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 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-poly-acrylamide gel electrophoresis (SDS-PAGE), the purified enzyme showed a band with molecular mass of approximately 29 kDa under reducing conditions, while 60 kDa under non-reducing conditions. The optimum temperature of the enzyme was 50 degrees 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 degrees C, while alpha-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.
Cao
M J
MJ
Graduate School of Marine Science and Engineering, Nagasaki University, Japan.
Osatomi
K
K
Hara
K
K
Ishihara
T
T
D020816:Amino Acid Motifs; D000818:Animals; D015153:Blotting, Western; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004795:Enzyme Stability; D005399:Fishes / Q000378:metabolism*; D018482:Muscle, Skeletal / Q000201:enzymology*; D009210:Myofibrils / Q000737:chemistry / Q000201:enzymology*; D010446:Peptide Fragments / Q000032:analysis; D011485:Protein Binding; D011489:Protein Denaturation; D017386:Sequence Homology, Amino Acid; D012697:Serine Endopeptidases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D013379:Substrate Specificity
D004791:Enzyme Inhibitors; D010446:Peptide Fragments; D012697:Serine Endopeptidases