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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

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
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

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Presently, several assays for SMase activity are known, including an in situ assay for acid SMase (10), a robotic assay for sphingomyelinase activity (11), a high-throughput technique (12), and several others (13, 14) that all require radioactive substrates.

Here we describe a fluorescence method for the accurate and fast determination of SMase-activity in a large number of samples. It is based on a fluorescently labeled sphingomyelin substrate that is cleaved hydrolytically by SMase to give fluorescent ceramide. This product can be separated from the remaining substrate on TLC plates followed by determination of fluorescence intensity using a CCD-camera [detection-limit: 0.2 pmol of the N-7-nitrobenz-2-oxa-1,3-diazol (NBD)-fluorophore]. The linearity range is between 0.1 pmol and at least 2,000 pmol label. This assay is suitable as a routine method for determination of SMase-activity in pure enzymes and crude enzyme preparations as well as in complex biological samples (arterial SMCs).

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Materials
Chemicals were purchased from Merck (Darmstadt, Germany). Egg yolk sphingomyelin, neutral sphingomyelinase from Bacillus cereus (347 U/mg Protein), and acid sphingomyelinase (177 U/mg protein) from human placenta were from Sigma (Vienna, Austria). Radioactive sphingomyelin (SM) (egg) ([³H]choline methyl) (81.5 Ci/mmol) was purchased from Perkin Elmer Life Sciences (Boston, MA). LCS Safety Cocktail for liquid scintillation counting was from Mallinckrodt Baker (Deventer, Holland). Plastic-ware for cell culture was purchased from Greiner, Frickenhausen, Germany. Media and supplements for cell culture were purchased from Paa Laboratories (Linz, Austria). Tumor necrosis factor (TNF) (human recombinant, over expressed in Escherichia coli) and [C₆]Ceramide (solution in EtOH) were from Calbiochem (Bad Soden, Germany).

Cell culture
Human transformed SMCs (ACBRI #443 Human Coronary Artery SMCs from Applied Cell Biology Research Institute, Kirkland WA) were routinely grown in DMEM supplemented with 10% FCS, 2 mM L-glutamine, 100 U/ml penicillin/streptomycin and 25 mM HEPES at 37°C in humidified CO₂ (8%) atmosphere. For SMase experiments cells were growth-arrested by incubation with DMEM + 0.1% FCS for 24 h.

Preparation of fluorescent sphingomyelin and ceramide
Sphingosylphosphocholine was prepared from egg sphingomyelin according to the method of Kaller (15). It was acylated by NBD-amino-dodecanoylhydroxysuccinimide ester in 5% aqueous NaHCO₃-Ethanol, 9:1 (v/v) to give crude N-(NBD-amino-dodecanoyl)-sphingomyelin. The pure product was obtained after purification by preparative TLC on silica gel using CHCl₃-CH₃OH-H₂O, 65:25:4 (v/v/v) as a solvent. It showed a single spot on TLC (R_F: 0.15; solvent as above).

N-(NBD-amino-dodecanoyl)-ceramide was obtained by hydrolytic cleavage of the corresponding sphingomyelin with sphingomyelinase from Bacillus cereus in 20 mM HEPES buffer, 1 mM MgCl₂, pH 7.4. The crude product was purified by column chromatography as described above. The pure compound showed a single spot on TLC (R_F: 0.85; conditions as above).

Isolation and oxidation of low-density lipoprotein
Human LDL (d = 1.019–1.063) was isolated from pooled fresh plasma using a Beckman NVT-rotor (16). LDL was oxidatively modified (oxLDL) as described by Watson (17). According to the latter procedure, mildly oxidized LDL was obtained. It is characterized by a moderate content of lipid peroxidation products [30–60 nmol of peroxide equivalents/mg of apolipoprotein B (apoB)] (18), and a slightly enhanced electrophoretic mobility (REM) (0.34) as compared with native LDL (0.30) in 0.8% agarose-gels.The protein content of isolated and modified LDL was determined by the method of Lowry (19).

