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PARTIAL PURIFICATION AND CHARACTERIZATION OF TROPOMYOSIN‐BOUND SERINE PROTEINASE FROM THE SKELETAL MUSCLE OF YELLOW CROAKER ( PSEUDOSCIAENA CROCEA )
Author(s) -
CAO MINJIE,
WENG LING,
LIU GUANGMING,
HARA KENJI,
SU WENJIN
Publication year - 2007
Publication title -
journal of food biochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.507
H-Index - 47
eISSN - 1745-4514
pISSN - 0145-8884
DOI - 10.1111/j.1745-4514.2007.00119.x
Subject(s) - tropomyosin , fast protein liquid chromatography , biochemistry , myofibril , serine , chemistry , high performance liquid chromatography , gel electrophoresis , serine proteinase inhibitors , chromatography , polyacrylamide gel electrophoresis , affinity chromatography , skeletal muscle , cysteine , enzyme , microbiology and biotechnology , biology , serine protease , myosin , protease , endocrinology
A myofibril‐bound serine proteinase (MBSP) in the skeletal muscle of yellow croaker ( Pseudosciaena crocea ) was isolated from myofibril by heat treatment and chromatographies on Sephacryl S‐200, fast performance liquid chromatography (FPLC) on Mono Q column and high performance liquid chromatography (HPLC) using a Bio‐Sil SEC‐125 column. A single protein band with a molecular weight of 34 kDa was observed on sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE). Optimum temperature and pH of the purified protein was 55C and 8.0. Inhibitor susceptibility analysis indicated that the enzymatic activity was effectively suppressed by serine proteinase inhibitors such as Pefabloc 4‐(2‐aminoethyl)‐benzenesulfonyl fluoride hydrochloride and aprotinin, while inhibitors for other proteinases (namely cysteine, asparatic and metallo) did not show any inhibitory effect. At the last purification stage, the electrophoretic study revealed that the proteinase associated with tropomyosin, as the N‐terminal amino acid sequence of the 34 kDa main band protein, exhibited high sequence homology (90%) to α‐tropomyosin from white croaker, human and rat. This result suggested that yellow croaker MBSP is an α‐tropomyosin‐binding proteinase.