THE CYTOSOL OF HUMAN-ERYTHROCYTES CONTAINS A HIGHLY CA-2+-SENSITIVE THIOL PROTEASE (CALPAIN I) AND ITS SPECIFIC INHIBITOR PROTEIN (CALPASTATIN)

The cytosol of human erythrocytes contained a Ca2+-dependent thiol protease (calpain) and its specific inhibitor (calpastatin) as demonstrated by DEAE-cellulose chromatography at pH 8.0, although no proteolytic activity toward casein was detected in the unfractionated hemolysate. The protease required only 40 .mu.M Ca2+ for 50% activation, indicating that it belongs to the highly Ca2+-sensitive type of calpain, namely, calpain I. It was not inactivated by heating at 58.degree. C for 10 min at pH 7.2, the optimal pH for its action on casein. The inhibitor comprised major and minor components, calpastatin H (MW = 280,000) and calpastatin L (MW = 48,000). Both were heat-stable proteins which were readily inactivated by tryptic digestion. The inhibition of erythrocyte calpain by erythrocyte calpastain H or L was not due to sequestering of Ca2+ from the reaction medium by the inhibitor protein. The calpain preparation preferentially digests bands III and IVa of human erythrocyte membrane proteins, with little or no cleavage of the bands corresponding to spectrin.