Preparation of an aqueous dispersion of the fluorescent substrate
A solution of NBD-sphingomyelin in tetrahydrofuran (20 nmol in 30 µl) was injected with a hamilton syringe into 2 ml acid reaction buffer (250 mM sodium-acetate, 1 mM EDTA, pH 5.0) for determination of acid SMase-activity, into 2 ml of neutral Mg²⁺-containing reaction buffer (20 mM HEPES, 1 mM MgCl₂, pH 7.4) for determination of neutral Mg²⁺-dependent SMase-activity or into 2 ml of neutral reaction buffer (20 mM HEPES, pH 7.4) for determination of neutral Mg²⁺-independent SMase-activity, under stirring at room temperature. The final substrate concentration was 10 µM.

Fluorescence sphingomyelinase assay
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 µl for 1 h at 37°C. The reaction was stopped by adding 600 µl CHCl₃-CH₃OH, 2:1 (v/v) for lipid extraction. The solvent of a 100 µl aliquot of the organic phase was removed under N₂, the residue redissolved in 10 µl CHCl₃, and the resultant solution was applied onto a TLC plate.

The labeled lipids were separated on silica TLC plates using CHCl₃-CH₃OH-H₂O, 65:25:4 (v/v/v) as solvent (20). Under these conditions, NBD-SM and NBD-ceramide appear as single spots at R_F = 0.15 and R_F = 0.85, respectively. The fluorescence intensities were determined with a Herolab CCD camera (excitation at 365 nm) using EasyWin^®-software for data acquisition and processing. Absolute amounts of ceramide/pmol were obtained from a calibration plot of the amount of NBD-ceramide (0.2 to 20 pmols) versus fluorescence intensity.

SMase-activity as a function of enzyme-concentration (0.05 to 1.6 U) was determined at nonlimiting amount substrate concentration (9.6 nmol lipid in the incubation mix).

Radioactive sphingomyelinase assay
Basically, the same procedure was used as described previously (21). A mixture of [³H]sphingomyelin (³H-labeled in the choline moiety, 81.5 Ci /mmol) and unlabeled egg yolk sphingomyelin served as a substrate. Experimental conditions (buffers, total lipid concentration) for assaying SMase activity were the same as indicated above for the fluorescence method. Radioactivity in a typical assay mixture was 1x10⁶ cpm. SMase activity was determined from the amount of radioactive choline released into the aqueous phase using LCS safety cocktail for liquid scintillation counting.

Determination of acid and neutral SMase-activity in SMC cell extracts
SMCs were incubated with 0.1 ng/ml TNF, native LDL, minimally modified LDL (60 µg Protein/ml each), or 10 µM [C₆]ceramide in six-well plates (9.6 cm²/well) for 15 min to 8 h. Cells were washed with ice-cold PBS, scraped from the multi-well plates, and isolated by centrifugation. Acid lysis buffer (250 mM sodium-acetate, 0.2% Triton X-100, pH 5.0) for analyzing acid SMase-activity, neutral Mg²⁺-containing lysis buffer (20 mM HEPES, 10 mM MgCl₂, 2 mM EDTA, 5 mM DTT, 1 mM NaF, 0.1 mM Na₂MoO₄, 1 mM PMSF, 10 µg/ml Leupeptine, 10 µg/ml Benzamidine, 10 µg/ml Soja-Trypsine inhibitor, 0.2% Triton X-100, pH 7.4) for analyzing neutral Mg²⁺-dependent SMase or neutral lysis buffer (20 mM HEPES, 2 mM EDTA, 5 mM DTT, 1 mM NaF, 0.1 mM Na₂MoO₄, 1 mM PMSF, 10 µg/ml Leupeptine, 10 µg/ml benzamidine, 10 µg/ml Soja-Trypsine inhibitor, 0.2% Triton X-100, pH 7.4) for analyzing neutral Mg²⁺-independent Smase were added (60 µl/9.6 cm² well), and samples were incubated on ice for 1 h (vortexing every 15 minutes). The samples were then subjected to centrifugation at 14,000 g at 4°C for 5 min. The supernatant was isolated and an aliquot containing 20 µg protein (determined by the method of Bradford) (22) was analyzed for acid and neutral SMase-activity, respectively, under the conditions described above. The reaction was stopped by addition of organic solvent. For the determination of acid SMase-activity, an 100 µl aliquot of the organic phase was evaporated under a nitrogen stream. The residue was dissolved in 10 µl CHCl₃ and applied to a TLC silica-gel plate. For TLC analysis of neutral SMase-activity a 300 µl aliquot of the organic phase was exposed to a N₂ stream and the residue was dissolved in 10 µl CHCl₃ for TLC analysis.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Determination of SMase-activity
Sphingomyelinases in intact cells hydrolyze sphingomyelin leading to the formation of ceramide and phosphatidylcholine (23). For determination of SMase-activity in vitro, 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. After the reaction the remaining substrate and the product are extracted by organic solvent and separated by TLC followed by fluorescence analysis using a CCD camera.

