This material was originally published in the Purdue Cytometry CD-ROM Series,volume 4

Sphingomyelinases
Luisa Di Marzio and M. Grazia Cifone
Department of Experimental Medicine,
University of L’Aquila, Coppito 2, 67100 L’Aquila, Italy.

1. Introduction


 

Sphingomyelinase (sphingomyelin choline phosphohydrolase) (SMase) catalyzes the hydrolytic cleavage of sphimgomyelin (SM) via reaction which lead to ceramide and phosphocholine generation (1). It has been shown that SM may be cleaved at both acid pH optima and neutral pH optima. Several different forms of mammalian SMases have been identified, including:

· a lysosomal SMase present in all tissues, acting optimally at low pH, and showing no dependence on divalent cations (2, 3);

· an acidic, Zn2+-stimulated SMase present in fetal bovine serum and to a lesser degree in newborn human serum (4), and recently reported to be secreted by several human cell types during several pathophysiological processes (5);

· a neutral, membrane-associated, Mg2+-stimulated SMase that is found predominantly in brain and kidney (6), and that is known to arise from a separate gene from lysosomal Smase (7);

· a cytosolic SMase that, like Mg2+-dependent neutral SMase, has a neutral pH optimum but no dependence on divalent cations (8).

 
The SMase activity is generally not difficult to measure, and a variety of methods are available. It is possible to assay the SMase activity in vivo through labeling the cells with a radioactive precursor of SM and then determining the label product levels. Here (see point 2A), a method based on the cellular SM labeling with radioactive methyl-choline is described since consensus exists that cellular biosynthesis of SM is mostly catalyzed by PC:ceramide phosphocholine transferase (SM synthase) which directly transfers the phosphocholine group from PC to ceramide (9).

 

The activity of SMase can be also determined in vitro either using radiolabelled SM, or a chromogenic analog of SM or colored and fluorescent derivatives of natural SM. The latter derivatives have been used for developing spectrophotometric or fluorometric assays of SMase. The methods to assay SMase activity in vitro are usually based on the release of the radioactive polar head group such as phosphocholine into a water phase of a lipid extraction. Others rely on the measurement of the lipid-soluble product of the SMase, ceramide, in the organic phase of suitable lipid extraction. Here (see point 2B), a radioactive method to assay in vitro acidic and neutral SMase activity is described.

 

 2A Protocol for in vivo SMase assay

 

2.1. Materials
 

 

2.2. Methodology

1. Cells (3-5x105/ml) in medium with 2-5% serum are labelled for 48-72 hrs with methyl-3H-choline (final specific activity 0.5 mCi/ml) in order to label SM pools.

2. Cells are washed three times with Ca-Mg free PBS.

3. Cells are then starved for 4 hrs in medium supplemented with 2% BSA.

4. Cells are washed again with PBS and resuspended in serum-free medium (5-10x106 cells/ml). It is important to verify if a good incorporation of radioactive precursor occurred in the analyzed cell system. Thus a little aliquot of cell suspension should be counted by liquid scintillation counting.

5. Cells are then treated for different times according to the experimental protocol of the researcher.

6. Incubation is stopped by immersion of the samples in methanol/dry ice (-70°C) for 10 sec.

7. Samples are centrifuged at 4°C in a microfuge at 1,200 rpm for 10 min.

8. Cell pellets are resuspended in ice-cold TRIS-HCl 20 mM pH 7.4 and lysed by sonication with a cell sonifier (1.5 min, 10 sec on and 10 sec off, 5W, and 80% output).

9. Protein concentrations are determined using a Pierce Protein Assay.

10. Lipids are extracted at 4°C as follows:

· Addition of 400 ml methanol
· Vortex-mix for 30 sec
· Addition of 500 ml chloroform
· Vortex-mix for 30 sec
· Centrifugation at 1000 X g for 15 min at 4°C
· The acqueous phase (upper) can be extracted 2-3 times again with 500 ml chloroform
· The organic phases (lower) obtained in the different extraction steps are collected and washed once with a synthetic upper phase consisting of 1 ml chloroform/methanol/water (3:48:47, v/v) to remove free radioactive choline
· The organic phases are dried under nitrogen, resuspended in 200 ml chloroform and applied to a silica gel TLC plate preferentially with an automatic applicator under N2. Samples corresponding to equal amounts of proteins (50-100 mg proteins) are loaded.

