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

A myofibril-bound serine proteinase (MBSP) from the skeletal muscle of liza rd fish (Saurida wanieso) was purified to homogeneity by a heating treatmen t followed by a series of column chromatographies on DEAE-Sephacel, Sephacr yl S-200, Q-Sepharose, Hydroxyapatite and Benzamidine-Sepharose 6B, and cha racterized enzymatically. On SDS-polyacrylamide gel electrophoresis (SDS-PA GE); the purified enzyme showed a band with molecular mass of approximate t o 29 kDa under reducing conditions, while 60 kDa under non-reducing conditi ons. The optimum temperature of the enzyme was 50 degrees C using t-butylox ycarbonyl-Phe-Ser-Arg-4-methylcoumaryl-7-amide (Boc-Phe-Ser-Arg-MCA) as a s ubstrate. Substrate specificity analysis both using MCA-substrates and pept ides showed that MBSP specifically cleaved at the carboxyl side of the argi nine residue. Inhibitor susceptibility analysis revealed that MBSP was inhi bited effectively by Pefabloc SC, soybean trypsin inhibitor (STI) and aprot inin, indicating the characteristic of a serine proteinase. When myofibril was incubated with the enzyme, it optically degraded myosin heavy chain at 55-60 degrees C, while alpha-actinin and actin were not at all hydrolyzed a s detected by immunoblotting. The N-terminal amino acid sequence of MBSP wa s partially determined as IVGGAEXVPY- and was very homologous to other seri ne proteases. (C) 2000 Elsevier Science Inc. All rights reserved.