Technical note: a rapid method for quantification of calpain and calpastatin activities in muscle.
Stepwise and continuous gradient ion-exchange chromatography were compared for yield of calpains and calpastatin from ovine muscle in a study designed to quantify their activities for comparative purposes. In Exp. 1, a continuous (25 to 400 mM NaCl) gradient and a two-step gradient method (200 mM NaCl to coelute mu-calpain and calpastatin together and then 400 mM NaCl to elute mu-calpain) were compared. For the two-step method, mu-calpain activities were determined by subtracting calpastatin activities before and after heat inactivation of mu-calpain. Both the two-step and the continuous gradient method yielded similar results over a broad range of activities. The stepwise gradient method does not require the use of fraction collectors and pumps, and it can be completed in a fraction of the time required for the continuous gradient method. In Exp. 2, the two-step method was compared with a three-step method (100 mM NaCl to elute calpastatin, then 200 mM NaCl to elute mu-calpain, and then 400 mM NaCl to elute m-calpain). Unlike the continuous gradient method, calpastatin and mu-calpain could not be completely separated using the three-step chromatography method. Thus, the three-step gradient method should not be used to quantify the components of the calpain proteolytic system. The present results indicate that the two-step gradient method is a fast and inexpensive method to determine calpain and calpastatin activities in studies designed to quantify the components of the calpain proteolytic system in skeletal muscle.
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Roman L. Hruska US Meat Animal Research Center, ARS, USDA, Clay Center, NE 68933-0166, USA.
Koohmaraie
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D000818:Animals; D002135:Calcium-Binding Proteins / Q000032:analysis*; D002154:Calpain / Q000032:analysis*; D002852:Chromatography, Ion Exchange / Q000379:methods; D007527:Isoenzymes / Q000032:analysis; D009132:Muscles / Q000737:chemistry*; D012756:Sheep / Q000378:metabolism*
D002135:Calcium-Binding Proteins; D007527:Isoenzymes; C033668:calpastatin; D002154:Calpain