Sphingomyelinase Assay

Two situations must be distinguished, (i) assays of pure enzyme and (ii) assays of cell extracts. When purified enzyme preparations are available, no labeled substrate is required. Natural or synthetic sphingomyelin is prepared, pure or mixed with other lipids, in the form of extruded large unilamellar vesicles (LUV) ca. 100 nm in diameter. When pure sphingomyelin vesicles are used extrusion must take place at a temperature close to or above the gel-fluid transition temperature of the lipid, i.e. often 45-50 °C. LUV and enzyme are mixed in the appropriate assay buffer and aliquots are removed at fixed time intervals. The aliquots are mixed with chloroform-methanol and, after phase separation, phosphorous (from phosphorylcholine) is assayed in the aqueous phase. The procedure has been described in detail by Ruiz-Arguello et al. [91]. 

Some commercial preparations of purified sphingomyehnase from B. cereus may be contaminated with phospholipase C. This is irrelevant when the substrate consists of pure sphingomyelin, but when the LUV contain glycerophospholipids, e.g. phosphatidylcholine, contamination is serious because both enzymes will give off the water-soluble end-product phosphorylcholine. In such cases, the assays include o-phenanthroline, a specific inhibitor of phospholipase C that is innocuous for SMase [92-94]. 

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Van Diggelen OP, Voznyi YV, Keulemans JLM, Schoonderwoerd K, Ledvinova J, Mengel E, Zschiesche M, Santer R, Harzer (2005) A new fluorimetric assay for the diagnosis of Niemann-Pick A/ , with specificity of natural sphingomyelinase substrate. J Inherit Metab Dis 28 733-741... 

Loidl, A. Claus, R. Deigner, H. P. Hermetter, A. High-precision fluorescence assay for sphingomyelinase activity of isolated enzymes and cell lysates. /. Lipid Res. 2002, 43, 815-823. 

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