View larger version (15K): [in this window] [in a new window]   Fig. 1. Fluorescence spectroscopic assay for the determination of sphingomyelinase (SMase)-activity. Fluorescent N-7-nitrobenz-2-oxa-1,3-diazol (NBD)-C12-sphingomyelin is hydrolyzed by sphingomyelinase, leading to the formation of corresponding fluorescent ceramide.

View larger version (12K): [in this window] [in a new window]   Fig. 2. Activity of acid SMase from human placenta. Effect of substrate concentration. A: Acid SMase from human placenta (1.6 units) was incubated with increasing amounts (0.2 to 9.6 nmol) of NBD-sphingomyelin in acid reaction buffer (250 mM Sodium-Acetate, 1 mM EDTA, pH 5.0) in a total volume of 200 µl. After incubation at 37°C for 1 h the reaction was stopped by addition of 600 µl CHCl3-CH3OH, 2:1 (v/v). Lipids were extracted and an aliquot of the organic lipid phase (100 µl) was evaporated under a N2 stream. The residue was dissolved in 10 µl CHCl3. NBD-ceramide was separated from the remaining substrate by TLC using CHCl3-CH3OH-H2O, 65:25:4 (v/v/v) as a solvent. The remaining substrate [NBD-sphingomyelin (SM)] and the product (NBD-ceramide) appeared as single spots at RF = 0.15 and RF = 0.85, respectively. NBD-ceramide was used as a reference lipid. The fluorescence intensities of the NBD-ceramide spots formed were determined using a CCD camera followed by software-based data processing. The amounts of pmol ceramide were obtained from the respective fluorescence intensities using the calibration plot shown in Fig. 3. B: Plot of pmol NBD-ceramide generated from NBD-SM by acid SMase versus the amount of substrate in the reaction mixture. The indicated values are means ± SD (n 3).

View larger version (15K): [in this window] [in a new window]   Fig. 3. Calibration of NBD-ceramide fluorescence on TLC plates. A: Increasing amounts of fluorescent ceramide were applied onto a TLC plate followed by chromatography using the same solvent as indicated in Fig. 2. Under these conditions NBD-ceramide appeared as a single spot at RF = 0.85. The emission intensities of the fluorescent ceramide spots were measured as described in the legend to Fig. 2. A representative chromatogram is shown. B: Calibration plot of NBD-ceramide concentration versus fluorescence intensity as determined on silica gel plates. Indicated values are means ± SD (n 3).

Figure 4 shows the results of a typical fluorescence analysis when NBD-SM substrate was subjected to various amounts of acid SMase. The amount of the fluorescent reaction product (ceramide, R_F = 0.85) was determined as a measure for enzyme activity, and showed a linear relationship with enzyme concentration. Activities based on the fluorescent substrate were 85% and 82.5% of the activities that were determined using the radiolabeled substrate for aSMase in cell lysates and isolated aSMase from human placenta, respectively (Fig. 5) .

View larger version (14K): [in this window] [in a new window]   Fig. 4. Activity of acid sphingomyelinase (aSMase). Effect of enzyme conzentration. A: Increasing amounts of acid SMase from human placenta (0.05 units to 1.6 units) were incubated with 9.6 nmol NBD-SM in acid reaction buffer (250 mM sodium-acetate, 1 mM EDTA, pH 5.0) in a total volume of 200 µl. Enzyme activities were determined as indicated in the legend to Fig. 2A. B: Plot of pmol NBD-ceramide generated from NBD-SM by acid SMase vs. the amount of enzyme in the reaction mixture. The indicated values are means ± SD. (n 3).

