Abstract
Sphingomyelinases are important enzymes of signal transduction. They catalyze the hydrolysis of sphingomyelin, giving rise to the intracellular formation of biologically active ceramide and phosphatidylcholine. Here we report on a fluorescence method for the fast and accurate determination of this enzyme in biological samples. The assay is based on a fluorescent sphingomyelin analog carrying fluorescent 7-nitro-2-1,3-benzooxadiazolyl amino-dodecanoic acid instead of an aliphatic acyl chain at the nitrogen atom. The fluorescent substrate is hydrolysed by sphingomyelinases to form fluorescent ceramide, which can be separated from the remaining substrate using TLC on silica gel. The fluorescence intensity pattern obtained on the TLC plate can accurately be determined using a CCD camera. Typically, a large number of samples can be analyzed simultaneously. Examples for the quantitative analysis of sphingomyelinases from freshly prepared cellular homogenates as well as from commercial sources are given.—Loidl, A., R. Claus, H. P. Deigner, and A. Hermetter. High-precision fluorescence assay for sphingomyelinase activity of isolated enzymes and cell lysates. J. Lipid Res. 2002. 43: 815–823.
Highlights
Sphingomyelinases are important enzymes of signal transduction
We have developed a high-throughput method based on a fluorescent substrate analog (NBD-sphingomyelin) (Fig.1) that is hydrolyzed to form the corresponding ceramide
The described fluorescence assay was applied to the determination of neutral and acid SMase activities in human smooth muscle cell (SMC) after incubation with various agents, namely native LDL, minimally modified LDL, ceramide (10 M), or TNF␣ (0.1 ng/ml) for 1 h
Summary
Sphingomyelinases are important enzymes of signal transduction. They catalyze the hydrolysis of sphingomyelin, giving rise to the intracellular formation of biologically active ceramide and phosphatidylcholine. For determination of SMase-activity as a function of substrate concentration, 1.6 units of acid SMase from human placenta were incubated with NBD-sphingomyelin (absolute amounts between 0.2 and 9.6 nmol) in a total volume of 200 l for 1 h at 37ЊC. Experimental conditions (buffers, total lipid concentration) for assaying SMase activity were the same as indicated above for the fluorescence method.
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