 

 11. Lipids are separated by TLC using a solvent system containing chloroform:methanol:acetic acid: water (100:60:20:5, v/v). The labeled compounds are lyso-PC, SM, and PC. Thus, unlabeled lyso-PC, SM, and PC are used as standards and visualized in iodine vapour (Rf= 0.1, 0.26, 0.6, respectively). The radioactive spots are visualized by autoradiography, scraped from the plate and counted by liquid scintillation. The plates are developed overnight with Kodak X-Omat AR film to identify the bands in comparison with autentic standards. When methyl 3H-choline is used, the plates are previously sprayed with EN3HANCE and then developed as above.

12. Radioactivity measurements are converted to pmol product by using the specific activity of substrate. SMase activity is expressed as pmol SM hydrolysed/mg protein.
 

 2B. Protocol for in vitro acidic and neutral SMase assays.

 

2.1. Materials
 

 

2.2A. Methodology for acidic SMase assay (10)

 

1. After treatment, the cells (5-10x106) are placed on ice, washed twice with ice-cold PBS and pelletted in microfuge tubes at 4°C for 10 min at 3000 rpm.

2. The cellular pellet is resuspended in 300 ml of 0.2% Triton X-100.

3. The cells are incubated at 4°C for 15 min.

4. The cells are disrupted by brief sonication (1.5 min, 10 sec on and 10 sec off, 5W, and 80% output) and spun in a microfuge at 14,000 rpm at 4°C for 10 min.

5. Protein content in supernatant containing the cytosolic and microsomal fractions is measured with a Pierce Protein Assay with bovine serum albumine as standard.

6. 50-60 mg of proteins/50 ml are incubated for 0.5-2 hrs at 37°C in the assay buffer (110 ml) containing 250 mM sodium acetate, 1 mM EDTA, pH 5.0. The reaction is initiated by the addition of 40 ml of substrate (50-100 pmol 3H-sphingomyelin or 50-100 pmol 14C-sphingomyelin) in the assay buffer containing 3% Triton X-100, solubilized by sonication (6 min, 1 min on and 1 min off, 6W, 80% output) and vortexing. The final volume of the assay system is 200 ml.

7. The reaction is stopped by the addition of 250 ml of chloroform:methanol (2:1, v/v). The samples are pulse-vortexed three times for 30 sec.

· Radioactive phosphorylcholine is then extracted as follows:
· Add 800 ml of chloroform:methanol (2:1, v/v) and 250 ml of H2O;
· Vortex-mix for 30 sec;
· Centrifugation at 1000 X g for 15 min at 4°C
· The acqueous phase (upper) can be extracted 2-3 times again with 500 ml chloroform 8. The organic phases (lower) obtained in the different extraction steps are collected and washed once with a synthetic upper phase consisting of 1 ml chloroform/methanol/water (3:48:47, v/v) to remove free radioactive phosphorylcholine

9. the acqueus phases (upper) are collected and transferred to scintillation vials and routinely counted by liquid scintillation counting.

10. The organic phase (lower) (containing the SM) is evaporated to dryness under nitrogen, dissolved in a smaller volume (20-40 ml) of chloroform/methanol (2:1, v/v) and also counted for radioactivity. Increments of radioactive phosphorylcholine in the acqueus phase should be correspond to decrements of radioactive SM in the organic phase.

11. An aliquot of organic phase can also be analysed in TLC. Samples are run on a TLC plate in chloroform:methanol:ammonia hydroxide (7 N):water (85:15:0.5:0.5, v/v)

12. Plates are then developed overnight with KODAK X-Omat AR film for autoradiography to identify the band of SM (only a radioactive band corresponding to SM should be present). Band can be scraped for liquid scintillation counting.

13. Radioactivity measurements are converted to pmol product by using the specific activity of substrate. SMase activation is expressed as pmol SM hydrolysed/mg protein/min or pmol phosphorylcholine produced/mg protein/min.

 
 

 2.2B. Methodology for in vitro neutral SMase assays (10).

Neutral SMase is measured as above described for acidic SMase except that:

 

1. The cells are resuspended and sonicated in Hepes 20 mM, pH 7.4, 10 mM, MgCl2, 2 mM EDTA, 5 mM DTT, 0.1 mM Na3VO4, 0.1 mM Na2MoO4, 30 mM p-nitrophenylphosphate, 10 mM b-glycerophosphate, 750 mM ATP, 1 mM PMSF, 10 mM leupeptin, 10 mM pepstatin A and 0.2% Triton X-100.

