A fluorescent assay for ceramide synthase activity

Kim, Hyun Joon; Qiao, Qiao; Toop, Hamish D.; Morris, Jonathan C.; Don, Anthony S.

doi:10.1194/jlr.d025627

Cited by 32 publications

(22 citation statements)

References 22 publications

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“…1 ) has been used previously to assay CerS ( 17,18,21 ), no attempts have been made to minimize reaction volumes to allow the use of small amounts of biological material or to improve the mode of separating NBD-lipid substrates and products. We have now been able to decrease the reaction volume to a minimum of 20 µl, rather than the 100-250 µl used previously with [4, H]Sph ( 22 ) or with NBD-Sph ( 21 ), permitting the use of small amounts of protein for the assay. Thus, we could assay CerS5 activity using as little as 1 µg of protein and 5 min reaction time, and CerS4 activity using 10 ( Fig.…”

Section: Optimizing Conditions For Use Of Nbd-sph In the Cers Assaymentioning

confidence: 99%

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A rapid ceramide synthase activity using NBD-sphinganine and solid phase extraction

Tidhar

Sims

Rosenfeld-Gur

et al. 2015

Journal of Lipid Research

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For many years, CerS activity was assayed using radioactive substrates [either 3 H-Sph ( 7 ) or 14 C-labeled acyl-CoAs ( 20 )], with separation of the substrate and products performed by TLC. Recently, a fl uorescent Sph analog has become available, (7-nitro-2-1,3-benzoxadiazol-4-yl) (2 S ,3 R )-2-aminooctadecane-1,3-diol (NBD-Sph), which is a substrate for CerS ( 18,21 ). Clearly, the use of fl uorescent substrates is preferable to use of radioactive substrates as it avoids radiation hazards ( 21 ).We now describe an assay using NBD-Sph, in which the fl uorescent lipid products are separated by solid phase extraction (SPE) C18 chromatography using a 96-well plate. This assay has a number of advantages over other assays, including short assay times, the use of small reaction volumes (20 µl) and small amounts of protein. It alleviates the use of TLC for lipid separation, which often results in degradation of Sph on the TLC plate. We suggest that this assay will permit the rapid assay of CerS activity in large numbers of samples and will prove particularly useful for laboratories that do not have access to facilities available in more lipid-oriented laboratories. MATERIALS AND METHODS MaterialsMethanol gradient grade for liquid chromatography, water for chromatography, and silica gel 60 TLC plates were from Merck (Darmstadt, Germany). Formic acid (purity >98%) was from Sigma-Aldrich (St. Louis, MO). Chloroform for spectrophotometry was from J. T. Baker (Center Valley, PA). Ammonium acetate was from Macron Chemicals (Center Valley, PA). Strata end-capped silica-based C18-E (15 mg/well) 96-well plates and the vacuum manifold for SPE with vacuum gauge and cover mat were from Phenomenex (Torrance, CA). The vacuum pump was an Alcatel 2002 from Ideal Vacuum Products (Albuquerque, NM).Abstract Ceramides are synthesized by six mammalian ceramide synthases (CerSs), each of which uses fatty acylCoAs of different chain lengths for N -acylation of the sphingoid long-chain base. We now describe a rapid and reliable CerS assay that uses a fl uorescent N-[6-[(7-nitrobenzo-2-oxa-1,3-diazol-4-yl) (NBD ) sphinganine substrate followed by separation of the NBD-lipid substrate and products using solid phase extraction (SPE) C18 chromatography. SPE chromatography is a quick and reliable alternative to TLC, and moreover, there is no degradation of either NBD-sphinganine or NBD-ceramide. We have optimized the assay for use with minimal amounts of protein in a minimal volume. This assay will prove useful for the analysis of CerS activity, which is of particular importance in light of the growing involvement of CerS in cell regulation and in the pathology of human diseases. ( 1 ), is synthesized by the N -acylation of sphingoid long-chain bases by ceramide synthases (CerSs). Mammals contain six distinct CerSs, with each using a subset of fatty acylCoAs for the N -acylation of sphinganine (Sph) to generate dihydroceramides or of sphingosine to generate ceramides ( 2, 3 ). CerSs have become the focus of great interest due to the realizatio...

