Identification of a myofibril-bound serine proteinase (MBSP) in the skeletal muscle of lizard fish Saurida wanieso which specifically cleaves the arginine site
Mj. Cao et al., Identification of a myofibril-bound serine proteinase (MBSP) in the skeletal muscle of lizard fish Saurida wanieso which specifically cleaves the arginine site, COMP BIOC B, 125(2), 2000, pp. 255-264
Citations number
24
Categorie Soggetti
Biochemistry & Biophysics
Journal title
COMPARATIVE BIOCHEMISTRY AND PHYSIOLOGY B-BIOCHEMISTRY & MOLECULAR BIOLOGY
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.