BINDING-SITES FOR CALCIUM-ACTIVATED NEUTRAL PROTEASE ON ERYTHROCYTE-MEMBRANES ARE NOT MEMBRANE PHOSPHOLIPIDS

In order to explore the binding sites for calcium-activated neutral protease (CANP) with high calcium sensitivity (μCANP) on the inner surface of human erythrocyte membranes, we analyzed the binding of μCANP to two kinds of membranes modified by treatment with phospholipase C or Triton X-100. Binding analyses were performed using an immunoblot technique. The amount of μCANP bound to phospholipase C-treated inside-out vesicles was essentially the same as that bound to untreated inside-out vesicles. It was also observed that μCANP binds to Triton X-100-treated membranes, in which most of the integral proteins and glycerophospholipids are removed while the lining proteins remain intact. In both types of modified membrane, the bound μCANP was rapidly converted to an active form by autolysis at physiological free Ca2+ concentrations. These results indicate that the binding sites for μCANP on the inner surface of erythrocyte membranes consist of components other than membrane phospholipids. In addition, it is suggested that one of the binding sites for μCANP is some lining protein. © 1990.