2. The assay buffer is Hepes 20 mM, pH 7.4, 1 mM MgCl2.
 

 3. Commentary

 

3.1. Background Information

 

The described methods have the objective to assay SMase activity in vivo and in vitro. Both can be used to assay SMase activity in the cells and both are generally used to demonstrate an activation of these enzymes in a particular experimental condition or after a treatment with exogenous agents. In tissue homogenates, only the in vitro method is available. In the cellular systems, the metabolic labeling of SM could be useful mainly for kinetics studies. To discriminate between neutral SMase and acidic SMase, it is suggested the treatment of the cells with agents like monensin and ammonium chloride that raise the pH in endolysosomal compartments. These agents have been reported to inhibit the activation of acidic SMase, leaving the neutral SMase unaffected (10). On the other hand, the in vitro assay allows to more specifically discriminate between the acidic and neutral SMases. Indeed:

· the enzymes have different optimal pH;
· the neutral SMase is Mg2+-dependent, whereas acidic SMase is Mg2+-independent;
· the presence of ATP and b-glycerophosphate in the neutral SMase assay completely block the residual activity of acidic SMase at pH 7.4;
· the absence of protease and phosphatase inhibitors in the acidic SMase assay permits to exclude the residual neutral SMase at low pH since the latter is proved rather sensitive to proteases and phosphatases.

 

 To discriminate the activity of Mg2+-dependent and Mg2+-independent neutral SMase, it is sufficient to perform the test with the neutral SMase assay buffer described in paragraph 2.2B. with or without MgCl2, respectively (8).

 

3.2 Troubleshooting

The most critical steps in both experimental procedures can be the follows:

· the extraction procedure, which should be extremely accurate
· the scintillation counting of the organic phase (amounts of chloroform higher that 60 ml has quenching effects).
  3.3 Key References

 

1. Brady, R.O., Kanfer, J.N., Moek, M.B., and Fredrickson, D.S. 1966. The metabolism of sphingomyelin. II. Evidence of an enzymatic deficiency in Niemann-Pick disease. Proc. Natl. Acad. Sci. USA 55:366.

2. Kanfer, J.N., Young, O., Shapiro, D., and Brady, R.O. 1966. The metabolism of sphingomyelin. I. Purification and properties of a sphingomyelin-cleaving enzyme from rat liver tissue. J. Biol. Chem. 241:1081.

3. Levade, T., Salvayre, R., and Blazy-Douste, L. 1986. Sphingomyelinases and Niemann-Pick disease. J. Clin. Chem. Biochem. 24:205-220.

4. Spence, M.W., Byers, D.M., Palmer, F.B.St. C., and Cook, H.W. 1989. A new Zn2+-stimulated sphingomyelinase in fetal bovine serum. J. Biol. Chem. 264:5358-5363.

5. Schissel, S.L., Shuchman, E.H., Williams, K.J., and Tabas, I. 1996. Zn2+-stimulated sphingomyelinase is secreted by many cell types and is a product of the acid sphingomyelinase gene. J. Biol. Chem. 271:18431-18436.

6. Spence, M.W. 1993. Sphingomyelinases. Adv. Lipid Res. 26:3-23.

7. Gatt, S., Dinur, T., and Kopolovic, J. 1978. Niemann Pick disease: presence of the magnesium-dependent sphingomyelinase in brain of the infantile form of the disease. J. Neurochem. 31:547-550.

8. Okazaki, T., Bielawska, A., Domae, N., Bell, R.M., and Hannun, Y.A. 1994. Characteristics and partial purification of a novel cytosolic magnesium-independent, neutral sphingomyelinase activated in the early signal transduction of 1a,25-dihydroxyvitamin D3-induced HL-60 cell differentiation. J. Biol. Chem. 269:4070-4077.

9. Andrieu, N., Salvayre, R., and Levade, T. 1996. Comparative study of the metabolic pools of sphingomyelin and phosphatidylcholine sensitive to tumor necrosis factor. Eur. J. Biochem. 236:738-745.

10. Wiegman, K., Schutze, S., Machleidt, T., Witte, D., and Kronke, M. 1994. Functional dichotomy of neutral and acidic sphingomyelinases in tumor necrosis factor signaling. Cell 78:1005-1015.

 

 Appendix 1: Stock solutions

Solution Preparation Storage
 

 

Appendix 2: Equipment