Effect of Ca2+ on binding of the calpains to calpastatin.
Autolyzed mu-calpain, unautolyzed mu-calpain, autolyzed m-calpain, and unautolyzed m-calpain (mu-calpain is the micromolar Ca2+-requiring proteinase, m-calpain is the millimolar Ca2+-requiring proteinase) were passed through a calpastatin-affinity column at different free Ca2+ concentrations, and binding of the calpains to calpastatin was compared with proteolytic activity of that calpain at each Ca2+ concentration. Unautolyzed m-calpain, autolyzed m-calpain, and autolyzed mu-calpain required less Ca2+ for half-maximal binding to calpastatin than for half-maximal activity. Unautolyzed mu-calpain, however, required slightly more Ca2+ for half-maximal binding to calpastatin than for half-maximal activity. Half-maximal binding of oxidatively inactivated mu- or m-calpain to calpastatin required approximately the same Ca2+ concentrations as half-maximal binding of unautolyzed mu- or m-calpain, respectively, to calpastatin. Binding of unautolyzed m-calpain and autolyzed mu-calpain to calpastatin occurred over a wide range of Ca2+ concentrations, and it seems likely that two or more Ca2+-binding sites with different Ca2+-binding constants are involved in binding of the calpains to calpastatin. Proteolytic activity occurs at different Ca2+ concentrations than calpastatin binding, suggesting a second set of Ca2+-binding sites associated with proteolytic activity. Third and fourth sets of Ca2+-binding sites may be involved in autolysis and in binding to phosphatidylinositol or cell membranes; these four Ca2+-dependent properties of the calpains may require the eight potential Ca2+-binding sites that amino acid sequences predict are present in the calpain molecules.
Kapprell
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HP
Department of Animal Sciences, University of Arizona, Tucson 85721.
Goll
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DE
D000818:Animals; D002118:Calcium / Q000494:pharmacology*; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D002154:Calpain / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D002417:Cattle; D002846:Chromatography, Affinity; D004492:Edetic Acid / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D008970:Molecular Weight; D009132:Muscles / Q000201:enzymology; D011480:Protease Inhibitors / Q000378:metabolism*; D011485:Protein Binding
D002135:Calcium-Binding Proteins; D011480:Protease Inhibitors; C033668:calpastatin; D004492:Edetic Acid; D002154:Calpain; D002118:Calcium