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 contin uous (25 to 400 mM NaCl) gradient and a two-step gradient method (200 mM Na Cl to coelute mu-calpain and calpastatin together and then 400 mM NaCl to e lute m-calpain) were compared. For the two-step method, mu-calpain activiti es were determined by subtracting calpastatin activities before and after h eat inactivation of mu-calpain. Both the two-step and the continuous gradie nt method yielded similar results over a broad range of activities. The ste pwise gradient method does not require the use of fraction collectors and p umps, and it can be completed in a fraction of the time required for the co ntinuous 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 cont inuous 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 calpai n proteolytic system. The present results indicate that the two-step gradie nt method is a fast and inexpensive method to determine calpain and calpast atin activities in studies designed to quantify the components of the calpa in proteolytic system in skeletal muscle.