Isolation and characterization of μ-calpain, m-calpain, and calpastatin from postmortem muscle. I. Initial steps1

Abstract

Evidence has indicated that mu-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 mu-calpain both rapidly lose their activity during postmortem storage, so that proteolytic activity of mu-calpain is nearly zero after 3 d postmortem, even when assayed at pH 7.5 and 25 degrees 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 Ca(2+) 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 mu-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 mu-calpain even though very little mu-calpain activity can be detected in zymogram assays of muscle extracts from 11- to 13-d postmortem muscle; this mu-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 mu-calpain whose large subunit is autolyzed to a 76-kDa polypeptide and whose small subunit contains polypeptides similar to the proteolytically active mu-calpain. Hence, loss of calpastatin activity in postmortem muscle is due to its degradation, but the cause of the loss of mu-calpain activity remains unknown.

Identification of calpastatin, μ-calpain and m-calpain in Atlantic salmon (Salmo salar L.) muscle

“…This is, however, in contrast to findings in rainbow trout (Oncorhynchus mykiss), where the glycine rich part of domain V was lacking and therefore making the calpain molecule less hydrophobic (Salem, Nath, & Killefer, 2004b). Moreover, inclusion of a non ionic detergent (Brij 35) in the elution buffer is shown to contribute to a complete elution of the calpains (Camou et al, 2007). In our experiment, calpain eluted of the hydrophobic column with Brij 35 included in the buffer and was in this way separated from calpastatin.…”

“…The calpain peaks eluted off the DEAE-sephacel column at a lower salt concentration than reported for both mammalian (Camou et al, 2007) and rainbow trout calpains (Saito et al, 2007). This indicates that calpains from Atlantic salmon have a weaker ionic interaction with the column media (DEAE-sephacel), which could result from differences in their amino acid composition compared to their mammalian counterparts.…”

“…Salmon calpastatin eluted off the DEAE-sephacel column over a broad range of salt concentrations as seen in other experiments (Camou et al, 2007;Geesink et al, 2000;Saito et al, 2007). Specifically, two separate peaks with differing inhibitory activity were found in salmon muscle in this experiment.…”

A expressão da isoforma de calpastatina responsiva à ractopamina altera a maciez da carne, com implicações na eficiência de crescimento de suínos

“…O sistema das calpaínas (µ-calpaína, m-calpaína e calpastatina) foi primeiramente identificado como composto de proteinases cisteínicas, cálcio dependentes presente em todas as células, e desde então tem sido descrito com grande detalhamento (CAMOU et al, 2007;GOLL et al, 2003;SORIMACHI, H.;SUZUKI, 2001). As isoformas µ-calpaína e m-calpaína catalisam a proteólise no citoesqueleto e em proteínas de membrana e são reguladas pela concentração de íons de cálcio e pela calpastatina que funciona como inibidor.…”

Enzymes in Tenderization of Meat – The System of Calpains and Other Systems

“…Calpastatine participates in meat tenderization and is negatively correlated with tenderness [Kent et al, 2004;Neath et al, 2007]. Therefore it is claimed that calpains I and II and calpastatine play an important role in meat tenderization [Camou et al, 2007].…”