Properties of myofibril-bound calpain activity in longissimus muscle of callipyge and normal sheep

Properties of the calpain bound to myofibrils in longissimus muscle from ca llipyge or noncallipyge sheep were examined after 0, 1, 3, and 10 d of post mortem storage at 4 degreesC. Western analysis has shown that most of this calpain is L-calpain, although the sensitivity of the antibodies used in th e earlier studies could not eliminate the possibility that up to 10% of the calpain was m-calpain. The calpain is bound tightly, and very little is re moved by washing with the detergent Triton X-100; hence, it is not bound to phospholipids in the myofibril. Over 25% of total V-calpain was bound to m yofibrils from at-death muscle, and this increased to similar to 40% after 1 d postmortem. The amount of myofibril-bound V-calpain increased only slig htly between 1 and 10 d of postmortem storage. The percentage of autolyzed mu -calpain increases with time postmortem until after 10 d postmortem, whe n all myofibril-bound L-calpain is autolyzed. The specific activity of the myofibril-bound calpain is very low and is only 6 to 13% as high as the spe cific activity of extractable L-calpain from the same muscle. It is unclear whether this low specific activity is the result of unavailability of the active site of the myofibril-bound calpain to exogenous substrate. The myof ibril-bound calpain degrades desmin, nebulin, titin, and troponin T in the myofibrils, and also releases undegraded alpha -actinin and undergoes addit ional autolysis when incubated with Ca2+; all these activities occurred slo wly considering the amount of myofibril-bound calpain. Activity of the myof ibril-bound calpain was partly (58 to 67%) inhibited by the calpain inhibit ors, E-64 and iodoacetate; was more effectively inhibited by a broader-base d protease inhibitor, leupeptin (84 to 89%); and was poorly inhibited (43 t o 45%) by calpastatin. Release of undegraded a-actinin and autolysis are pr operties specific to the calpains, and it is unclear whether some of the my ofibril-bound proteolytic activity originates from proteases other than the calpains or whether the active site of myofibril-bound calpain is shielded from the inhibitors. Activities and properties of the myofibril-bound calp ain were identical in longissimus muscle from callipyge and normal sheep, a lthough previous studies had indicated that the "normal" longissimus was mu ch more tender than the callipyge longissimus. Hence, it seems unlikely tha t the myofibril-bound calpain has a significant role in postmortem tenderiz ation of ovine longissimus.