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Journal of Lipid Research, Vol. 43, 815-823, May 2002
Copyright © 2002 by Lipid Research, Inc.

Methods

High-precision fluorescence assay for sphingomyelinase activity of isolated enzymes and cell lysates

A. Loidl^*, R. Claus, H. P. Deigner and A. Hermetter^1,*

^* Department of Biochemistry, Technische Universität Graz, Petersgasse 12, A-8010 Graz, Austria
Clinic for Anaesthesiology and Intensive Care Therapy, FSU Jena, Bachstrasse 18, D-07740 Jena, Germany
Zentrallaboratorium Deutscher Apotheker, Carl-Mannich-Strasse 20, D-65760 Eschborn, Germany

¹ To whom correspondence should be addressed. e-mail: albin.hermetter{at}tugraz.at

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.

Abbreviations: mmLDL, minimally modified LDL; natLDL, native LDL; oxLDL, oxidized LDL; SM, sphingomyelin; SMase, sphingomyelinase; aSMase, acid sphingomyelinase; SMC, smooth muscle cell; TNF, tumor necrosis factor ; NBD, N-7-nitrobenz-2-oxa-1,3-diazol

Supplementary key words intracellular signaling • ceramide • lipid second messenger • atherosclerosis • smooth muscle cells • low density lipoprotein • apoptosis • cell proliferation

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