An improved purification protocol for heart and skeletal muscle calpastatin reveals two 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 with apparent molecular weights of 125 and 145 kDa. 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 micro- 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.
Geesink
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Roman L. Hruska U.S. Meat Animal Research Center, ARS, USDA, Clay Center, Nebraska 68933-0166, USA.
Nonneman
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Koohmaraie
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D017398:Alternative Splicing; D000818:Animals; D015153:Blotting, Western; D002135:Calcium-Binding Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification*; D002417:Cattle; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D006358:Hot Temperature; D007536:Isomerism; D018482:Muscle, Skeletal / Q000737:chemistry* / Q000378:metabolism; D009206:Myocardium / Q000737:chemistry* / Q000378:metabolism; D016133:Polymerase Chain Reaction; D012194:RNA-Directed DNA Polymerase; D013552:Swine
D002135:Calcium-Binding Proteins; C033668:calpastatin; D012194:RNA-Directed DNA Polymerase