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Quantitative determination of sphingomyelin I

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Introduction Protocol References Credit lines
Category
Glycolipids and related compounds
Protocol Name

Quantitative determination of sphingomyelin I

Authors
 Nozomu Okino *
Department of Bioscience and Biotechnology, Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu University

Makoto Ito
Department of Bioscience and Biotechnology, Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu University
*To whom correspondence should be addressed.
Keywords

Sphingomyelin

Sphingomyelinase

Diacylglycerol kinase (DGK)

Reagents
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n-octylglucoside (MERCK, #494459)

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Cardiolipin (Sigma-Aldrich, #C0563)

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Diethylenetriaminepenta acetic acid (DETAPAC) (Sigma-Aldrich, #D1133)

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E. coli DGK (Merck, #266726 or 266724)

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Sphingomyelinase from Bacillus cereus (Sigma-Aldrich, #9396)

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TLC plate (Silicagel 60, 20 x 20 cm, Merck)

Instruments
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Speed Vac concentrator (Savant)
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Imaging analyzer (FLA5000, Fujifilm)
Methods
1.

Quantitative determination of sphingomyelin [I]

1) 

 Dissolve the lipids in 10 μl of 7.5% n-octyl-β-D-glucopyranoside, 5 mM cardiolipin, and 1 mM DETAPAC.

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2) 

 Mix with 30 μl of 0.1 M imidazole–HCl, pH 6.6, containing 0.1 M NaCl, 25 mM MgCl2, 4 mM DTT, and 2 mM EGTA.

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3) 

 Add 5 μl of enzyme (5 mU of E. coli DAG kinase and 100 mU of B. cereus sphingomyelinase) in 10 mM imidazole–HCl, pH 6.6, containing 1 mM DETAPAC, 1 mM DTT, and 10% glycerol.

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4) 

 Initiate the reaction by adding 5 μl of 5 mM “cold” ATP and 0.1 μCi [γ-32P]ATP in the buffer described in 3) (the final 50-μl reaction mixture contains 0.5 mM ATP and 0.01 μCi [γ-32P]ATP).

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5) 

 Incubate at 25°C for 1 h.

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6) 

 Add 0.6 ml of CHCl3/MeOH (1/1, v/v).

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7) 

 Vortex for a few seconds and add 250 μl of 1 M KCl in 20 mM Mops, pH 7.0.

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8) 

 Centrifuge at 10,000 x g for 5 min.

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9) 

 Transfer the organic phase to an Eppendorf tube and dry.

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10) 

 Dissolve the sample in an aliquot of CHCl3/ MeOH (1/1, v/v) and spot on a TLC plate.

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11) 

 Develop the plate with CHCl3/acetone/MeOH/acetic acid/water (10/4/3/2/1, v/v/v/v/v).

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12) 

 Dry the TLC plate and cover with a wrap.

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13) 

 Expose the TLC plate to an imaging plate.

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14) 

 Determine the radioactivity of the bands corresponding to [32P]Cer-1-P with an imaging analyzer.

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Discussion

To determine the sphingomyelin content of the samples, the amount of endogenous ceramide, determined in a control experiment in which the reaction is conducted without Bacillus sphingomyelinase, should be subtracted. With this procedure, 10 – 1000 pmol of sphingomyelin can be determined.

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Date of registration：2012-11-13 14:33:03
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