Kumamoto T, Kleese W C, Cong J Y, Goll D E, Pierce P R, Allen R E
Muscle Biology Group, University of Arizona, Tucson 85721.
Anat Rec. 1992 Jan;232(1):60-77. doi: 10.1002/ar.1092320108.
Immunofluorescence and immunogold localization studies show that the two Ca(2+)-dependent proteinases (mu-calpain for the micromolar Ca(2+)-requiring proteinase and m-calpain for the millimolar Ca(2+)-requiring proteinase) and their protein inhibitor (calpastatin) are located exclusively intracellularly in normal rat soleus muscle. Quantitative immunogold studies indicate that binding of antibodies to both calpains and to calpastatin is approximately two times greater at the Z-disk of myofibrils than it is at the I-band or A-band regions. Mitochondria and nuclei in muscle cells contain both calpains and calpastatin at concentrations approximately one-tenth and one-fifth, respectively, of the concentration at the Z-disk, as estimated by antibody binding. Very little calpain or calpastatin was seen in the cytoplasmic intermyofibrillar spaces, and most of the calpain and calpastatin in muscle cells is associated with intracellular structures. Immunofluorescence results suggest that concentration of m-calpain but not mu-calpain or calpastatin is, in some instances, slightly higher near the intracellular surface of the plasma membrane than elsewhere in the muscle cell. Most m-calpain, however, is distributed throughout the interior of mature rat skeletal muscle cells. Denervation, or fasting and refeeding increases the concentration of the calpains and calpastatin in the muscle cell but does not change their distribution. Some mu- and m-calpain and calpastatin is found extracellularly in denervated soleus muscle or soleus muscle from fasting rats, but the extracellular calpains and calpastatin seem to originate from "leakage" of these proteins out of the cell because serum creatine kinase levels are much higher than normal in denervated or fasting rats.
免疫荧光和免疫金定位研究表明，两种钙依赖性蛋白酶（微摩尔钙需求蛋白酶的μ-钙蛋白酶和毫摩尔钙需求蛋白酶的m-钙蛋白酶）及其蛋白抑制剂（钙蛋白酶抑制蛋白）在正常大鼠比目鱼肌中仅位于细胞内。定量免疫金研究表明，肌原纤维Z盘处针对钙蛋白酶和钙蛋白酶抑制蛋白的抗体结合量比I带或A带区域大约高两倍。通过抗体结合估计，肌肉细胞中的线粒体和细胞核所含钙蛋白酶和钙蛋白酶抑制蛋白的浓度分别约为Z盘处浓度的十分之一和五分之一。在肌原纤维间的细胞质空间中几乎看不到钙蛋白酶或钙蛋白酶抑制蛋白，肌肉细胞中的大多数钙蛋白酶和钙蛋白酶抑制蛋白与细胞内结构相关。免疫荧光结果表明，在某些情况下，m-钙蛋白酶的浓度在质膜细胞内表面附近略高于肌肉细胞中的其他位置，而μ-钙蛋白酶或钙蛋白酶抑制蛋白则不然。然而，大多数m-钙蛋白酶分布在成熟大鼠骨骼肌细胞内部。去神经支配、禁食和再喂食会增加肌肉细胞中钙蛋白酶和钙蛋白酶抑制蛋白的浓度，但不会改变它们的分布。在去神经支配的比目鱼肌或禁食大鼠的比目鱼肌中，细胞外发现了一些μ-钙蛋白酶、m-钙蛋白酶和钙蛋白酶抑制蛋白，但细胞外的钙蛋白酶和钙蛋白酶抑制蛋白似乎源于这些蛋白质从细胞中“泄漏”，因为去神经支配或禁食大鼠的血清肌酸激酶水平远高于正常水平。
