Doumit M E, Koohmaraie M
Roman L. Hruska U.S. Meat Animal Research Center, ARS, USDA, Clay Center, NE 68933-0166, USA.
J Anim Sci. 1999 Jun;77(6):1467-73. doi: 10.2527/1999.7761467x.
A negative correlation exists between calpastatin activity and meat tenderness. Therefore, it is important to determine the mechanism of calpastatin inactivation in postmortem skeletal muscle. Western immunoblot analysis was performed to determine the protease(s) responsible for degradation of muscle calpastatin during postmortem storage. To accomplish this, purified calpastatin was digested with different proteases in vitro, and their pattern of calpastatin degradation was compared with that of calpastatin degradation in postmortem muscle. Polyclonal antibodies raised in mice against recombinant bovine skeletal muscle calpastatin were used to monitor calpastatin degradation. Lamb longissimus was stored at 4 degrees C and sampled at 0, 6, 12, 24, 72, 168, and 336 h postmortem. Postmortem storage produced a discrete pattern of calpastatin degradation products that included immunoreactive bands at approximately 100, 80, 65, 54, 32, and 29 kDa. Undegraded calpastatin (130 kDa) was barely detectable after 72 h of postmortem storage at 4 degrees C, and no immunoreactive calpastatin was observed by 336 h postmortem. For in vitro proteolysis, lamb longissimus calpastatin (0 h postmortem) was purified using Affi-Gel Blue chromatography. Calpastatin was digested with m-calpain, mu-calpain, cathepsin B, proteasome, trypsin, or chymotrypsin. Each of these enzymes degraded calpastatin. Immunoreactive fragments resulting from digestion of calpastatin with m- and mu-calpain were similar to each other and closely resembled those observed during postmortem aging of lamb longissimus at 4 degrees C. Digestion of calpastatin with mu-calpain reduced calpastatin activity. Degradation of calpastatin by other proteases resulted in unique patterns of immunoreactive fragments, distinct from that observed in longissimus. Thus, m- and(or) mu-calpain seem to be responsible for calpastatin degradation during postmortem storage of meat.
钙蛋白酶抑制蛋白活性与肉的嫩度之间存在负相关。因此，确定宰后骨骼肌中钙蛋白酶抑制蛋白失活的机制很重要。进行了蛋白质免疫印迹分析，以确定负责宰后储存期间肌肉钙蛋白酶抑制蛋白降解的蛋白酶。为实现这一目的，在体外使用不同的蛋白酶消化纯化的钙蛋白酶抑制蛋白，并将其钙蛋白酶抑制蛋白降解模式与宰后肌肉中的钙蛋白酶抑制蛋白降解模式进行比较。用针对重组牛骨骼肌钙蛋白酶抑制蛋白的小鼠多克隆抗体来监测钙蛋白酶抑制蛋白的降解。将羊的背最长肌在4℃下储存，并在宰后0、6、12、24、72、168和336小时取样。宰后储存产生了一种离散的钙蛋白酶抑制蛋白降解产物模式，包括在约100、80、65、54、32和29 kDa处的免疫反应条带。在4℃下宰后储存72小时后，几乎检测不到未降解的钙蛋白酶抑制蛋白（130 kDa），并且在宰后336小时未观察到免疫反应性钙蛋白酶抑制蛋白。对于体外蛋白水解，使用Affi-Gel Blue层析法纯化宰后0小时的羊背最长肌钙蛋白酶抑制蛋白。用微钙蛋白酶、μ-钙蛋白酶、组织蛋白酶B、蛋白酶体、胰蛋白酶或胰凝乳蛋白酶消化钙蛋白酶抑制蛋白。这些酶中的每一种都能降解钙蛋白酶抑制蛋白。用微钙蛋白酶和μ-钙蛋白酶消化钙蛋白酶抑制蛋白产生的免疫反应性片段彼此相似，并且与在4℃下羊背最长肌宰后成熟期间观察到的片段非常相似。用μ-钙蛋白酶消化钙蛋白酶抑制蛋白会降低钙蛋白酶抑制蛋白的活性。其他蛋白酶对钙蛋白酶抑制蛋白的降解导致了独特的免疫反应性片段模式，与在背最长肌中观察到的不同。因此，微钙蛋白酶和（或）μ-钙蛋白酶似乎是肉类宰后储存期间钙蛋白酶抑制蛋白降解的原因。
