Festoff B W, Patterson M R, Romstedt K
J Cell Physiol. 1982 Feb;110(2):190-5. doi: 10.1002/jcp.1041100213.
Clonal mouse skeletal muscle cells which differentiate in culture and form synapses with neuronal cells were found to secrete high levels of protease activity as measured with an 125I-fibrin assay. The secreted proteolytic activity was more than 90% dependent upon the presence of plasminogen in the medium, and had a pH optimum at 7 to 8. This activity was not inhibited by n-ethylmaleimide, pepstatin, EDTA, or EGTA. At millimolar concentrations, greater than 90% inhibition was obtained with either soybean trypsin inhibitor, epsilon aminocaproic acid, Trasylol, or leupeptin. Almost complete inhibition occurred with 1 mM diisopropylfluorophosphate suggesting the presence of a serine residue at the catalytic site. In contrast to the high levels of secreted activity, a lower steady-state level of cell-associated protease activity was detected in cell lysates. The high level of plasminogen activator secreted into the medium of cultured muscle cells suggests a role for such extracellular protease activity in myogenesis during development and remodeling following muscle injury. Such information may be useful in understanding the initial degeneration of neuromuscular contacts in experimental and pathologic denervation.
在培养过程中分化并与神经元细胞形成突触的克隆小鼠骨骼肌细胞，经125I-纤维蛋白测定法检测，发现其分泌高水平的蛋白酶活性。所分泌的蛋白水解活性超过90%依赖于培养基中纤溶酶原的存在，最适pH值为7至8。该活性不受N-乙基马来酰亚胺、胃蛋白酶抑制剂、乙二胺四乙酸（EDTA）或乙二醇双四乙酸（EGTA）的抑制。在毫摩尔浓度下，大豆胰蛋白酶抑制剂、ε-氨基己酸、抑肽酶或亮抑酶肽均可产生大于90%的抑制作用。1 mM二异丙基氟磷酸几乎可完全抑制，这表明催化位点存在丝氨酸残基。与分泌的高水平活性相反，在细胞裂解物中检测到较低的细胞相关蛋白酶活性稳态水平。分泌到培养肌肉细胞培养基中的高水平纤溶酶原激活剂表明，这种细胞外蛋白酶活性在发育过程中的肌生成以及肌肉损伤后的重塑中发挥作用。这些信息可能有助于理解实验性和病理性去神经支配中神经肌肉接触的初始退化。
