessing protein surface hydrophobicity and electrophoresis. Sarcoplasmic protein was prepared following the description of Veiseth et al (2001). All samples were homogenized with extraction solutions (10 mM ethylene diamine tetraacetic acid, 100 mM Tris-HCl, and 0.1 % β-mercaptoethanol (v/v), pH 8.3).…”

Eriobotrya japonica improves hyperlipidemia and reverses insulin resistance in high‐fat‐fed mice

“…Protein extractions and immunoblots for the determination of AMPK phosphorylation were carried out on frozen liver and adipose tissue from mice according to a previous report (Shen et al, 2005). Briefl y, liver and adipose samples (0.1 g, 0.1 g, respectively) were powdered under liquid nitrogen and homogenized for 20 s in 500 μL buffer containing 20 mm Tris-HCl (pH 7.4 at 4°C), 2% SDS, 5 mm EDTA, 5 mm EGTA, 1 mm DTT, 100 mm NaF, 2 mm sodium vanadate, 0.5 mm phenylmethylsulfonyl fl uoride, 10 μg/mL leupeptin and 10 μL/ mL pepstatin (Raser et al, 1995;Veiseth et al, 2001). 40 μg of each homogenate was mixed with an equal amount of 2 × standard SDS sample loading buffer containing 125 mm Tris-HCl (pH 6.8), 4% SDS, 20% glycerol, 10% β-mercaptoethanol and 0.25% bromophenol blue, and boiled for 10 min before electrophoresis.…”