Averna Monica, Stifanese Roberto, De Tullio Roberta, Defranchi Enrico, Salamino Franca, Melloni Edon, Pontremoli Sandro
Department of Experimental Medicine (DIMES), Section of Biochemistry and Centre of Excellence for Biomedical Research (CEBR), University of Genova, Italy.
FEBS J. 2006 Apr;273(8):1660-8. doi: 10.1111/j.1742-4658.2006.05180.x.
Conformational changes in the calpain molecule following interaction with natural ligands can be monitored by the binding of a specific monoclonal antibody directed against the catalytic domain of the protease. None of these conformational states showed catalytic activity and probably represent intermediate forms preceding the active enzyme state. In its native inactive conformation, calpain shows very low affinity for this monoclonal antibody, whereas, on binding to the ligands Ca(2+), substrate or calpastatin, the affinity increases up to 10-fold, with calpastatin being the most effective. This methodology was also used to show that calpain undergoes similar conformational changes in intact cells exposed to stimuli that induce either a rise in intracellular [Ca(2+)] or extensive diffusion of calpastatin into the cytosol without affecting Ca(2+) homeostasis. The fact that the changes in the calpain state are also observed under the latter conditions indicates that calpastatin availability in the cytosol is the triggering event for calpain-calpastatin interaction, which is presumably involved in the control of the extent of calpain activation through translocation to specific sites of action.
钙蛋白酶分子与天然配体相互作用后构象的变化，可以通过一种针对该蛋白酶催化结构域的特异性单克隆抗体的结合来监测。这些构象状态均未显示出催化活性，可能代表活性酶状态之前的中间形式。在其天然无活性构象中，钙蛋白酶对这种单克隆抗体的亲和力非常低，而在与配体Ca(2+)、底物或钙蛋白酶抑制蛋白结合时，亲和力会增加至10倍，其中钙蛋白酶抑制蛋白最为有效。该方法还用于表明，在完整细胞中，当暴露于诱导细胞内[Ca(2+)]升高或钙蛋白酶抑制蛋白大量扩散到细胞质中而不影响Ca(2+)稳态的刺激时，钙蛋白酶会发生类似的构象变化。在后一种条件下也观察到钙蛋白酶状态的变化，这一事实表明细胞质中钙蛋白酶抑制蛋白的可利用性是钙蛋白酶 - 钙蛋白酶抑制蛋白相互作用的触发事件，这可能参与通过转运到特定作用位点来控制钙蛋白酶激活的程度。
