Identification of a myofibril-bound serine proteinase (MBSP) in the skeletal muscle of lizard fish Saurida wanieso which specifically cleaves the arginine site

Abstract

A myofibril-bound serine proteinase (MBSP) from the skeletal muscle of lizard fish ( Saurida wanieso) was purified to homogeneity by a heating treatment followed by a series of column chromatographies on DEAE-Sephacel, Sephacryl S-200, Q-Sepharose, Hydroxyapatite and Benzamidine-Sepharose 6B, and characterized enzymatically. On SDS-polyacrylamide gel electrophoresis (SDS-PAGE), the purified enzyme showed a band with molecular mass of ≈29 kDa under reducing conditions, while 60 kDa under non-reducing conditions. The optimum temperature of the enzyme was 50°C using t-butyloxycarbonyl-Phe-Ser-Arg-4-methylcoumaryl-7-amide (Boc-Phe-Ser-Arg-MCA) as a substrate. Substrate specificity analysis both using MCA-substrates and peptides showed that MBSP specifically cleaved at the carboxyl side of the arginine residue. Inhibitor susceptibility analysis revealed that MBSP was inhibited effectively by Pefabloc SC, soybean trypsin inhibitor (STI) and aprotinin, indicating the characteristic of a serine proteinase. When myofibril was incubated with the enzyme, it optically degraded myosin heavy chain at 55–60°C, while α-actinin and actin were not at all hydrolyzed as detected by immunoblotting. The N-terminal amino acid sequence of MBSP was partially determined as IVGGAEXVPY- and was very homologous to other serine proteases.