Characterization of Calpain I-Binding Proteins in Human Erythrocyte Plasma Membrane

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The calpain-binding components on the plasma membrane were characterized using calpain I. ¹²⁵I-labeled calpain was bound to inside-out membrane vesicles from human erythrocyte in a Ca²⁺-dependent manner, but not to right-side-out membrane vesicles. The maximum binding was observed at more than 5μM Ca²⁺. The binding amount of calpain to the inside-out membrane vesicles was decreased when the vesicles were pretreated with 100μg/ml of trypsin, chymotrypsin, elastase, or pronase P for 30 min at 37°C, although the binding to the vesicles pretreated with 200μg/m1 of phospholipase A₂ or C was not affected. Calpain-binding proteins in the membrane were analyzed by using a modified immunoblot-ting for calpain. Immunostained bands of 240, 220, 89, 72, 52, and 36 kDa were detected as the calpain-binding proteins in the native membrane. All of these bands had disappeared in trypsin-treated membrane. The disappearance of bands was dose-dependent with respect to trypsin and paralleled the reduction of binding amount of calpain to the trypsinized membrane. In calpain-treated membrane, the 240 and 36 kDa bands were retained in the blotting, though the other bands disappeared dose-dependently with respect to calpain. These results suggested that the significant component in the inner surface of plasma membrane for binding of calpain was proteinaceous and the calpain-binding proteins could be classified into two species, i. e. substrates of calpain (220, 89, 72, and 52 kDa protein) and non-substrates (240 and 36 kDa protein).