Chromogenic Assay for the Activity of Sphingomyelinase from Bacillus cereus and Its Application to the Enzymatic Hydrolysis of Lysophospholipids(Analytical Biochemistry)
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概要
- 論文の詳細を見る
We developed a convenient chromogenic assay method for the activity of sphingomyelinase (SMase) from Bacillus cereus. SMase reaction was quenched by Zn^<2+>, 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 Zn^<2+> 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 (K_m and k_<cat>) 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.
- 社団法人日本薬学会の論文
- 2004-11-01
著者
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Fujii Shinobu
Department of Mathematics, Hiroshima University
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藤井 忍
大阪薬大・生化
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池田 潔
School Of Pharmaceutical Sciences University Of Shizuoka
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Yoshida A
Department Of Biochemistry Osaka University Of Pharmaceutical Sciences
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池田 潔
Tanabe Seiyaku Co. Ltd. Saitama Jpn
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YOSHIDA Ayumi
Department of Biochemistry, Osaka University of Pharmaceutical Sciences
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SAKURAI Saori
Department of Biochemistry, Osaka University of Pharmaceutical Sciences
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MORITA Michio
Department of Biochemistry, Osaka University of Pharmaceutical Sciences
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TSUKAMOTO Kikuo
Department of Molecular Biology, Graduate School of Pharmaceutical Sciences, Nagoya City University
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IKEZAWA Hiroho
Department of Molecular Biology, Graduate School of Pharmaceutical Sciences, Nagoya City University
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IKEDA Kiyoshi
Department of Biochemistry, Osaka University of Pharmaceutical Sciences
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Sakurai Shigeru
Department Of Environmental Health Faculty Of Pharmaceutical Sciences Hokuriku University:department
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Ikezawa H
Department Of Molecular Biology Graduate School Of Pharmaceutical Sciences Nagoya City University
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Yoshida Ayumi
Department Of Biochemistry Osaka University Of Pharmaceutical Sciences
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Ikeda Kiyoshi
Department Of Biochemistry Osaka University Of Pharmaceutical Sciences
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Fujii Shinobu
Department Of Dermatology Osaka Red Cross Hospital
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Morita Michio
Department Of Biochemistry Osaka University Of Pharmaceutical Sciences
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Sakurai Saori
Department Of Biochemistry Osaka University Of Pharmaceutical Sciences
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Tsukamoto K
Department Of Molecular Biology Graduate School Of Pharmaceutical Sciences Nagoya City University
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池田 かおり
Developmental Research Laboratories Shionogi & Co. Ltd.
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Ikeda Kaori
Developmental Research Laboratories Shionogi & Co. Ltd.
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MORITA MICHIO
Department of Applied Chemistry, Graduate School of Engineering, Nagoya University
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