title: Purification, characterization, and cDNA cloning of a myofibril-bound serine proteinase from the skeletal muscle of crucian carp (Carassius auratus).
abstract: A myofibril-bound serine proteinase (MBSP) was highly purified from the skeletal muscle of crucian carp (Carasius auratus) by acidic treatment of myofibril solution and chromatographies on Q-Sepharose and benzamidine-Sepharose 6B. MBSP revealed a main protein band of approximately 28 kDa on SDS-polyacrylamide gel electrophoresis (PAGE) and was particularly inhibited by serine proteinase inhibitors. Substrate-specificity analysis revealed that the enzyme specifically cleaved at the carboxyl side of arginine and lysine residues, suggesting the characteristics of a trypsin-type serine proteinase. MBSP gene was cloned on the basis of the N-terminal sequence and the conserved active site peptide of serine proteinases together with 5'-rapid amplification of cDNA ends (5'-RACE) and 3'-RACE. The coding region gave an amino acid sequence of 242 residues including the initiation methionine and a signal peptide of 20 residues. Amino acid residues of His60, Asp106, and Ser196 consisting of the catalytic triad of serine proteinases were conserved in the sequence. Crucian carp MBSP shared relatively high identities with other serine proteinases, especially in well-conserved regions.
authors.affiliation: College of Biological Engineering, The Key Laboratory of Science and Technology for Aquaculture and Food Safety, Jimei University, 361021 Xiamen, China.
authors.forename: Chuan
authors.initials: C
authors.lastname: Guo
authors.forename: Min-Jie
authors.initials: MJ
authors.lastname: Cao
authors.forename: Guang-Ming
authors.initials: GM
authors.lastname: Liu
authors.forename: Xiong-Shui
authors.initials: XS
authors.lastname: Lin
authors.forename: Kenji
authors.initials: K
authors.lastname: Hara
authors.forename: Wen-Jin
authors.initials: WJ
authors.lastname: Su
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics*; D006054:Goldfish; D008969:Molecular Sequence Data; D018482:Muscle, Skeletal / Q000201:enzymology*; D009210:Myofibrils / Q000201:enzymology*; D016415:Sequence Alignment; D012697:Serine Endopeptidases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism
chemical_list: D018076:DNA, Complementary; D012697:Serine Endopeptidases