title: Calcium-activated neutral proteases (calpains) are carbohydrate binding proteins.
abstract: 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.
authors.affiliation: Department of Pathology and Laboratory Medicine, University of Pennsylvania Medical School, Philadelphia 19104.
authors.forename: U J
authors.initials: UJ
authors.lastname: Zimmerman
authors.forename: W W
authors.initials: WW
authors.lastname: Schlaepfer
mesh_terms: D002118:Calcium / Q000494:pharmacology*; D002135:Calcium-Binding Proteins / Q000378:metabolism / Q000494:pharmacology; D002154:Calpain / Q000378:metabolism*; D050260:Carbohydrate Metabolism; D002845:Chromatography; D004187:Disaccharides / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D019855:Ethylene Glycol; D005026:Ethylene Glycols; D007976:Leupeptins / Q000494:pharmacology; D017830:Octoxynol; D011092:Polyethylene Glycols; D012685:Sepharose / Q000378:metabolism; D013861:Thiocyanates / Q000494:pharmacology; D014508:Urea
chemical_list: D002135:Calcium-Binding Proteins; D004187:Disaccharides; D005026:Ethylene Glycols; D007976:Leupeptins; D013861:Thiocyanates; D011092:Polyethylene Glycols; C056853:6-O-galactopyranosylgalactose; C024553:sodium thiocyanate; C033668:calpastatin; D014508:Urea; D017830:Octoxynol; D012685:Sepharose; D002154:Calpain; D019855:Ethylene Glycol; C032854:leupeptin; D002118:Calcium