Purification and characterization of paralytic shellfish toxin transforming enzyme from Mactra chinensis

Hsi Pin Lin, Yuko Cho, Hitoshi Yashiro, Tomoko Yamada, Yasukatsu Oshima

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    44 Citations (Scopus)


    A carbamoylase, which catalyzes hydrolysis of the carbamoyl (or N-sulfocarbamoyl) moiety of paralytic shellfish toxins, was purified from the digestive glands of the Japanese clam Mactra chinensis. Using five steps of column chromatography, 290 μg of Carbamoylase I showing homogeneity on SDS-PAGE was obtained. Carbamoylase I was revealed to be a glycoprotein, having estimated molecular weight of 190 kDa. Observation of single band equivalent to 94 kDa on SDS-PAGE under reducing conditions suggested it to be a homodimer. The optimal temperature and pH were 20°C and 7.0. Carbamoylase I did not require a divalent cation and its activity was inhibited by the serine proteinase inhibitors, benzenesulfonyl fluoride and 4-(2-aminoethyl)- benzenesulfonyl fluoride. Carbamoylase I hydrolyzed both carbamate and N-sulfocarbamate toxins. The presence or absence of a hydroxyl moiety at the N-1 position of the substrate toxins did not significantly alter the reaction rate, but the stereochemistry of sulfate esters at C-11 greatly affected it. The Km was 3.02 μM for saxitoxin as a substrate. Nineteen amino acids of the N-terminal sequence were identified by the Edman method. MALDI-TOF-MS/MS spectra of 18O-labeled tryptic peptides indicated the possible internal amino acid sequences of five peptides.

    Original languageEnglish
    Pages (from-to)657-668
    Number of pages12
    Issue number6
    Publication statusPublished - 2004 Nov


    • Carbamoylase
    • Digestive glands
    • GTX
    • Mactra chinensis
    • PSP
    • Saxitoxin

    ASJC Scopus subject areas

    • Toxicology


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