Isolation and characterization of μ-calpain, m-calpain, and calpastatin from postmortem muscle. I. Initial steps1
| Autor: | J. A. Marchello, S. W. Mares, Marlon D Taylor, Darrel E. Goll, Valery F. Thompson, J. P. Camou, R. Vazquez |
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| Rok vydání: | 2007 |
| Předmět: | |
| Zdroj: | Journal of Animal Science. 85:3400-3414 |
| ISSN: | 1525-3163 0021-8812 |
| DOI: | 10.2527/jas.2007-0356 |
| Popis: | 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 |
| Databáze: | OpenAIRE |
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