Bovine skeletal muscle calpastatin: cloning, sequence analysis, and steady-state mRNA expression.
Calpastatin is a specific inhibitor of the calpains. Calpains play a key role in postmortem tenderization of meat and have been hypothesized to be involved in muscle protein degradation in living tissue. Isolation, cloning of complementary DNA, and nucleotide sequencing of bovine calpastatin from the longissimus muscle have been completed. Two clones were identified that encompass the entire coding sequence. Clone pCR41, derived by reverse transcription-PCR, covers domains L and 1; clone pBSA1, obtained from cDNA library screening, covers domains 2 through 4 in addition to the 3'-nontranslated region. Nucleotide sequence analysis of the cDNA for bovine calpastatin revealed an average nucleotide sequence identity of approximately 70 to 80% compared with published 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 calpastatin as determined by SDS-PAGE (M(r) 68,000). Northern blot analysis revealed the presence of multiple calpastatin mRNA transcripts having estimated sizes of 3.8, 3.0, and 1.5 kb in beef and 3.8, 3.0, 2.5, and 1.5 kb in sheep. Calpastatin mRNA expression was increased with beta-adrenergic agonist-induced muscle hypertrophy.
Killefer
J
J
Roman L. Hruska U.S. Meat Animal Research Center, ARS, USDA, Clay Center, NE 68933.
Koohmaraie
M
M
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D002135:Calcium-Binding Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D002154:Calpain / Q000037:antagonists & inhibitors*; D002417:Cattle / Q000378:metabolism*; D003001:Cloning, Molecular; D016384:Consensus Sequence; D004247:DNA / Q000737:chemistry; D017931:DNA Primers / Q000737:chemistry; D005786:Gene Expression Regulation; D015723:Gene Library; D008297:Male; D008969:Molecular Sequence Data; D009132:Muscles / Q000737:chemistry* / Q000378:metabolism; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000096:biosynthesis*; D017422:Sequence Analysis, DNA
D002135:Calcium-Binding Proteins; D017931:DNA Primers; D012333:RNA, Messenger; C033668:calpastatin; D004247:DNA; D002154:Calpain