A novel type of myofibril-bound serine protease from white croaker (Argyrosomus argentatus).
Myofibril-bound serine protease (MBSP) was purified from the myofibril fraction of white croaker (Argyrosomus argentatus) muscle and its enzymatic properties were compared with other fish MBSPs. White croaker MBSP was extracted by the heat treatment of myofibrils and then purified by a series of column chromatographies on Q-Sepharose, Sephacryl S-300, hydroxyapatite and Benzamidine Sepharose. The purified MBSP migrated as a single protein band at 67 kDa in SDS-PAGE under both reducing and non-reducing conditions. It was inhibited by Pefabloc SC, soybean trypsin inhibitor (STI), aprotinin and benzamidine, and was not affected by E-64, pepstatin A and EDTA. The enzyme was most active against Boc-Phe-Ser-Arg-MCA at pH 7.0 and 50 degrees C, and preferentially hydrolyzed Boc-Val-Pro-Arg-MCA and Boc-Asp-Pro-Arg-MCA. Unlike other marine fish MBSPs, white croaker MBSP considerably hydrolyzed Boc-Val-Leu-Lys-MCA and Boc-Glu-Lys-Lys-MCA. Some enzymatic characteristics including the molecular structure and the substrate specificity for a lysine residue at the P(1) position are quite different not only from other fish MBSPs but also from soluble serine protease obtained from white croaker muscle (MSSP). White croaker MBSP could be therefore classified into a novel type of fish muscle MBSP.
Ohkubo
Makoto
M
Department of Biological and Environmental Sciences, Miyazaki University, Miyazaki 889-2192, Japan. sansouko@hotmail.com
Osatomi
Kiyoshi
K
Hara
Kenji
K
Ishihara
Tadashi
T
Aranishi
Futoshi
F
D000818:Animals; D002845:Chromatography; D006863:Hydrogen-Ion Concentration; D009124:Muscle Proteins / Q000302:isolation & purification* / Q000378:metabolism*; D009210:Myofibrils / Q000737:chemistry*; D009842:Oligopeptides / Q000378:metabolism; D010473:Perciformes; D012697:Serine Endopeptidases / Q000302:isolation & purification* / Q000378:metabolism*; D015842:Serine Proteinase Inhibitors / Q000494:pharmacology; D013379:Substrate Specificity; D013696:Temperature
D009124:Muscle Proteins; D009842:Oligopeptides; D015842:Serine Proteinase Inhibitors; D012697:Serine Endopeptidases