Sphingomyelin is hydrolyzed by Bacillus cereus sphingomyelinase and the amount of ceramide thus obtained is determined using E. coli diacylglycerol (DAG) kinase as described in "Analysis of ceramide and sphingomyelin ~Quantitative determination of ceramide using E. coli diacylglycerol kinase". |
Category | Glycolipids and related compounds |
Protocol Name | Quantitative determination of sphingomyelin I |
Authors
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Okino, Nozomu
*
Department of Bioscience and Biotechnology, Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu University
Ito, Makoto
Department of Bioscience and Biotechnology, Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu University
*To whom correspondence should be addressed.
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KeyWords |
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Reagents
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n-octylglucoside (Merck Millipore, Billerica, MA, #494459) |
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Cardiolipin (Sigma-Aldrich, St. Louis, MO, #C0563) |
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Diethylenetriaminepenta acetic acid (DETAPAC) (Sigma-Aldrich, #D1133) |
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E. coli DGK (Merck Millipore, #266726 or 266724) |
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Sphingomyelinase from Bacillus cereus (Sigma-Aldrich, #9396) |
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TLC plate (Silicagel 60, 20 × 20 cm: Merck Millipore) |
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Instruments
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Speed Vac concentrator (Thermo Fisher Scientific Inc., Waltham, MA) |
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Imaging analyzer (FLA5000, Fujifilm, Tokyo, Japan) |
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Methods |
1. |
Quantitative determination of sphingomyelin [I]
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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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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 × 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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This work is released underCreative Commons licenses
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Date of registration:2014-07-31 10:00:40 |
- He, X., Chen, F., Gatt, S., and Schuchman, E.H. (2001) An enzymatic assay for quantifying sphingomyelin in tissues and plasma from humans and mice with Niemann-Pick disease. Anal. Biochem. 293, 204–211 [PMID : 11399033]
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Okino, Nozomu,
Ito, Makoto,
(2014). GlycoPOD https://jcggdb.jp/GlycoPOD.
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Ito, Makoto,
(2014).
Quantitative determination of sphingomyelin I.
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Ito, Makoto,
(2014).
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