Delgado E F, Geesink G H, Marchello J A, Goll D E, Koohmaraie M
Roman L. Hruska U.S. Meat Animal Research Center, USDA, ARS, Clay Center, Nebraska 68933-0166, USA.
J Anim Sci. 2001 Aug;79(8):2097-107. doi: 10.2527/2001.7982097x.
Properties of the calpain bound to myofibrils in longissimus muscle from callipyge or noncallipyge sheep were examined after 0, 1, 3, and 10 d of postmortem storage at 4 degrees C. Western analysis has shown that most of this calpain is mu-calpain, although the sensitivity of the antibodies used in the 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 removed by washing with the detergent Triton X-100; hence, it is not bound to phospholipids in the myofibril. Over 25% of total mu-calpain was bound to myofibrils from at-death muscle, and this increased to approximately 40% after 1 d postmortem. The amount of myofibril-bound mu-calpain increased only slightly between 1 and 10 d of postmortem storage. The percentage of autolyzed mu-calpain increases with time postmortem until after 10 d postmortem, when all myofibril-bound mu-calpain is autolyzed. The specific activity of the myofibril-bound calpain is very low and is only 6 to 13% as high as the specific activity of extractable mu-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 myofibril-bound calpain degrades desmin, nebulin, titin, and troponin T in the myofibrils, and also releases undegraded alpha-actinin and undergoes additional autolysis when incubated with Ca2+; all these activities occurred slowly considering the amount of myofibril-bound calpain. Activity of the myofibril-bound calpain was partly (58 to 67%) inhibited by the calpain inhibitors, E-64 and iodoacetate; was more effectively inhibited by a broader-based protease inhibitor, leupeptin (84 to 89%); and was poorly inhibited (43 to 45%) by calpastatin. Release of undegraded alpha-actinin and autolysis are properties specific to the calpains, and it is unclear whether some of the myofibril-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 calpain were identical in longissimus muscle from callipyge and normal sheep, although previous studies had indicated that the "normal" longissimus was much more tender than the callipyge longissimus. Hence, it seems unlikely that the myofibril-bound calpain has a significant role in postmortem tenderization of ovine longissimus.
在4℃下对臀肌肥大或非臀肌肥大绵羊的背最长肌肌原纤维结合钙蛋白酶的特性进行了0、1、3和10天的死后储存研究。蛋白质免疫印迹分析表明，这种钙蛋白酶大部分是μ-钙蛋白酶，尽管早期研究中使用的抗体的敏感性不能排除高达10%的钙蛋白酶是m-钙蛋白酶的可能性。钙蛋白酶紧密结合，用去污剂Triton X-100洗涤只能去除很少一部分；因此，它不与肌原纤维中的磷脂结合。超过25%的总μ-钙蛋白酶与死亡时的肌肉中的肌原纤维结合，死后1天这一比例增加到约40%。死后储存1至10天期间，肌原纤维结合的μ-钙蛋白酶的量仅略有增加。自溶的μ-钙蛋白酶的百分比随死后时间增加，直到死后10天，此时所有肌原纤维结合的μ-钙蛋白酶都发生自溶。肌原纤维结合的钙蛋白酶的比活性非常低，仅为同一肌肉中可提取的μ-钙蛋白酶比活性的6%至13%。目前尚不清楚这种低比活性是由于肌原纤维结合的钙蛋白酶的活性位点对外源底物不可用所致。肌原纤维结合钙蛋白酶可降解肌原纤维中的结蛋白、伴肌动蛋白、肌联蛋白和肌钙蛋白T，与Ca2+孵育时还会释放未降解的α-辅肌动蛋白并发生额外的自溶；考虑到肌原纤维结合钙蛋白酶的量，所有这些活性都发生得很缓慢。肌原纤维结合钙蛋白酶的活性部分（58%至67%）受到钙蛋白酶抑制剂E-64和碘乙酸的抑制；更有效地受到更广泛的蛋白酶抑制剂亮抑蛋白酶肽（84%至89%）的抑制；而受到钙蛋白酶抑制蛋白的抑制较弱（43%至45%）。释放未降解的α-辅肌动蛋白和自溶是钙蛋白酶特有的特性，目前尚不清楚一些肌原纤维结合的蛋白水解活性是否源自钙蛋白酶以外的蛋白酶，或者肌原纤维结合钙蛋白酶的活性位点是否被抑制剂屏蔽。臀肌肥大绵羊和正常绵羊的背最长肌中肌原纤维结合钙蛋白酶的活性和特性是相同的，尽管先前的研究表明“正常”背最长肌比臀肌肥大背最长肌更嫩。因此，肌原纤维结合钙蛋白酶似乎不太可能在绵羊背最长肌的死后嫩化中起重要作用。
