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ANIMAL PRODUCTS |
Department of Animal Science, Iowa State University, Ames 50011
2 Correspondence: 2372 Kildee Hall (phone: 515-294-9126; fax: 515-294-9143; e-mail: slonerga{at}iastate.edu).
The objectives of this study were to determine the extent to which pH and ionic strength influence µ- and m-calpain activity and the inhibition of calpains by calpastatin. Calpastatin, µ-calpain, and m-calpain were purified from at-death porcine semimembranosus. µ-Calpain or m-calpain (0.45 U) were incubated with the calpain substrate Suc-Leu-Leu-Val-Tyr-7-amino-4-methyl coumarin in the presence of calpastatin (0, 0.15, or 0.30 U of calpain inhibitory activity) under the following pH and ionic strength conditions: pH 7.5 and 165 mM NaCl or 295 mM NaCl; pH 6.5 and 165 mM NaCl or 295 mM NaCl; and pH 6.0 and 165 mM NaCl or 295 mM NaCl. The reactions were initiated with addition of 100 µM (µ-calpain) or 1 mM CaCl2 (m-calpain), and calpain activity was recorded at 30 and 60 min. µ-Calpain had the greatest (P < 0.01) activity at pH 6.5 at each ionic strength. Higher ionic strength decreased µ-calpain activity (P < 0.01) at all pH conditions. Inhibition percent of µ-calpain by calpastatin was not affected by pH; however, it was influenced by ionic strength. Inhibition of µ-calpain by calpastatin was higher (P < 0.01) at 295 mM NaCl than at 165 mM NaCl when 0.3 units of calpastatin were included in the assay. Activity of m-calpain was greater (P < 0.01) at pH 7.5 than at pH 6.5. m-Calpain activity was not detected at pH 6.0. Inhibition of m-calpain was greater (P < 0.01) when 0.15 and 0.3 U calpastatin were added at pH 6.5 than 7.5 at 165 mM NaCl, whereas percentage inhibition of m-calpain was greater (P < 0.01) at 295 mM than 165 mM NaCl at pH 7.5 and 6.5. These observations provide new evidence that defines further the influence of pH decline and increased ionic strength on µ-calpain, m-calpain, and calpastatin activity, thereby helping to more accurately define a role for these enzymes in the process of postmortem tenderization.
Key Words: Calpain • Calpastatin • Ionic Strength • Proteolysis • pH
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