Development,Validation, and Application of a Novel Method for Mammalian Sphingomyelin Synthase Activity Measurement

Sphingomyelin synthase (SMS) is a key enzyme for the synthesis of mammalian sphingomyelin. The use of SMS plays diverse roles in physiology and pathology; thus, it could be a useful disease marker and/or drug target. We report here a novel and sensitive method for SMS activity measurement. Using a HPLC column (C18-RP), SMS activity was monitored by measuring a decrease of the fluorescent substrate C6-4-nitrobenzo-2-oxa-1,3-diazole (NBD)-ceramide (C6-NBD-Cer) and increase of the product (C6-NBD-SM). Time- and protein-dependent formation of C6-NBD-SM was investigated and enzyme kinetics was determined K _m  = 7.49 ± 0.48 µM (C6-NBD-Cer) and V _max  = 27.86 ± 0.73fmol/h/mg homogenate protein]. This method is feasible, rapid, accurate, highly reproducible, and suitable for quantifying SMS enzyme activity in SMS inhibitor screening studies. A known SMS inhibitor, D609, was employed to evaluate the assay and its IC₅₀ value has been determined.

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