Isolation and characterization of a 25K serine proteinase from bovine lens cortex.

A lens serine proteinase with trypsin-like specificity has been purified to homogeneity. This is one of two serine proteinases associated with the alpha-crystallin fraction from bovine lens. The purification was accomplished by a combination of isoelectric precipitation, activation to release the proteinase, gel-filtration and affinity chromatography. The purified proteinase showed a single protein band of 25 000 daltons on SDS-PAGE. A single protein band was also seen on non-denaturing gels which correlated with the location of the proteinase activity. The proteinase had a pH optimum between 7.2 and 8.2, was stable between pH 5.8 and 8.6 but was unstable above 40 degrees C upon heating. The enzyme lacked any requirement for metal ions and hydrolyzed arginine, lysine and asparagine substrates. alpha-Crystallin, and especially the B-chain of alpha-crystalline, was rapidly hydrolyzed by the proteinase compared to other lens crystallins. Metallo- and cysteine-proteinase inhibitors had no effect upon the enzyme activity whereas three different serine-proteinase inhibitors completely abolished all activity. A number of protein and peptide trypsin inhibitors also completely inhibited the lens 25K serine proteinase.