Chromogenic Assay for the Activity of Sphingomyelinase from Bacillus cereus and Its Application to the Enzymatic Hydrolysis of Lysophospholipids

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  • Fujii Shinobu
    Department of Biochemistry, Osaka University of Pharmaceutical Sciences
  • Yoshida Ayumi
    Department of Biochemistry, Osaka University of Pharmaceutical Sciences
  • Sakurai Saori
    Department of Biochemistry, Osaka University of Pharmaceutical Sciences
  • Morita Michio
    Department of Biochemistry, Osaka University of Pharmaceutical Sciences
  • Tsukamoto Kikuo
    Department of Molecular Biology, Graduate School of Pharmaceutical Sciences, Nagoya City University
  • Ikezawa Hiroho
    Department of Molecular Biology, Graduate School of Pharmaceutical Sciences, Nagoya City University
  • Ikeda Kiyoshi
    Department of Biochemistry, Osaka University of Pharmaceutical Sciences

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We developed a convenient chromogenic assay method for the activity of sphingomyelinase (SMase) from Bacillus cereus. SMase reaction was quenched by Zn2+, and the released phosphocholine was converted into a choline by the action of alkaline phosphatase. After that, the choline was converted into a chromogenic dye by the actions of choline oxidase and peroxidase in the presence of EDTA to trap the added Zn2+ which could interfere with the choline oxidase/peroxidase reactions. Triton X-100 also was added to the reaction mixture, in order to remove turbidity generated from ceramide which had been produced by the SMase reaction. To test a large number of samples in a short period of time, this assay was performed using 96-well microtiter plates. This method proved to be applicable not only to the measurement of the hydrolysis of sphingomyelin but also to those of lysophosphatidylcholine (lysoPC) and lyso platelet-activating factor by B. cereus SMase. Using this method, the kinetic parameters (Km and kcat) for B. cereus SMase toward various types of substrates were then determined, and the effect of Triton X-100 on the hydrolysis of lysoPC was examined.

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