Calcium-activated neutral proteases (calpains) are carbohydrate binding proteins.

Calcium-activated neutral proteases (calpain, EC 3.4.22.17) bind to agarose matrices (Bio-Gel A-150m, Sepharose 4B, and Ultrogel AcA 34) with high affinity in the presence of calcium. 6-O-beta-Galactopyranosyl-D-galactose, a disaccharide which closely resembles the repeating unit of the agarose matrices, completely blocks the binding of calpains and can release agarose-bound enzymes in the presence of calcium. At least 1 microM level of free calcium is required for binding. Other calcium binding proteins, including calmodulin, calpastatin, casein, and neurofilament proteins, fail to bind under the same conditions. Both calpain I and calpain II can be readily purified from crude enzyme preparations by agarose chromatography in the presence of calcium and leupeptin. Agarose-bound enzymes are eluted with calcium-free solutions or can be released in the presence of calcium by 1% Triton X-100, but not by 1 M urea or 20% ethylene glycol. Enzymes eluted from agarose are activated, as evidenced by the appearance of faster migrating forms (76 and 78 kDa) of the 80-kDa catalytic subunit of calpain I upon electrophoresis and by the increased sensitivity of calpain II to activation by micromolar levels of calcium. The electrophoretic migration of the 30-kDa regulatory subunit is, however, unaltered in enzyme fractions eluted from an agarose column. When the enzyme subunits are dissociated in 1 M NaSCN, only the 30-kDa subunit binds to the agarose matrix. Furthermore, neither calpain I nor calpain II binds to agarose when their 30-kDa subunit is autocatalyzed to an 18-kDa fragment, indicating that the NH2-terminal of the 30-kDa subunit is important for the binding of calpains to an agarose matrix.