Procalpain is activated on the plasma membrane and the calpain acts on the membrane.
The mechanism of activation of human erythrocyte calpain was investigated using the immunoblotting technique with anticalpain monoclonal antibody. The purified calpain underwent a Ca2+-induced fragmentation of the 80 kDa subunit to 76 kDa and 36 kDa fragments. The behavior of the 76 kDa fragment in electrophoresis corresponded to the proteinase activity of calpain, whereas the behavior of the 80 kDa subunit and the 36 kDa fragment did not. When inside-out membrane vesicles were added to the reaction mixture of calpain and Ca2+ and the vesicles were separated from the supernatant solution by centrifugation, the 80 kDa subunit and 76 kDa fragment were found in the vesicle fraction. No other fragments were found in this fraction. On the other hand, the 80 kDa subunit and 36 kDa fragment were found in the supernatant fraction. When right-side-out membrane vesicles were added to the reaction mixture and the vesicles were separated from the supernatant fraction, no fragment was found in the vesicle fraction, while only the 36 kDa fragment was found in the supernatant fraction. These results indicate that the 80 kDa subunit of procalpain was bound in a Ca2+-dependent manner to the cytosolic surface of the plasma membrane and then underwent fragmentation to produce the 76 kDa fragment (active form) and that it expressed its proteinase activity at the surface of the membrane.
D000911:Antibodies, Monoclonal; D002118:Calcium / Q000097:blood; D002154:Calpain / Q000097:blood* / Q000276:immunology; D003600:Cytosol / Q000201:enzymology; D004789:Enzyme Activation; D004792:Enzyme Precursors / Q000097:blood* / Q000276:immunology; D004910:Erythrocyte Membrane / Q000201:enzymology*; D006801:Humans; D007163:Immunosorbent Techniques; D008970:Molecular Weight
D000911:Antibodies, Monoclonal; D004792:Enzyme Precursors; D002154:Calpain; C047018:procalpain; D002118:Calcium