Purification and Characterization of Serine Proteinase from White Croaker Skeletal Muscle

Abstract

A trypsin-type proteinase was purified about 8, 000-fold from white croaker skeletal muscle with a 6% recovery. The enzyme was most active at pH 8.0 and 9.0 toward Boc-Val-Leu-Lys-MCA and casein, respectively. The enzyme was inactivated by DFP, STI, TLCK, and leupeptin, but not by E-64 and EDTA. It hydrolyzed only the substrates for trypsin-type proteinase. The purified enzyme gave a single protein band on Disc-PAGE and SDS-PAGE. The molecular weight of the final preparation was estimated to be about 680, 000 by gel permeation chromatography, and to be about 32, 000 and 71, 000 by SDS-PAGE with or without a reducing reagent, respectively. On the other hand, the molecular weight of the partially purified enzyme was about 123, 000 by gel filtration on Sephadex G-150. These results suggest that the purified enzyme consists of homologous subunits of molecular weight 32, 000. The caseinolytic activity of the purified enzyme was most active at 40°C. but that of the partially purified enzyme was observed with a maximum at 55°C and was not detected below 40°C. When the activities of both enzymes were measured at 55°C in the presence of 0.5_M NaCl, the activities were about 50% of those in the absence of NaCl. The purified enzyme also hydrolyzed myosin heavy chains at 55°C in the presence of 3% NaCl. These results suggest that the serine proteinase may participate in “modori” in kamaboko processing.