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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 #175485
Title: AN ASSESSMENT OF EXTRACTION AND ASSAY TECHNIQUES FOR QUANTIFICATION OF CALPAIN AND CALPASTATIN FROM SMALL TISSUE SAMPLES

Author
item KENT, MATTHEW - LINGVITAE GENOMICS
item VEISETH, EVA - AGRIC UNIV OF NORWAY
item THERKILDSEN, MARGRETHE - DANISH INST AGRIC SCI
item Koohmaraie, Mohammad

Submitted to: Journal of Animal Science
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/17/2005
Publication Date: 9/20/2005
Citation: Kent, M.P., Veiseth, E., Therkildsen, M., Koohmaraie, M. 2005. An assessment of extraction and assay techniques for quantification of calpain and calpastatin from small tissue samples. Journal of Animal Science. 83:2182-2188.

Interpretive Summary: It has been hypothesized that the calpain proteolytic system plays an important role in muscle growth. It is now clear that the calpain proteolytic system is the enzyme system that is responsible for degradation of proteins and that their degradation causes meat tenderization during postmortem aging (storage of meat cuts at refrigerated temperatures). For these reasons there is a great interest in appropriate methodology for quantification of the activities of the components of the calpain system in muscle. The traditional and standard method requires a rather large sample size (25 g of muscle or more). Over the years there has been a great interest in miniaturizing the assay (using a small or very small amount of muscle). We conducted a series of experiments in an attempt to determine if miniaturization is possible. Results indicated that there were no acceptable miniaturization methods and that accurate results can only be obtained when a large amount of muscle is used.

Technical Abstract: Our objective was to evaluate whether biopsies could be used to measure calpain and calpastatin activities in skeletal muscle. The accuracy of different separation and assay methods for the quantification of calpains and calpastatin from small (1.0-g and 0.2-g) skeletal muscle samples was tested. In Exp. 1, longissimus was removed from 6 lambs and a 50-g sub-sample was processed using the Standard Method (DEAE-Sephacel chromatography and casein assay). Sub-samples (1.0 g and 0.2 g) were also processed using the two-step separation (1 mL DEAE-Sephacel and bulk elution using 200 mM and 400 mM NaCl) and heated calpastatin methods; in both cases fractions were assayed with Bodipy-labeled and [14C]-labeled casein microassays. Finally, casein zymography was used to separate and quantify the calpain proteases from 1.0-g and 0.2-g samples. The values obtained after processing a 50 g sample using the Standard Method were judged most accurate and the alternative approaches were contrasted against these. For each extraction and assay approach we considered (1) the effect of the sample size upon the mean activity, (2) increased or decreased variation of data, and (3) the correlation relative to the Standard Method. Where possible we compared the ratio of calpain to calpastatin activities determined using the alternative approaches with the ratios found using the Standard Method. These methodologies were further investigated in Exp. 2 where single homogenates from different tissues (heart, spleen, lung, muscle) were assayed using the alternative approaches. Experiment 1 established that none of the approaches seemed reliable relative to the Standard Method due to poor correlations (0.23 - 0.91) and unacceptable variation. By using a large, homogenous sample in Exp. 2 however, we determined that this error was not due to the methodologies themselves. Therefore, the unacceptable variation found in Exp. 1 resulted from the small sample size, and we recommend that large tissue samples (e.g., 50 g) should be used for calpain and calpastatin activity measurements in skeletal muscle instead of small tissue biopsies (e.g., 0.2 and 1.0 g).