A novel serine protease complexed with alpha2-macroglobulin from skeletal muscle of lizard fish (Saurida undosquamis).
A novel fish muscle serine protease named muscle soluble serine protease (MSSP) was purified from the soluble fraction of lizard fish (Saurida undosquamis: Synodontidae) muscle by ammonium sulfate fractionation followed by four steps of column chromatographies. In native-PAGE, the purified enzyme appeared as a single band with an estimated mol. mass of approximately 380 kDa by gel filtration. In SDS-PAGE under reducing conditions, the purified enzyme migrated as two protein bands at 110 and 100 kDa, named subunits A and B, respectively. The 20 residues of N-terminal amino acid sequence of subunit B showed 70% of homology to beta-chain of carp alpha(2)-macroglobulin-1. Moreover, both subunits A and B showed immunoreactivity with anti carp alpha(2)-macroglobulin antibody. Purified MSSP was inactivated by Pefabloc SC, aprotinin, benzamidine and TLCK, but not by alpha(1)-antitrypsin. After acid treatment (pH 2, 24 h), however, the enzyme activity eluted at 14 kDa from Sephacryl S-200 carried out under acidic conditions was inhibited by alpha(1)-antitrypsin. Lizard fish MSSP most rapidly hydrolyzed Boc-Val-Pro-Arg-MCA and Boc-Gln-Arg-Arg-MCA, but did not hydrolyzed Suc-Leu-Leu-Val-Tyr-MCA and Suc-Ala-Ala-Pro-Phe-MCA, and was not suppressed either by E-64, pepstatin A and ethylenediaminetetraacetic acid (EDTA). These results indicate that the purified MSSP is a serine protease complexed with alpha(2)-macroglobulin, and the entrapped protease was dissociated by the acid treatment. Purified and free MSSPs were most active at pH 10.0 and 9.0, respectively. Purified MSSP degraded myofibrillar proteins and casein but time courses of degradation of these substrates by the enzyme differed.
Ohkubo
Makoto
M
Graduate School of Science and Technology, Nagasaki University, Bunkyo Machi 1-14, Nagasaki 852-8521, Japan. sansouko@hotmail.com
Miyagawa
Kourin
K
Osatomi
Kiyoshi
K
Hara
Kenji
K
Nozaki
Yukinori
Y
Ishihara
Tadashi
T
D000595:Amino Acid Sequence; D000818:Animals; D002845:Chromatography; D004492:Edetic Acid / Q000737:chemistry; D004586:Electrophoresis; D004789:Enzyme Activation; D005399:Fishes / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D018482:Muscle, Skeletal / Q000201:enzymology*; D011485:Protein Binding; D021122:Protein Subunits / Q000302:isolation & purification / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D012697:Serine Endopeptidases / Q000302:isolation & purification / Q000378:metabolism*; D000511:alpha-Macroglobulins / Q000378:metabolism*
D021122:Protein Subunits; D000511:alpha-Macroglobulins; D004492:Edetic Acid; D012697:Serine Endopeptidases