Submitted to: Archives of Biochemistry and Biophysics
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/13/1998
Publication Date: N/A
Interpretive Summary: Meat tenderness is an important component of meat quality, and inconsistency in meat tenderness has been identified as one of the major problems facing the meat industry. Degradation of structural muscle proteins by calcium dependent neutral proteases (calpains) is responsible for meat tenderization during postmortem storage of meat. The expression of calpain activity is in part regulated by the presence of an inhibitor of the calpains, calpastatin. Calpastatin activity at 24 h postmortem is inversely proportional to postmortem tenderization and accounts for a greater proportion of the variation in beef tenderness (approximately 40%) than any other single variable. To study inhibition of calpain by calpastatin, these proteins need to be purified from tissues. The aim of the present study was to develop an efficient method to purify calpastatin. The method used yielded large quantities of essentially pure calpastatin from muscle and heart. Heart muscle is a rich source of calpastatin, and during purification of calpastatin from this tissue it was noted that two forms of calpastatin were present in heart, but only one in muscle. These results were confirmed by Western blotting of tissue extracts and direct sequencing of calpastatin cDNA.
Technical Abstract: Employment of a new protocol for efficient purification of calpastatin revealed the presence of two forms of calpastatin in porcine heart with apparent molecular weights of 125 and 145 kDa, respectively. Purification from bovine muscle resulted in a single species with an apparent molecular weight of 125 kDa. The presence of multiple species of calpastatin in porcine heart does not appear to be an artifact of the purification procedure since Western blotting revealed the presence of two types of calpastatin in ovine and porcine heart, but only one type in bovine heart, and bovine, ovine and porcine muscle. The origin of the two species in porcine heart was examined by RT-PCR and direct sequencing of calpastatin cDNA. This analysis revealed that porcine skeletal muscle exclusively produces transcripts lacking exon 3, while porcine heart produces transcripts that include or lack exon 3, consistent with the presence of two isoforms of the protein. The 125 kDa form of porcine calpastatin, therefore, appears to be a result of alternative splicing of the calpastatin transcript. The biological significance of the heart specific isoform is not clear, however, its ability to inhibit u-, or m-calpain does not differ considerably. The present purification protocol yielded 4.9 and 1.8 mg calpastatin per kg tissue from porcine heart and bovine skeletal muscle, respectively.