View larger version (47K): [in this window] [in a new window]   Fig. 5. Activity of acid SMase. Comparison of fluorescent and radiolabeled substrate. B: Determination of aSMase activity in smooth muscle cell (SMC) lysates. Fluorescent NBD-sphingomyelin (2 nmol) or a mixture of unlabeled sphingomyelin with radiolabeled [3H]SM (total lipid 2 nmol) in acid reaction buffer (250 mM sodium-acetate, 1 mM EDTA, pH 5.0) were added to lysates of SMC containing 30 µg protein. Lysates were prepared and enzyme activities were determined as described in Materials and Methods. A: Determination of activity of acid SMase from human placenta. Fluorescent NBD-SM or radiolabeled [3H]choline methyl SM in acid reaction buffer were incubated with 1.6 U aSMase. SMase activities were measured as described above. Indicated SMase activities are means ± SD (n 4).

SMase-activity in SMC was determined as pmol ceramide formed/µg protein (Figs. 6–8) . Absolute levels of SMases in untreated cells were 386 pmol/min/µg protein for acid, 39.2 pmol/min/µg protein for neutral Mg²⁺-dependent Smase, and 53 pmol/min/µg for Mg²⁺-independent neutral SMase (Fig. 6).

View larger version (19K): [in this window] [in a new window]   Fig. 6. Activities of acid, neutral Mg2+-dependent, and neutral Mg2+-independent SMase in SMC lysates. SMCs were lyzed in lysis buffer for assaying neutral Mg2+-independent (1), neutral Mg2+-dependent (2), and acid (3) SMases. Enzyme activities were determined as described under Materials and Methods. For TLC, NBD-SM (4) and NBD-ceramide (5) were used as reference lipids. Indicated enzyme activities are means ± SD (n = 3).

View larger version (22K): [in this window] [in a new window]   Fig. 8. Neutral SMase activity in SMC lysates. Effects of LDL, TNF, and ceramide. A: TLC analysis of lipids. Cells were stimulated by TNF (0.1 ng/ml), natLDL (60 µg Protein/ml), mmLDL (60 µg Protein/ml), 10 µM ceramide in DMEM + 0.1% FCS or DMEM + 0.1% FCS only (control) for 1 h followed by cell lysis in neutral Mg2+-containing lysis buffer. Activites were determined as described in Materials and Methods. For TLC, NBD-SM and NBD-ceramide (5 pmol each) were used as reference lipids. B: Neutral SMase activities were determined from the formation of fluorescent ceramide. All indicated values are means ± SD (n 3).

View larger version (22K): [in this window] [in a new window]   Fig. 7. Acid SMase activity in SMC lysates. Effects of LDL, tumor necrosis factor (TNF), and ceramide. A: TLC analysis of lipids. Cells were stimulated by TNF (0.1 ng/ml), native (nat)LDL (60 µg Protein/ml), minimally modified (mm)LDL (60 µg Protein/ml) or 10 µM ceramide in DMEM + 0.1% FCS or DMEM + 0.1% FCS only (control) for 1 h followed by cell lysis in acid lysis buffer. Enzyme activities were determined as described under Materials and Methods. For TLC, NBD-SM and ceramide (5 pmol each) were used as reference lipids. B: Acid SMase activities (pmol/min/µg). SMase activities of stimulated SMCs were determined from the formation of fluorescent ceramide (upper panel). Indicated values are means ± SD (n 3).

Modified LDL at a concentration that is mitogenic for SMC (60 µg/ml of apoB) induced an increase in acid (Fig. 7) and neutral (Fig. 8) SMase-activities, whereas natLDL produced similar but much lower effects. MmLDL (60 µg protein/ml) activated acid SMase (741 pmol/min/µg, 192% of control) nearly as effectively as a stimulation by 0.1 ng/ml TNF (Fig. 7). Neutral SMase in SMC was also activated by mmLDL (Fig. 8)

Stimulation of SMC with 10 µM ceramide for 1 h resulted in an increase of acid SMase-activity by 170% of the control (656 pmol/min/µg, Fig. 8A). On the other hand, neutral SMase remained unaffected in the presence of 10 µM ceramide after 1 h (Fig. 8B). Activation of neutral SMase by 10 µM ceramide was only observed after incubation of SMC for 7–8 h (data not shown).

