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.

Purification and characterization of myofibril-bound serine protease from lizard fish (Saurida undosquamis) muscle.

Myofibril-bound serine protease (MBSP) from lizard fish (SAURIDA UNDOSQUAMIS: Synodontidae) skeletal muscle was purified to homogeneity with higher purification (1260-fold) and higher recovery (7%) than our previous report in lizard fish (Saurida wanieso). The new purification method combines a heat-treatment for dissociation from washed myofibrils, acid-treatment at pH 5.0 before and after lyophilization, and alcohol-treatment, followed by two column chromatographies. The molecular mass of the enzyme was estimated to be 50 kDa under non-reducing conditions and 28 kDa under reducing conditions by SDS-PAGE. The N-terminal amino acid sequence of the MBSP was determined to be 22 residues (IVGGYEXEAYSKPYQVSINLGY) and the sequence showed high homology to carp and other fish trypsins (64-77%), but did not show high homology to carp MBSP (41%). The enzyme activity was inhibited by serine protease inhibitors such as Pefabloc SC, leupeptin, TLCK and native protein inhibitors (soybean trypsin inhibitor, alpha(1)-antitrypsin and aprotinin). The purified enzyme specifically hydrolyzed at the carboxyl side of the arginine residue of synthetic 4-methyl-coumaryl-7-amide substrate. When purified MBSP was stored at -35 degrees C in the presence of 50% ethylene glycol (V/V), the enzyme activity was entirely preserved over 6 months and stable against freezing and thawing. Activities for both casein and the synthetic substrate were most active at pH 9.0, and the enzyme was most active approximately 55 degrees C with casein and between 35 and 45 degrees C for synthetic substrate. When myofibrils were incubated with purified MBSP, myosin heavy chain was mostly degraded approximately 55 degrees C, but the degradation of actin was very slow.