BOVINE SKELETAL-MUSCLE CALPASTATIN - CLONING, SEQUENCE-ANALYSIS, AND STEADY-STATE MESSENGER-RNA EXPRESSION

Calpastatin is a specific inhibitor of the calpains. Calpains play a k ey role in postmortem tenderization of meat and have been hypothesized to be involved in muscle protein degradation in living tissue. Isolat ion, cloning of complementary DNA, and nucleotide sequencing of bovine calpastatin from the longissimus muscle have been completed. Two clon es were identified that encompass the entire coding sequence. Clone pC R41, derived by reverse transcription-PCR, covers domains L and 1; clo ne pBSA1, obtained from cDNA library screening, covers domains 2 throu gh 4 in addition to the 3'-nontranslated region. Nucleotide sequence a nalysis of the cDNA for bovine calpastatin revealed an average nucleot ide sequence identity of approximately 70 to 80% compared with publish ed calpastatin nucleotide sequences of human, rabbit, and pig. Exon 3, corresponding to a highly conserved 22-amino acid region, was deleted from bovine calpastatin domain L. The calculated molecular weight of bovine skeletal muscle calpastatin of 706 amino acid residues (M(r) 75 ,842) corresponds-to the value of purified bovine skeletal muscle calp astatin as determined by SDS-PAGE (M(r) 68,000). Northern blot analysi s revealed the presence of multiple calpastatin mRNA transcripts havin g estimated sizes of 3.8, 3.0, and 1.5 kb in beef and 3.8, 3.0, 2.5, a nd 1.5 kb in sheep. Calpastatin mRNA expression was increased with bet a-adrenergic agonist-induced muscle hypertrophy.