ABSTRACT
The sulfur analogue of sphingomyelin was designed and stereoselectively synthesized from S-benzyl-N-Boc-cysteine. The introduction of the phosphoryl choline moiety was successfully achieved by our own method using 2-bromoethyl dimethyl phosphite and carbon tetrabromide followed by a trimethylamine treatment. The synthesized compound proved to be a useful substrate for monitoring the enzyme activity of sphingomyelinase by detecting the liberated thiol group with a thiol-sensitive reagent.
Subject(s)
Sphingomyelin Phosphodiesterase/metabolism , Sphingomyelins/chemical synthesis , Sulfur Compounds/chemical synthesis , Bacillus cereus/drug effects , Bacillus cereus/enzymology , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/pharmacology , Sphingomyelin Phosphodiesterase/antagonists & inhibitors , Sphingomyelins/metabolism , Sulfur Compounds/metabolismABSTRACT
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.
Subject(s)
Bacillus cereus/enzymology , Lysophospholipids/chemistry , Platelet Activating Factor/analogs & derivatives , Sphingomyelin Phosphodiesterase/analysis , Hydrolysis , Kinetics , Lysophosphatidylcholines/chemistry , Octoxynol/chemistry , Phosphorylcholine/chemistry , Platelet Activating Factor/chemistry , Sphingomyelin Phosphodiesterase/chemistry , Time FactorsABSTRACT
Phosphate analogs have been known to inhibit competitively various phosphatases and phospholipase C and D. We found for the first time that only beryllium fluoride (BeF(x)) among the phosphate analogs studied inhibits Bacillus cereus sphingomyelinase (SMase) activity. The active inhibitory species proved to be not BeF(3)(-) but BeF(2) by the measurement of SMase activity and of (19)F NMR spectroscopy in the presence of a fixed concentration of BeCl(2) and different concentrations of NaF, although both the species have been reported for other kinds of enzymes. The result of kinetic experiment also indicated that the BeF(x) binds in the vicinity of the essential binding site for the substrate and that the Mg(2+) binding to SMase is essential for the binding of BeF(x) to the enzyme.
