An improved purification procedure for calpastatin, the inhibitor protein specific for the intracellular calcium-dependent proteinases, calpains.
The specific inhibitor protein (calpastatin) for the calcium-dependent intracellular proteinases (calpains) is an important regulator of these enzymes. In this communication we describe a one day procedure for purifying 3 to 5 mg of calpastatin from a kilogram of bovine myocardium. This represents a substantial improvement over previously described methods, and should facilitate future studies of calpastatin structure and function. A key, novel step in the purification was dye-matrix chromatography on an Affi-Gel Blue column. Contrary to previous indications, calpastatin purified by the new method did not contain significant amounts of carbohydrate. However, the presence of covalently bound phosphate in purified bovine myocardial calpastatin was confirmed and co-migration of phosphate and calpastatin activity was demonstrated on Bio-Gel A-1.5m chromatography. Thus, it is possible that calpastatin function is regulated by phosphorylation.
Mellgren
R L
RL
Department of Pharmacology and Therapeutics, Medical College of Ohio, Toledo 43699.
Nettey
M S
MS
Mericle
M T
MT
Renno
W
W
Lane
R D
RD
D000818:Animals; D002135:Calcium-Binding Proteins / Q000302:isolation & purification*; D002154:Calpain / Q000037:antagonists & inhibitors*; D002241:Carbohydrates / Q000032:analysis; D002417:Cattle; D004591:Electrophoresis, Polyacrylamide Gel; D006023:Glycoproteins / Q000302:isolation & purification*; D007120:Immunochemistry; D009206:Myocardium / Q000032:analysis; D010710:Phosphates / Q000032:analysis
D002135:Calcium-Binding Proteins; D002241:Carbohydrates; D006023:Glycoproteins; D010710:Phosphates; C039304:calpain inhibitors; C033668:calpastatin; D002154:Calpain