Isolation and characterization of μ-calpain, m-calpain, and calpastatin from postmortem muscle. I. Initial steps

Evidence has indicated that μ-calpain, m-calpain, and calpastatin have important roles in the proteolytic degradation that results in postmortem tenderization. Simple assays of these 3 proteins at different times postmortem, however, has shown that calpastatin and μ-calpain both rapidly lose their activity during postmortem storage, so that proteolytic activity of μ-calpain is nearly zero after 3 d postmortem, even when assayed at pH 7.5 and 25°C, and ability of calpastatin to inhibit the calpains is 30% or less of its ability when assayed at death. m-Calpain, however, retains much of its proteolytic activity during postmortem storage, but the Ca²⁺ requirement of m-calpain is much higher than that reported to exist in postmortem muscle. Consequently, it is unclear how the calpain system functions in postmortem muscle. To clarify this issue, we have initiated attempts to purify the 2 calpains and calpastatin from bovine semitendinosus muscle after 11-13 d postmortem. The known properties of the calpains and calpastatin in postmortem muscle have important effects on approaches that can be used to purify them. A hexyl-TSK hydrophobic interaction column is a critical first step in separating calpastatin from the 2 calpains in postmortem muscle. Dot-blot assays were used to detect proteolytically inactive μ-calpain. After 2 column chromatographic steps, 5 fractions can be identified: 1) calpastatin I that does not bind to an anion-exchange matrix, that does not completely inhibit the calpains, and that consists of small polypeptides <60 kDa; 2) calpastatin II that binds weakly to an anion-exchange matrix and that contains polypeptides <60 kDa; all these polypeptides are smaller than the native 115- to 125-kDa skeletal muscle calpastatin; 3) proteolytically active μ-calpain even though very little μ-calpain activity can be detected in zymogram assays of muscle extracts from 11- to 13-d postmortem muscle; this μ-calpain has an autolyzed 76-kDa large subunit but the small subunit consists of 24-, 26- and a small amount of unautolyzed 28-kDa polypeptides; 4) proteolytically active m-calpain that is not autolyzed; and 5) proteolytically inactive μ-calpain whose large subunit is autolyzed to a 76-kDa polypeptide and whose small subunit contains polypeptides similar to the proteolytically active μ-calpain. Hence, loss of calpastatin activity in postmortem muscle is due to its degradation, but the cause of the loss of μ-calpain activity remains unknown.