Synthesis of sphingomyelin sulfur analogue and its behavior toward sphingomyelinase.

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.

Chromogenic assay for the activity of sphingomyelinase from Bacillus cereus and its application to the enzymatic hydrolysis of lysophospholipids.

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.

Novel inhibition mechanism of Bacillus cereus sphingomyelinase by beryllium fluoride.

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.