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.