Purification and characterization of sulfotransferase specific to O-22 of 11-hydroxy saxitoxin from the toxic dinoflagellate Gymnodinium catenatum (dinophyceae) |
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Authors: | Takashi YOSHIDA Yoshihiko SAKO Aritsune UCHIDA Tomonori KAKUTANI Osamu ARAKAWA Tamao NOGUCHI Yuzaburo ISHIDA |
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Affiliation: | Faculty of Biotechnology, Fukui Prefectural University, Obama, Fukui 917-0003;, Graduate School of Agriculture, Kyoto University, Kyoto, Kyoto 606-8502;, Faculty of Fisheries, Nagasaki University, Nagasaki, Nagasaki 852-8521 and; Faculty of Engineering, Fukuyama University, Fukuyama, Hiroshima 729-0292, Japan |
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Abstract: | ABSTRACT: A novel sulfotransferase (O-ST), which transferred the sulfate group of 3'-phosphoadenosine 5'-phosphosulfate (PAPS) to O-22 of 11-α,β-hydroxy saxitoxin (STX) and produced GTX2 + 3, was purified to homogeneity from the cytosolic fraction of clonal-axenic vegetative cells of the toxic dinoflagellate Gymnodinium catenatum GC21V. After four purification steps, including affinity chromatography and anion exchange chromatography, the enzyme was purified 500-fold and the yield was 4%. On affinity chromatography with a PAP-agarose column, O-ST was observed in the bound fraction, and N-ST specific to N-21 of STX and GTX2 + 3 was found in the unbound fraction. The molecular mass of the purified enzyme was determined by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) to be 65 kDa. Gel filtration chromatography showed a native molecular mass of 67 kDa, indicating that O-ST is a monomeric enzyme. The enzyme was optimally active at pH 6.0 and 35°C. O-ST did not require metal cations for its activity. O-ST required PAPS as the sole source of sulfate. O-ST transferred a sulfate group from PAPS to only O-22 of 11-α,β-hydroxy STX and not to N-21 of these toxins. These observations suggested that two ST, N-ST and O-ST, participate in the sulfation of PSP toxins. |
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Keywords: | 3'-phosphoadenosine 5'-phosphosulfate Dinophyceae gonyautoxin Gymnodinium catenatum paralytic shellfish poisoning saxitoxin sulfotransferase toxic dinoflagellate |
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