Detecting Sphingomyelinase Activity

Sphingomyelinase (SMase) is an enzyme that is responsible for cleaving sphingomyelin (SM) to phosphocholine and ceramide. The activation of SMases in cells plays an important role in cellular responses. Five types of sphingomyelinase (SMase) have been identified based on their cation dependence and pH optima of action. Among the five types, the lysosomal acidic SMase and the magnesium-dependent neutral SMase are considered major candidates for the production of ceramide in the cellular response to stress.

Amplite^® Sphingomyelinase Assay Kits and provide sensitive methods for detecting neutral SMase activity or screening its inhibitors. They can be used for measuring the SMase activity in blood, cell extracts, or other solutions. For Kit 13620, the absorbance of light at 595 nm is proportional to the formation of phosphocholine, therefore to the SMase activity. The kit is an optimized 'mix and read' assay that is compatible with HTS liquid handling instruments. For Kit 13621, it uses Amplite^® Red as a fluorogenic probe to indirectly quantify the phosphocholine produced from the hydrolysis of sphingomyelin (SM) by sphingomyelinase (SMase). The fluorescence intensity of Amplite^® Red is proportional to the formation of phosphocholine, therefore to the SMase activity. Amplite^® Red enables the assay readable either in fluorescence intensity or absorption mode.