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ARS Home » Plains Area » Clay Center, Nebraska » U.S. Meat Animal Research Center » Meat Safety and Quality » Research » Publications at this Location » Publication #99153
Title: TECHNICAL NOTE: A RAPID METHOD FOR QUANTIFICATION OF CALPAIN AND CALPASTATIN ACTIVITIES IN MUSCLE

Author
item Geesink, Gerrit
item Koohmaraie, Mohammad

Submitted to: Journal of Animal Science
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/20/1999
Publication Date: N/A
Citation: N/A

Interpretive Summary: The calpain proteolytic system plays a key role in the tenderization process that occurs during postmortem storage of meat under refrigerated conditions. Therefore, quantification of the components of this system, i.e., calpastatin, u-calpain, and m-calpain is frequently an essential element of studies aimed at explaining differences in tenderization of meat from different breeds or muscles. Separation of all three components of the calpain system can be achieved in a single step using ion-exchange chromatography. However, complete separation of calpastatin and u-calpain can only be obtained by elution with a very shallow gradient, requiring long elution times. As a result, the number of samples that can be processed in a given time is dependent on the amount of chromatography equipment available. In the present paper, we present a method which takes advantage of the fact that calpastatin and u-calpain can be separated from m-calpain using stepwise chromatography, eliminating the need for expensive chromatography equipment, and that calpastatin is resistant to denaturation of heat. The present results indicate that u-calpain activity in eluates containing both u-calpain and calpastatin can be reliably estimated from calpastatin measurements before and after heat treatment of the eluates. Stepwise ion-exchange chromatography, therefore, presents a relatively fast and inexpensive method to determine calpain and calpastatin activities in postmortem muscle.

Technical Abstract: Stepwise and continuous gradient ion-exchange chromatography were compared for yield of calpains and calpastatin from ovine muscle extracts. In the first experiment, continuous gradient (25-400 mM NaCl) chromatography and a two-step gradient method (200 mM to co-elute u-calpain and calpastatin together, and then 400 mM NaCl to elute m-calpain) were compared. For the two-step method, u-calpain activities were determined by subtracting calpastatin activities before and after heat inactivation of u-calpain. Both the two-step and the continuous gradient method yielded similar results over a broad range of activities. In the second experiment, the two-step method was compared with a three-step separation method (100 mM NaCl to elute calpastatin, 200 mM NaCl to elute u-calpain, and 400 mM to elute m-calpain). Unlike the continuous gradient method, calpastatin and u-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 two-step gradient ion-exchange is a relatively 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.