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.

Purification, Characterization, and Analysis of the Allergenic Properties of Myosin Light Chain in Procambarus clarkii

“…Total RNA was prepared from crayfish (P. clarkii) muscle based on the TRIzol method. 29 The first-strand cDNA was synthesized with the TIANScript RT kit (Tiangen, Beijing, China), according to the manufacturer's instructions, and oligo(dT) 18 primers were designed according to the manufacturer's instructions. Based on the MALDI-TOF-MS results and the alignment of the MLC cDNA sequences of C. crangon and P. monodon, degenerate oligonucleotide primers were designed as the sense primers for PCR.…”

Effect of a Myofibril-Bound Serine Proteinase on the Deg of Giant Protein Titin and Nebulin

“…As MBSPs from different fish species showed similar enzymatic characteristics (Cao et al. 2000, 2005; Guo et al. 2007), in the present study, purified crucian carp MBSP was used instead to investigate its degradation effect on titin and nebulin from marine fish sea bream.…”

“…More recently, a trypsin‐type myofibril‐bound serine proteinase (MBSP) that effectively degrades myofibrillar proteins has been purified from the skeletal muscle of crucian carp ( Carassius auratus ), and its primary sequence was determined (Cao et al. 2006; Guo et al. 2007).…”

Identification of an aminopeptidase from the skeletal muscle of grass carp (Ctenopharyngodon idellus)

“…Lysosomal cathepsins participate in further proteolysis of postmortem muscle at a pH of 5.5-5.7 (Ladrat et al 2003). A recently identified myofibril-bound serine proteinase was responsible for the degradation of myofibrillar proteins such as myosin heavy chain, a-actinin and tropomyosin optimally at pH 8.0 (Cao et al 2006;Guo et al 2007). The physiological functions of aminopeptidases can be divided into three main categories: processing/maturation, activation and degradation.…”

“…Initial degradation of myofibrillar proteins into oligopeptides is performed by endopeptidases including cathepsin B and L (Ladrat et al 2003), calpains (Bartoli and Richard 2005) and myofibril-bound serine proteinases (Cao et al 2006;Guo et al 2007), resulting in the breakdown of myofibrillar proteins and causing muscle softening. Exopeptidases such as aminopeptidases and carboxypeptidases are responsible for further degradation of oligopeptides into free amino acids (Toldrá and Flores 1998).…”