AN IMPROVED PURIFICATION PROTOCOL FOR HEART AND SKELETAL-MUSCLE CALPASTATIN REVEALS 2 ISOFORMS RESULTING FROM ALTERNATIVE SPLICING

Employment of a new protocol for efficient purification of calpastatin revealed the presence of two forms of calpastatin in porcine heart wi th apparent molecular weights of 125 and 145 kDa. Purification from bo vine muscle resulted in a single species with an apparent molecular we ight of 125 kDa. The presence of multiple species of calpastatin in po rcine heart does not appear to be an artifact of the purification proc edure since Western blotting revealed the presence of two types of cal pastatin 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 calpa statin cDNA. This analysis revealed that porcine skeletal muscle exclu sively produces transcripts lacking exon 3, while porcine heart produc es transcripts that include or lack exon 3, consistent with the presen ce of two isoforms of the protein. The 125-kDa form of porcine calpast atin therefore appears to be a result of alternative splicing of the c alpastatin transcript. The biological significance of the heart specif ic isoform is not clear; however, its ability to inhibit mu- or m-calp ain does not differ considerably. The present purification protocol yi elded 4.9 and 1.8 mg calpastatin per kg tissue from porcine heart and bovine skeletal muscle, respectively. (C) 1998 Academic Press.