Purification and characterization of calpain and calpastatin from rainbow trout, Oncorhynchus mykiss.
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.
Saito
Masataka
M
Laboratory of Food Science and Technology, Kagawa Nutrition University, 3-9-21 Chiyoda, Saitama, Japan.
Li
Hongqi
H
Thompson
Valery F
VF
Kunisaki
Naomichi
N
Goll
Darrel E
DE
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
D002135:Calcium-Binding Proteins; D018076:DNA, Complementary; C033668:calpastatin; D002154:Calpain; C449000:m-calpain; C449002:mu-calpain; D002118:Calcium