Calpain and calpastatin activity in porcine longissimus and the red and white portions of the semitendinosus

摘要

The objective of this study was to determine fi-calpain, m-calpain, and calpastatin activity in porcine longissimus (LD) (n=22) and semitendinosus muscles. The semitendinosus was divided into red (RST) (n=19) and white (WST) (n=22) portions. Samples were taken immediately postmortem, and sarcoplasmic proteins were quantitatively extracted and equilibrated on a Q-Sepharose ion exchange column. Protein was eluted with a linear gradient of KCl. Calpastatin eluted in two separate peaks (calpastatin I and II, 50-90mA/ KCl and 120-190mM KCl, respectively), followed by u-calpain (180-240mM KCl) and m-calpain (300-400mA/ KCl). Calpastatin Ⅰ and Ⅱ inhibition of μ-and m-calpain from the same muscle was determined in one LD. Western blots (ID and 2D) and 2D gels were also used to compare the peaks. The LD had greater u-calpain activity (p<0.0001) and less m-calpain activity (p<0.001) than the RST and WST. While the LD had less total calpastatin and calpastatin Ⅱ than both the RST and WST (p<0.0001), LD calpastatin I activity was similar (p>0.5) to that of the WST. Both the LD and WST had less (p<0.05) calpastatin I activity compared to the RST. Calpastatin II was more active than calpastatin I against both μ- and m-calpain. Each calpastatin was more active against m-calpain than u-calpain. Results suggest that conditions in the LD may be more favorable for proteolysis than in the semitendinosus, due to greater μ-calpain activity and lesser calpastatin activity. The proportion of calpastatin present as each form indicates a previously undefined source of variation for postmortem proteolysis.