Effect of postmortem storage on activity of µ- and m-calpain in five different bovine muscles

¹ Muscle Biology Group, University of Arizona, Tucson, Arizona 85721 USA

^* To whom correspondence should be addressed. E-mail: darrel.goll{at}arizona.edu.

	Abstract

An "in situ" system involving incubation of 60-80-g pieces of muscle at 4 °C under different conditions was used to determine the effects of time of postmortem storage, of pH, and of temperature on activities of µ- and m-calpain activity in bovine skeletal muscle. Casein zymograms were used to allow measurement of calpain activity with a minimum of sample preparation and to ensure that the calpains were not exposed to ionic strengths of 100 or higher before assay of their activities. In four of the five muscles (longissimus dorsi, lumbar; longissimus dorsi, thoracic; psoas major, semimembranosus, and triceps brachii) studied, µ-calpain activity deceased nearly to zero within 48 h postmortem. Activity of m-calpain also decreased in the in situ system used, but at a much slower rate. Activities of both µ- and m-calpain decreased more slowly in the triceps brachii muscle than in the other four muscles during postmortem storage. Although previous studies have indicated that µ-calpain but not m-calpain is proteolytically active at pH 5.8, these studies have used calpains obtained from at-death muscle. Both µ- and m-calpain are proteolytically inactive if their activities are measured at pH 5.8 and after incubating the muscle pieces for 24 h at pH 5.8. Western analysis suggested that neither the large 80-kDa subunit nor the small, 28-kDa subunit of m-calpain was autolyzed during postmortem storage of the muscle pieces. As has been reported previously, the 80-kDa subunit of µ-calpain was autolyzed to 78- and then to a 76-kDa polypeptide after 7 days postmortem, but the 28-kDa small subunit was not autolyzed; hence, autolyzed µ-calpain molecule in postmortem muscle is a 76-/28-kDa molecule and not a 76-/18-kDa molecule as previously assumed. Because both subunits were present in the postmortem calpains, loss of µ-calpain activity during postmortem storage is not due to dissociation of the two subunits and inactivation. Although previous studies have shown that the 76-/18-kDa µ-calpain molecule is completely active proteolytically, it is possible that the 76-/28-kDa µ-calpain molecule in postmortem muscle is proteolytically inactive and that this accounts for the loss of µ-calpain activity during postmortem storage. Because neither µ- nor m-calpain is proteolytically active at pH 5.8 after being incubated at pH 5.8 for 24 h, other proteolytic systems such as the caspases may contribute to postmortem proteolysis in addition to the calpains.

Key Words: µ-calpain, m-calpain, meat tenderness, zymograms, postmortem muscle