Molinari M, Anagli J, Carafoli E
Institute of Biochemistry, Swiss Federal Institute of Technology, Zurich.
J Biol Chem. 1994 Nov 11;269(45):27992-5.
The aim of this study was to investigate the process leading to Ca(2+)-activated neutral protease (CANP) activation in vivo. The unautolyzed form of CANP has been targeted to the erythrocyte membrane by increasing, in a controlled way, the Ca2+ concentration in the cells; this was achieved by incubating erythrocytes with the Ca2+ ionophore A23187 and fixed Ca2+ concentrations. After isolation of the CANP-bearing erythrocyte membrane, we could observe that CANP remained bound to the membrane in the 80-kDa unautolyzed form in the presence of low Ca2+ concentrations (1.75 microM); under these conditions, the preferred CANP substrates (the Ca(2+)-ATPase and Band 3) were cleaved. That the cleavage was due to CANP was shown by the finding that the two substrates were not degraded in the presence of a membrane-permeable irreversible CANP inhibitor, Cbz-Leu-Leu-Tyr-CHN2, nor when the free Ca2+ concentration was decreased to sub microM levels with EDTA. The findings suggest an activation mechanism of CANP based on its translocation to the membrane rather than on its autolysis. In this mechanism, CANP would become reversibly activated on the membrane and would return to the quiescent state after dissociating from it when the cell Ca2+ concentration has returned to the physiological, submicromolar level.
本研究的目的是调查体内导致钙激活中性蛋白酶（CANP）激活的过程。通过以可控方式增加细胞内的Ca2+浓度，将未自溶形式的CANP靶向红细胞膜；这是通过将红细胞与Ca2+离子载体A23187和固定的Ca2+浓度孵育来实现的。分离出携带CANP的红细胞膜后，我们可以观察到，在低Ca2+浓度（1.75 microM）下，CANP以80 kDa的未自溶形式与膜结合；在这些条件下，CANP的首选底物（Ca(2+)-ATP酶和带3蛋白）被切割。两个底物在膜通透性不可逆CANP抑制剂Cbz-Leu-Leu-Tyr-CHN2存在时未被降解，以及当用EDTA将游离Ca2+浓度降低至亚 microM水平时也未被降解，这一发现表明切割是由CANP引起的。这些发现提示了一种基于CANP转位至膜而非自溶的激活机制。在这种机制中，CANP在膜上可逆性激活，当细胞Ca2+浓度恢复到生理亚微摩尔水平并从膜上解离后，它将恢复到静止状态。