A more detailed analysis was carried out to study the time-dependence of the activation of acid and neutral SMase by mmLDL and native LDL in SMCs (Figs. 9A, B) .

View larger version (28K): [in this window] [in a new window]   Fig. 9. Time-dependent activation of SMase-activity in SMCs by mmLDL and natLDL. A: Acid SMase. B: Neutral SMase. SMCs were incubated with LDL or mmLDL (60 µg Protein/ml each) or DMEM + 0.1% FCS (control) for different periods of times followed by analysis of enzyme activity as described in Figs. 7 and 8. Indicated activities are means ± SD (n 4).

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Sphingomyelinase is an important enzyme of signal transduction (27). It generates ceramide, which is an important second messenger involved in cell proliferation (28) as well as apoptosis (29, 30, 31). Whether ceramide induces proliferation or apoptosis does not only depend on the cell type but also on the localisation and pH-optimum of the respective SMase isoform (32). Whereas lysosomal acid SMase is likely to play an important role in induction of cell death (22, 33), activation of Mg²⁺-dependent neutral SMase may lead to proliferation (33, 34).

Our assay, which is based on a fluorescent substrate, is reliable since it measures very similar activities as compared with established assays based on radioactive substrates. It is suitable for determination of all isoforms of SMase. It replaces the common radioactive methods (10–14) and avoids obvious expenditures for waste and contamination.

A linear correlation was found between enzyme mass and enzyme-activity between 0.05 and 1 unit, determined under conditions of non-limiting substrate concentrations.

We examined the activities of neutral and acid SMase in stimulated SMC using the new assay and found an increase in SMase activities depending on the isoform of the enzyme and the stimulus. TNF is a cytokine known to be a signal for cells to undergo apoptosis (27). NatLDL represents a growth signal, whereas oxLDL is a pathophysiologic stimulus of proliferation and/or death of vascular cells. All these agents exerted stimulating effects on the activity of aSMase and neutral SMase in SMC, leading to the formation of ceramide, a lipid second messenger. Ceramide interferes with several down-stream targets, which are part of the pathways leading to apoptosis and proliferation, respectively (35).

In contrast to results obtained with other cell types (36), we found an increase of enzyme activities of both SMase isoforms in SMCs with a maximum after 1 h for both isoforms.

MmLDL but not natLDL led to a pronounced activation of acid and neutral SMase already between 30 and 60 min incubation time. Freshly prepared LDL showed much lower effects under these conditions. In addition, a second increase of acid and neutral SMase-activities was reached after several hours of incubation with mmLDL. However, the same long-term effect was obtained with natLDL.

It is striking that acid and neutral SMase are similarly affected by mmLDL after very short time. Thus, it is likely that immediate physical interactions between the lipoproteins (lipids) and the cell surface membrane or forthcoming oxidative modifications are involved in these phenomena. This would be in agreement with the hypothesis that both isoforms are associated with the plasma membrane and thus are subject to modulations by membrane-active components (6, 37). Research is in progress to define the molecular basis for SMase activation by mmLDL lipids.

In this paper we have described the principle and useful applications of the fluorescence SMase-assay for the analysis of SMase activities in vitro and in cells (SMC). The described new method may be useful for determination of SMase activity in various fields of pathobiochemistry, where these enzymes may play a role, e.g., atherosclerosis, cancer, and immunology (4, 35, 38). For this purpose, the described assay for the determination of SMase-activity provides a sensitive, reproducible, and very simple method in the research laboratory and for routine laboratory practice.

	ACKNOWLEDGMENTS

 
The authors are grateful for financial support by Österreichische Nationalbank (project No. 6939), Österreichischer Herzfonds, and Österreichischer Forschungsfonds (FWF project No. P14714-PHA).

Manuscript received November 19, 2001 and in revised form February 8, 2002.

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