title: Purification and characterization of calpain and calpastatin from rainbow trout, Oncorhynchus mykiss.
abstract: Although the calpain system has been studied extensively in mammalian animals, much less is known about the properties of mu-calpain, m-calpain, and calpastatin in lower vertebrates such as fish. These three proteins were isolated and partly characterized from rainbow trout, Oncorhynchus mykiss, muscle. Trout m-calpain contains an 80-kDa large subunit, but the approximately 26-kDa small subunit from trout m-calpain is smaller than the 28-kDa small subunit from mammalian calpains. Trout mu-calpain and calpastatin were only partly purified; identity of trout mu-calpain was confirmed by labeling with antibodies to bovine skeletal muscle mu-calpain, and identity of trout calpastatin was confirmed by specific inhibition of bovine skeletal muscle mu- and m-calpain. Trout mu-calpain requires 4.4+/-2.8 microM and trout m-calpain requires 585+/-51 microM Ca(2+) for half-maximal activity, similar to the Ca(2+) requirements of mu- and m-calpain from mammalian tissues. Sequencing tryptic peptides indicated that the amino acid sequence of trout calpastatin shares little homology with the amino acid sequences of mammalian calpastatins. Screening a rainbow trout cDNA library identified three cDNAs encoding for the large subunit of a putative m-calpain. The amino acid sequence predicted by trout m-calpain cDNA was 65% identical to the human 80-kDa m-calpain sequence. Gene duplication and polyploidy occur in fish, and the amino acid sequence of the trout m-calpain 80-kDa subunit identified in this study was 83% identical to the sequence of a trout m-calpain 80-kDa subunit described earlier. This is the first report of two isoforms of m-calpain in a single species.
authors.affiliation: Laboratory of Food Science and Technology, Kagawa Nutrition University, 3-9-21 Chiyoda, Saitama, Japan.
authors.forename: Masataka
authors.initials: M
authors.lastname: Saito
authors.forename: Hongqi
authors.initials: H
authors.lastname: Li
authors.forename: Valery F
authors.initials: VF
authors.lastname: Thompson
authors.forename: Naomichi
authors.initials: N
authors.lastname: Kunisaki
authors.forename: Darrel E
authors.initials: DE
authors.lastname: Goll
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002118:Calcium / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D002154:Calpain / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D002417:Cattle; D003001:Cloning, Molecular; D018076:DNA, Complementary; D008969:Molecular Sequence Data; D018482:Muscle, Skeletal / Q000737:chemistry; D017686:Oncorhynchus mykiss; D017386:Sequence Homology, Amino Acid
chemical_list: D002135:Calcium-Binding Proteins; D018076:DNA, Complementary; C033668:calpastatin; D002154:Calpain; C449000:m-calpain; C449002:mu-calpain; D002118:Calcium