A high throughput sphingomyelinase assay.

Publisher Summary Sphingomyelinases (SMases) are phophodiesterases that catalyze hydrolysis of the phosphodiester linkage of sphingomyelin (SM) to yield ceramide or ceramide-l-phosphate as the lipidic product and phosphorylcholine or choline as the headgroup product. SMases have been assayed using a variety of substrates. Radiolabeled sphingomyelin, prepared via incorporation of 3 H or 14 C into the headgroup moiety, is the most common substrate. Colorimetric and fluorescent assays have been developed that utilize SM analogs containing chromophores or fluors. However, these classes of substrates differ significantly from authentic SM; these are not widely used. Assays employing radiolabeled substrate require physical separation of product from substrate. Usually this separation is accomplished with liquid-liquid phase extractions that effectively separate lipophilic substrate and hydrophilic product into organic and aqueous phases, respectively. The reaction components can also be separated chromatographically. These methods are tedious and are not suitable for large numbers of samples. An alternate method employs headgroup radiolabeled SM with separation of the unreacted substrate from the labeled product by precipitation. In this strategy, the labeled substrate is selectively precipitated while the labeled headgroup product remains soluble. The latter can then be transferred easily for quantitation by liquid scintillation counting. The advantage of this method is the ease with which multiple samples can be processed simultaneously. This chapter describes methods of exploiting this separation technique to greatly increase the efficiency and throughput of SMase assays.