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Sphingomyelinase

Manufactured by Merck Group
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Sphingomyelinase is a laboratory product that catalyzes the hydrolysis of sphingomyelin, a type of phospholipid, into ceramide and phosphocholine. It is an important enzyme in the metabolism of sphingolipids, which are essential components of cell membranes.

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Lab products found in correlation

Ephrin a5 fc, by Merck Group (1 mentions) Epha3 fc, by Merck Group (1 mentions) Methyl β cyclodextrin, by Merck Group (1 mentions) Pitstop 2, by Abcam (1 mentions) Choline oxidase, by Merck Group (1 mentions) Amplex red, by Thermo Fisher Scientific (1 mentions) Alkaline phosphatase, by Merck Group (1 mentions) Horseradish peroxidase, by Merck Group (1 mentions)

4 protocols using sphingomyelinase

1

Ephrin-A5 and EphA3 Receptor Signaling

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The medium on explant cultures was replaced with warm F12-MC containing recombinant human ephrin-A5-Fc, mouse EphA3-Fc (both RnD Systems, Minneapolis, MN, USA) or human Fc fragment (Calbiochem, San Diego, CA, USA). For inhibitor experiments, 30 µM Pitstop2 (Abcam, Cambridge, UK) or 400 mU/ml sphingomyelinase (Sigma-Aldrich, St. Louis, MO, USA) in F12-MC was applied during a 15 or 30 min pre-incubation, respectively, and thereafter together with ephrin-A5-Fc, EphA3-Fc or Fc. Methyl-β-cyclodextrin (2 mg/ml; Sigma-Aldrich, St. Louis, MO, USA) was administered together with ephrin-A5-Fc, EphA3-Fc or Fc. After incubation for 20 or 120 min, cultures were fixed and stained with Alexa488 or Alexa568-phalloidin and the percentage of collapsed GCs was counted.
Fiederling F., Weschenfelder M., Fritz M., von Philipsborn A., Bastmeyer M, & Weth F. (2017). Ephrin-A/EphA specific co-adaptation as a novel mechanism in topographic axon guidance. eLife, 6, e25533.
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2

Sphingomyelin Quantification Assay

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Samples were incubated in a total volume of 80–110 μl containing 50 mM Tris-HCl, pH 8.0, 0.66 mM CaCl2, 1.2 U/ml choline oxidase (Sigma-Aldrich, St. Louis, Missouri), 25 U/ml horse radish peroxidase (Sigma-Aldrich), 0.6 U/ml sphingomyelinase (Sigma-Aldrich), 12 U/ml alkaline phosphatase (Sigma-Aldrich), and 50 μM Amplex Red (Molecular Probes). Dilutions of sphingomyelin (Biomol International - Enzo Life Sciences, Inc., Farmingdale, New York; dissolved in 2% TritonX-100 in ethanol) were used as standards. Negative control samples, which lack choline oxidase, sphingomyelinase, and alkaline phosphatase, were used in all assays. After the samples were incubated on a 96-well assay plate, they were read in a microplate reader using excitation and emission at 540±10 and 590±10 nm respectively. The assay was adapted from a previously described method using Amplex Red (Hojjati & Jiang, 2006 (link)).
Kao H.T., Ryoo K., Lin A., Janoschka S.R., Augustine G.J, & Porton B. (2017). Synapsins regulate BDNF-mediated synaptic potentiation and axon elongation by acting on membrane rafts. The European journal of neuroscience, 45(8), 1085-1101.
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3

LDL Aggregation by Sphingomyelinase

Cited 1 time
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LDL was aggregated by incubating native LDL (2 mg protein/mL) with sphingomyelinase (Bacillus cereus, catalogue number S9396-25UN, Sigma) at 10 mU/mL (Walters and Wrenn, 2010 (link)) until the attenuance (absorbance plus light scattering) at 680 nm increased from about 0.0017–0.027. sphingomyelinase-aggregated LDL (SMase-LDL) was dialysed against a phosphate buffer containing EDTA and sterilised by membrane filtration (0.45 μm).
Ahmad F, & Leake D.S. (2018). Antioxidants inhibit low density lipoprotein oxidation less at lysosomal pH: A possible explanation as to why the clinical trials of antioxidants might have failed. Chemistry and Physics of Lipids, 213, 13-24.
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4

Time-Lapse TIRF Imaging with Drug Stimulation

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For all time‐lapse TIRF imaging experiments with drug stimulation, drugs were added to the cells 5 min after the initiation of the imaging except for methyl‐β‐cyclodextrin (MCD) treatment, where 5 mM MCD (Sigma‐Aldrich/Merck) was added to the cells as indicated in Fig EV6B. Other drugs were used with the following concentration: 200 μM cholesterol/MCD complex generated as described previously (Brown et al, 2002); 100 mU/ml sphingomyelinase (SMase) (Sigma‐Aldrich/Merck). For ECFP‐D4H binding assay, cells were pre‐incubated with 3 μM purified ECFP‐D4H proteins for 30 min at 37°C before imaging; cells were maintained in the presence of 3 μM purified ECFP‐D4H proteins throughout the imaging.
Ercan B., Naito T., Koh D.H., Dharmawan D, & Saheki Y. (2021). Molecular basis of accessible plasma membrane cholesterol recognition by the GRAM domain of GRAMD1b. The EMBO Journal, 40(6), e106524.
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