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This Article Full Text (PDF) All Versions of this Article: jas.2008-1317v1 87/6/2096    most recent Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Weaver, A. D. Articles by Gerrard, D. E. Search for Related Content PubMed PubMed Citation Articles by Weaver, A. D. Articles by Gerrard, D. E.

Sarcomere length influences µ–calpain mediated proteolysis of bovine myofibrils

A. D. Weaver^*, B. C. Bowker and D. E. Gerrard^*

^* Department of Animal Sciences, Purdue University, West Lafayette, IN 47907 USDA-ARS, Food Quality Laboratory, Beltsville, MD 20705

dgerrard{at}purdue.edu

Abstract

Muscle shortening and postmortem proteolysis both influence beef tenderness, but their interacting effects on tenderness are relatively unknown. Inherent myofibril structure and the extent of overlap between myosin and actin filaments are hypothesized to affect the availability of substrates for degradation by calpains. The objective of this study was to determine the influence of sarcomere length on the extent of calpain-induced proteolysis of bovine myofibrils in vitro. Bovine semitendinosus (ST) muscles were excised within 20 min postmortem and dissected into strips which were stretched and attached to applicator sticks or allowed slack to generate samples with different sarcomere lengths upon rigor completion. Samples were allowed to undergo rigor in a neutral pH buffer containing a protease inhibitor. Myofibrils were isolated and incubated at room temperature with excess exogenous µ-calpain at a ratio of 1:800 (wt/wt; enzyme:myofibrillar protein) at pH 6.8 for 0, 2, 60, 1440 or 2880 min. Purified troponin was subjected to the same digestion conditions. Proteolysis of troponin T (TnT) was monitored using SDS-PAGE and western blotting. Sarcomere length was greater (P < 0.0001) in stretched versus shortened samples (2.99 µm ± 0.03 vs. 2.12 ± 0.03 µm respectively, means ± SE). Western blots for both stretched and shortened samples exhibited bands corresponding to intact TnT and TnT fragments. The abundance of intact TnT decreased (P < 0.0001) with incubation time across both treatments. At 1440 and 2880 min, less (P < 0.05) intact TnT was detected in samples with long sarcomeres. These data indicate proteolysis of TnT occurs to a greater extent in samples with longer sarcomeres possibly due to easier access of proteases to their targeted substrates. Degradation patterns of TnT were qualitatively similar between myofibrils and purified troponin after incubation with µ–calpain. Therefore it is unlikely that the mechanism by which proteolysis is limited in short sarcomeres involves an actomyosin-mediated interference of TnT.

Key Words: beef • µ-calpain • sarcomere length • tenderness • troponin-T