Expression of calpastatin isoforms in muscle and functionality of multiple calpastatin promoters |
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Authors: | Parr Tim Jewell Kirsty K Sensky Paul L Brameld John M Bardsley Ronald G Buttery Peter J |
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Affiliation: | Division of Nutritional Biochemistry, School of Biosciences, University of Nottingham, Sutton Bonington Campus, Loughborough, Leicestershire LE12 5RD, UK. tim.parr@nottingham.ac.uk |
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Abstract: | Calpastatin is the specific endogenous inhibitor of calpain proteinase that is encoded by a single gene. Transient transfection assays in both a non-fusing skeletal muscle and non-muscle cell-line demonstrated that the putative porcine calpastatin promoter regions 5' to exons 1xa, 1xb, and 1u were functional. Both real-time quantitative and semi-quantitative RT-PCR on porcine skeletal muscle total RNA indicated that steady-state expression of Type I and III mRNAs containing exons 1xa and 1u, respectively, was at equivalent levels whilst the expression of Type II mRNA containing exon 1xb was significantly less (p<0.001). Immunoprobing of Western blotted muscle extracts with an antibody raised against a peptide sequence encoded by exon 1xa indicated that Type I protein was expressed and that there was significantly more Type I protein in cardiac than skeletal muscle (p<0.001). The results suggest that the expression of the single calpastatin gene was differentially controlled at several levels. |
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Keywords: | Calpain Calpastatin Promoter analysis Skeletal muscle Cardiac muscle |
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