Milli-calpain from Sea Bass (Dicentrarchus labrax) White Muscle: Purification, Characterization of Its Activity and Activation In Vitro

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• 作者：Christine Ladrat ; V¨¦ronique Verrez-Bagnis ; Jo?l No?l and Jo?l Fleurence
• 关键词：Key words ; calcium ; dependent cysteine protease ; calpain ; properties ; cleavage specificity ; autolysis ; fish muscle ; postmortem degradation.
• 刊名：Marine Biotechnology
• 出版年：2002
• 出版时间：January 2002
• 年：2002
• 卷：4
• 期：1
• 页码：51-62
• 全文大小：972 KB

文摘

A calcium-activated neutral cysteine protease was purified to homogeneity from Dicentrarchus labrax white muscle using three steps: hydrophobic interaction, anion exchange, and gel filtration chromatographies. The purified enzyme showed a native molecular weight of 124 kDa with an oligomeric structure (large subunit of 80 kDa and small subunit of 24 kDa). It has been classified as a milli-calpain from its calcium sensitivity. Activity was maximal at pH 7.0, 24¡ãC in Tris buffer without NaCl as determined by means of a two-level experimental design and response surface methodology. Sea bass calpain is neither glycosylated nor phosphorylated and shared some common cleavage specificities and activation and autolysis mechanisms with other typical mammalian or invertebrates calpains. Calcium-induced activation and autolysis of calpain has been characterized together with the effect of the strontium cation acting as a calcium analog. On the basis of its in vitro properties, the contribution of the sea bass milli-calpain to the process of postmortem deterioration of fish muscle is discussed, even though further information such as in vivo regulation or in vitro effects on myofibrils is required.