Effect of meat-bleaching and dilution-precipitation procedures on the removal of cathepsin L-like contained in the actomyosin of various fish species

In a previous report, it was found that cathepsins B, H, L and trypsin-like proteases still remained in the natural actomyosin (AM). Cathepsin L-like was found to be present as a detached form from AM, as studied by gel filtration, despite that the activity could not be removed by repeated bleaching of meat. It was also found that the activity varied from fish species to species. Hu et al. found that spotted chub mackerel, true lizardfish and redbanded searobin showed the highest, middle and the lowest cathepsin L-like activities, respectively. Actomyosin used in the study was prepared from fish mince bleached three times, and purified by a cycle of dilution-precipitation procedures. It might be necessary to investigate if the above treatments would affect the remaining activity of cathepsin L-like. The objective of this present study was to investigate if the activity differences among fish species of cathepsin L-like were affected by bleaching and dilution-precipitation. Spotted chub mackerel Scomber australasicus (38.5 cm, 729.0 g), true lizardfish Saurida undosquamis (three fishes with mean fork length of 35.7 cm, mean body weight of 159.8 g) and redbanded searobin Lepidotrigla guentheri (seven fisheswithmean fork length of 14.7 cm,meanbody weight of 42.3 g) were used as materials. The fresh fishes were purchased from Kochi Central Fish Market (Kochi prefecture, Japan) and were brought to the laboratory in ice and were used immediately. Actomyosin was prepared according to the method of Takashi and Arai. All of the procedures were performed at 4°C unless otherwise indicated. Approximately 40 g of dorsal muscle was homogenized with 4 volumes of I = 0.05 phosphate buffer (buffer A, pH 7.5) using a non-bubbling homogenizer (Diameter 3.6 cm; Nihonseiki Kaisha Ltd, Tokyo Japan) and centrifuged at 3000 ¥g for 10 min (Hitachi CR21E; Hitachi, Tokyo, Japan). This treatment was defined as a single bleaching of mince. The same procedures were repeated three times. The resulting supernatant by the procedures was referred to as SP with the numbers of bleaching. The pellet at the stage was collected and was dispersed in buffer A containing 0.6 M NaCl (buffer B). After standing at 4°C for 20 h, the sample was centrifuged at 10 000 ¥g for 30 min to obtain AM in the supernatant which was referred as AM with the numbers of bleaching. The NaCl concentration for SP fractions was adjusted to 0.6 M by adding solid NaCl. In order to purify AM, a dilution-precipitation procedure was applied. AM3 was used as a starting material. AM3 was diluted with 10 volumes of cold deionized water and centrifuged at 3000 ¥g for 10 min. This was defined as a single cycle of dilution-precipitation procedures. The procedures were repeated three times. The supernatant and the AM pellet were termed as DP and AM3 with the numbers of treatment. AM and DP fractions were both dissolved into 0.6 M NaCl. As a control, approximately 5 g of dorsal muscle was homogenized with six volumes of buffer B, and were left for 20 hat 4°C.The samplewascentrifuged *Corresponding author: Tel: 81-88-864-5155. Fax: 81-88-864-5155. Email: itoh@kochi-u.ac. jp Received 6 December 2006. Accepted 5 April 2007. FISHERIES SCIENCE 2008; 74: 696–698