Inomata M, Hayashi M, Nakamura M, Saito Y, Kawashima S
Department of Biochemistry, Tokyo Metropolitan Institute of Gerontology, Japan.
J Biol Chem. 1989 Nov 5;264(31):18838-43.
The binding of a calcium-activated neutral protease (CANP) with high calcium sensitivity (muCANP) to erythrocyte membranes and its subsequent autolytic activation on the membranes were analyzed by an immunoblot technique. In the presence of calcium ions, muCANP bound to the erythrocyte membranes as a heterodimer of 79- and 28-kDa subunits and was converted quickly on the membranes to an active form with a 76-kDa large subunit. The active form was then released from the membranes to the soluble fraction. These sequential reactions, however, were not specific to inside-out vesicles, but occurred also, except for some Ca2+-independent binding, on right side-out vesicles. A rapid degradation of some membrane proteins was observed after binding of muCANP to the membranes. The binding of muCANP to erythrocyte membranes was inhibited by substrates and the endogenous CANP inhibitor, which is also a suicide substrate. These results strongly suggest that muCANP binds to membranes by recognition of membrane proteins as substrates and not at a special site for activation. Thus, a possible mechanism for muCANP activation on membranes is that muCANP first binds to substrates on membranes, is activated, and then degrades the substrates to deform the membrane structures.
采用免疫印迹技术分析了高钙敏感性钙激活中性蛋白酶（μCANP）与红细胞膜的结合及其随后在膜上的自溶激活。在钙离子存在的情况下，μCANP作为79 kDa和28 kDa亚基的异二聚体与红细胞膜结合，并在膜上迅速转化为具有76 kDa大亚基的活性形式。然后，活性形式从膜释放到可溶性部分。然而，这些连续反应并非内翻囊泡所特有，除了一些不依赖Ca2+的结合外，在外翻囊泡上也会发生。在μCANP与膜结合后，观察到一些膜蛋白迅速降解。μCANP与红细胞膜的结合受到底物和内源性CANP抑制剂（也是一种自杀底物）的抑制。这些结果强烈表明，μCANP通过将膜蛋白识别为底物而与膜结合，而不是在一个特殊的激活位点结合。因此，μCANP在膜上激活的一种可能机制是，μCANP首先与膜上的底物结合，被激活，然后降解底物以使膜结构变形。
