Limited proteolysis of bovine lens α-crystallin by calpain, A Ca 2+-dependent cysteine proteinase, isolated from the same tissue

Abstract

A Ca 2+-dependent cysteine proteinase (calpain, EC 3.4.22.17) was found in the cytosolic fraction of bovine lens and purified to apparent homogeneity. The purified enzyme required 1 mM Ca 2+ for its full activation and was composed of two subunits of M r 80 000 and 29 000 as demonstrated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS). This enzyme, when activated by Ca 2+, degraded both A- and B-chains of α-crystallin, which were isolated also from bovine lens. SDS-gel electrophoresis of the digest revealed that the A-chain ( M r 19 000) was broken down to produce an 18-kDa polypeptide fragment and the B-chain ( M r 22 500) to produce a 19.50kDa polypeptide fragment. No further cleavage occurred even upon prolonged incubation or after the second addition of the enzyme, indicating the uniquely limited of each chain protein. The existence of calpastatin, an endogenous inhibitor protein specific for calpain, was also demonstrated in bovine lens cytosol.