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 \u3c 0.01) activity at pH 6.5 at each ionic strength. Higher ionic strength decreased μ-calpain activity (P\u3c 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 \u3c 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 \u3c 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 \u3c 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 \u3c 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
