Calpastatin in erythrocytes of young and old individuals

Abstract

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.

Calpain (Ca(2+)-dependent thiol protease) in erythrocytes of young and old individuals.

“…A slight increase in the amount of calpain was noted in membranes of NEMalkylated cells (Fig. 2, lane g) (17). Results obtained in old erythrocytes are shown in Fig.…”

“…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)(15)(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 old human individuals (>70 years old) as compared with that in erythrocyte membranes of young ones (20-30 years old) (17). The observed age-related enhanced sensitivity to calpain may be due to changes in band 3 protein, changes in other membrane components, and/or altered association with calpain.…”

“…Cytosol obtained from young individuals was used for the isolation and purification of calpain according to published procedures (2, 16), with units of calpain activity defined as described previously (16,17). Monoclonal antibodies specific to human erythrocyte W-calpain were generated according to established procedures (19).…”

“…Protein analysis was carried out by SDS/PAGE under reducing conditions, using 10%6 acrylamide in the gels. Gels were fixed and stained with Coomassie brilliant blue according to established methods (17). Other gels, run in parallel, were used for immunoblotting.…”

“…Calpain-induced membrane proteolysis may play a role in the shortening of erythrocyte life span in the aged (18). were prepared by established methods, as described previously (17).…”