Expression of calpastatin isoforms in muscle and functionality of multiple calpastatin promoters.
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.
Parr
Tim
T
Division of Nutritional Biochemistry, School of Biosciences, University of Nottingham, Sutton Bonington Campus, Loughborough, Leicestershire LE12 5RD, UK. tim.parr@nottingham.ac.uk
Jewell
Kirsty K
KK
Sensky
Paul L
PL
Brameld
John M
JM
Bardsley
Ronald G
RG
Buttery
Peter J
PJ
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D002135:Calcium-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D017931:DNA Primers; D018482:Muscle, Skeletal / Q000201:enzymology*; D011401:Promoter Regions, Genetic; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013552:Swine; D014162:Transfection
D002135:Calcium-Binding Proteins; D017931:DNA Primers; D020033:Protein Isoforms; D012333:RNA, Messenger; C033668:calpastatin