Limited proteolysis by calpain (Ca2+-activated protease; EC 3.4.22.17) is believed to regulate the function of membrane enzymes and modify the behavior of membrane structural proteins. Calpain is activated by autolysis. The degradation of band 3 protein by #-calpain is known to be enhanced in erythrocyte membranes from human individuals >70 years old (old) as compared with that from individuals 20-30 years old (young). In the present study, monoclonal antibody to IA-calpain was used to study the behavior of calpain in erythrocytes of young and old individuals. Less calpain was found in erythrocyte cytosol and membranes from old than in those from young. Increasing the erythrocyte Ca2+ induced translocation of calpain to the cell membrane and autolysis of the enzyme. Alkylation of erythrocyte thiols also promoted translocation of calpain to the membrane, especially in the presence of Ca2+. When calpain was added to erythrocyte membranes, initial binding was greater and subsequent autolysis faster in old than in young individuals, possibly arising from alterations in cell membranes of old individuals. The enhanced calpain autolysis was accompanied by enhanced degradation of band 3 protein in the old. The results suggest that calpain in old individuals is translocated to the cell membrane and is activated by autolysis, resulting in degradation of certain membrane proteins and loss of calpain. Enhanced calpain-induced membrane proteolysis may play a role in abnormal cell destruction (e.g., shortening the iffe span oferythrocytes in the aged, neuronal degeneration, etc). The erythrocyte membrane provides a convenient model for the study of age-associated alterations in cell membranes and in calpain behavior.Calpain is a neutral Ca2+-dependent thiol protease (EC 3.4.22.17), present in a wide variety of vertebrate cells. Two major isozymes are known, g-calpain and m-calpain, requiring juM and mM Ca2+ for activation, respectively (1-5). The isozyme types and amounts of the protease vary among cells and among species. Calpain is found mainly in the cell cytosol and is usually present in an inactive form. It has been suggested that, in the presence of Ca2+, calpain is activated by limited autolysis, a process that is enhanced by the translocation of calpain to the cell membrane (4-10). Activated calpain causes a limited degradation of certain membrane and cytoskeletal proteins and enzymes. The calpaininduced limited proteolysis may regulate enzyme activities and modulate the behavior of membrane structural proteins (3)(4)(5)(11)(12)(13).The mammalian erythrocyte contains the 1L-calpain isozyme (2). We have shown previously that calpain plays a role in membrane fusion: specifically, that degradation of certain membrane proteins by calpain is a prerequisite for membrane fusion induced by the membrane mobility agent A2C (14-16). In addition to calpain-induced changes associated with membrane fusion, we have also discovered that the degradation of band 3 protein by p.-calpain is enhanced in erythrocyte membranes of o...

To gain knowledge about the behaviour of calpastatin (the specific inhibitor of the Ca(2+)-dependent thiol protease calpain) in the intact cell, we analysed the inhibitor by specific antibodies and determined its activity in erythrocytes from individuals 20-34 years old (young) and 70-93 years old (old). Differences between old and young in the behaviour of erythrocyte calpastatin were observed. Erythrocytes of old individuals had lower amounts of calpastatin and less calpastatin activity than those of young ones. A difference between old and young was also found in the molecular-mass distribution of calpastatin subunits. Increasing the erythrocyte Ca2+ induced changes in calpastatin in young individuals, rendering it similar to calpastatin in cells of old individuals. When calpastatin (isolated from erythrocytes of a young individual) was added to erythrocyte membranes, the initial binding and subsequent association of calpastatin with the membrane were lower in old than in young individuals. We had previously found that calpain binding and activation were enhanced in erythrocyte membranes from old individuals, along with enhanced degradation of band 3 (a major erythrocyte transmembrane anion-transport protein). The overall results indicate an interaction of calpain with calpastatin in the intact cell. Enhanced activation of erythrocyte calpain and degradation of calpastatin occur under conditions of increased cellular Ca2+ and in cells of the aged.