title: Chromogenic assay for the activity of sphingomyelinase from Bacillus cereus and its application to the enzymatic hydrolysis of lysophospholipids.
abstract: 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.
authors.affiliation: Department of Biochemistry, Osaka University of Pharmaceutical Sciences, Nasahara, Takatsuki, Japan.
authors.forename: Shinobu
authors.initials: S
authors.lastname: Fujii
authors.forename: Ayumi
authors.initials: A
authors.lastname: Yoshida
authors.forename: Saori
authors.initials: S
authors.lastname: Sakurai
authors.forename: Michio
authors.initials: M
authors.lastname: Morita
authors.forename: Kikuo
authors.initials: K
authors.lastname: Tsukamoto
authors.forename: Hiroho
authors.initials: H
authors.lastname: Ikezawa
authors.forename: Kiyoshi
authors.initials: K
authors.lastname: Ikeda
mesh_terms: D001409:Bacillus cereus / Q000201:enzymology*; D006868:Hydrolysis; D007700:Kinetics; D008244:Lysophosphatidylcholines / Q000737:chemistry; D008246:Lysophospholipids / Q000737:chemistry*; D017830:Octoxynol / Q000737:chemistry; D010767:Phosphorylcholine / Q000737:chemistry; D010972:Platelet Activating Factor / Q000031:analogs & derivatives* / Q000737:chemistry; D013108:Sphingomyelin Phosphodiesterase / Q000032:analysis* / Q000737:chemistry; D013997:Time Factors
chemical_list: D008244:Lysophosphatidylcholines; D008246:Lysophospholipids; C029271:O-deacetyl platelet activating factor; D010972:Platelet Activating Factor; D010767:Phosphorylcholine; D017830:Octoxynol; D013108:Sphingomyelin Phosphodiesterase