Proteolytic cleavages of carp myosin by several proteases.

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  • Muramoto Manabu
    Department of Food Science, Faculty of Fisheries, Hokkaido University
  • Seki Nobuo
    Department of Food Science, Faculty of Fisheries, Hokkaido University

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Other Title
  • コイミオシンに対する各種プロテアーゼの作用
  • コイミオシン ニ タイスル カクシュ プロテアーゼ ノ サヨウ

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Abstract

Carp myosin was fluorescently labeled with 1, 5 IAEDANS at the SH1 thiol group to enable identification of the head portion of the heavy chain where this group is located. The labeled myosin was heated at 35°C for 10min to induce thermal denaturation (95% loss of myosin Ca-ATPase activity) or unheated, and then digested by five different proteases (i. e. calpain, a-chymotrypsin, trypsin, papain, and subtilisin) under the same conditions in 0.05M NaCl at neutral pH, respectively. The proteolytic products were analyzed by SDS-PAGE. The digestion of the unheated native myosin showed protease specific fragmentation patterns. It was, however, found from the denatured and aggregated myosin that all proteases used produced preferentially Mr=150k fragment, corresponding to myosin rod-like fragment due to the absence of the fluorescence, and many other small fragments derived from myosin head portion. The denatured myosin was rapidly digested than native one.<br>The cathepsin D from carp skeletal muscle exhibited dual PHoptima at pH3.0 and 4.5 when carp myosin was used as the substrate. At PH3.0, cathepsin D produced mainly Mr=140k and 160k fragments containing the fluorescence on SDS-PAGE from the denatured myosin which was soluble below pH4.0 even though in 0.05M NaCI, while in the denatured and aggregated form above pH4.0 myosin heavy chain was degraded to Mr=150k fragment without the fluorescence.<br>It was therefore concluded that the proteolytic cleavage of the denatured and aggregated myosin is remarkably influenced by the specific structural changes of the substrate rather than protease specificity.

Journal

  • NIPPON SUISAN GAKKAISHI

    NIPPON SUISAN GAKKAISHI 55 (9), 1605-1613, 1989

    The Japanese Society of Fisheries Science

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