Myosin heavy chain-degrading proteinase from spear squid muscle

Abstract

Trypsin-like proteinase responsible for textural deterioration of thermally induced gel was purified to apparent homogeneity from spear squid Loligo bleekeri mantle muscle by chromatography on Sephacryl S-400 and DEAE Sepharose. The enzyme gave a protein band with a molecular weight of 42 kDa on SDS-PAGE. The proteinase readily hydrolyzed casein, synthetic peptide butyloxycarbonyl-valine-leucine-lysine-4-methylcoumaryl-7-amide (Boc-Val-Leu-Lys-MCA) and myosin heavy chain. The proteolytic activity was effectively inhibited by serine protease inhibitors such as soybean trypsin inhibitor and N ga- p-tosyl- l-lysine-cnloromethyl ketone, but was not affected by the other types of protease inhibitors (e.g. EDTA, EGTA, iodoacetic acid, mercuric chloride and N-tosyl- l-phenylalanine-chloromethyl ketone). The optimal temperature was 40 °C for hydrolysis of both casein and Boc-Val-Leu-Lys-MCA. The optimal pH values were 6.8 and 7.9 for caseinolytic and peptide hydrolyzing activity, respectively. The proteolytic activity was increased 1.3-fold by addition of 0.25 m NaCl, but not by the addition of Ca 2+. Myosin heavy chain was shown to be cleaved into smaller fragments by incubation with the proteinase on SDS-PAGE. These results revealed that the enzyme was involved in degradation of myosin heavy chain from the squid mantle meat.