High-precision fluorescence assay for sphingomyelinase activity of isolated enzymes and cell lysates.
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
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A
Department of Biochemistry, Technische Universität Graz, Petersgasse 12, A-8010 Graz, Austria.
Claus
R
R
Deigner
H P
HP
Hermetter
A
A
D009327:4-Chloro-7-nitrobenzofurazan / Q000031:analogs & derivatives*; D000818:Animals; D002518:Ceramides; D004531:Eggs; D005456:Fluorescent Dyes; D006801:Humans; D007202:Indicators and Reagents; D008077:Lipoproteins, LDL / Q000097:blood; D013050:Spectrometry, Fluorescence / Q000379:methods; D013108:Sphingomyelin Phosphodiesterase / Q000032:analysis* / Q000378:metabolism
D002518:Ceramides; D005456:Fluorescent Dyes; D007202:Indicators and Reagents; D008077:Lipoproteins, LDL; C099252:oxidized low density lipoprotein; C037974:N-(7-(4-nitrobenzo-2-oxa-1,3-diazole))-6-aminocaproyl sphingosine; D013108:Sphingomyelin Phosphodiesterase; D009327:4-Chloro-7-nitrobenzofurazan