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Section: Optimizing Conditions For Use Of Nbd-sph In the Cers Assaymentioning

confidence: 99%

“…Recently, a fl uorescent Sph analog has become available, (7-nitro-2-1,3-benzoxadiazol-4-yl) (2 S ,3 R )-2-aminooctadecane-1,3-diol (NBD-Sph), which is a substrate for CerS ( 18,21 ). Clearly, the use of fl uorescent substrates is preferable to use of radioactive substrates as it avoids radiation hazards ( 21 ).…”

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confidence: 99%

A rapid ceramide synthase activity using NBD-sphinganine and solid phase extraction

Tidhar

Sims

Rosenfeld-Gur

et al. 2015

Journal of Lipid Research

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“…These isotope-based assays were then replaced by assays using fluorescently labeled lipids. Fluorescently labeled LCBs or fluorescently labeled ceramide have been employed to measure ceramide and IPC synthase activity in vitro (34)(35)(36). More recently, the development of lipid mass spectrometry has allowed the use of odd-chain lipids as tracers for in vitro assays (37,38).…”

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confidence: 99%

Following the flux of long-chain bases through the sphingolipid pathway in vivo using mass spectrometry

Martínez-Montañés

Schneiter

2016

Journal of Lipid Research

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This article is available online at http://www.jlr.org sphingolipids, and plants can make of three classes of sphingolipids (3-6).Sphingolipids and their biosynthetic precursors, longchain base (LCB) and ceramide, have structural as well as signal functions; their synthesis and turnover thus must be precisely controlled (7). The rate-limiting step of the pathway is under control of serine palmitoyltransferase (SPT), an endoplasmic reticulum (ER)-localized protein complex that catalyzes the condensation of palmitoyl-CoA with serine to yield keto-dihydrosphingosine, a LCB (8). SPT activity is negatively regulated by the Orm proteins, conserved integral ER membrane proteins that form a protein complex together with the phosphoinositide phosphatase, Sac1 (5, 9, 10). Orm proteins are subject to phosphorylation and this relieves their inhibition of SPT activity (9). The kinases that phosphorylate the Orm proteins integrate multiple signals to maintain sphingolipid homeostasis, including heat, ER stress, and nutritional availability via the target of rapamycin (TOR) signaling network (11)(12)(13)(14)(15).After reduction of the 3-keto-dihydrosphingosine to dihydrosphingosine (DHS) and its further hydroxylation to phytosphingosine (PHS), DHS or PHS are converted to ceramide by ceramide synthase (CerS), which catalyzes the formation of an amide bond between the LCB and a C26 very long-chain fatty acid (16,17). CerS activity is regulated by direct phosphorylation of the catalytic subunits, Lac1 and Lag1, through TORC1, TORC2, and casein kinase 2 (see Fig. 1 for an overview of the pathway and the nomenclature of the intermediates) (15,(18)(19)(20).Ceramide is then converted in the Golgi to inositol phosphorylceramide ( Sphingolipids are greatly enriched in the plasma membrane of eukaryotic cells and they have been implicated in the formation of lateral membrane domains important for the spatial segregations of proteins during their transport to the plasma membrane (1, 2). Whereas animal cells produce sphingolipids in which the ceramide anchor contains a hydrophilic head group composed of choline or carbohydrate moieties, fungi synthesize inositol-containing

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“…29. The fluorescence of the formed ceramides was detected using a Decon Science Tec documentation system (Hohengandern, Germany), equipped with a Q-Imaging Rolera MGi Plus EMCCD camera (Surrey, Canada).…”

Section: Methodsmentioning

confidence: 99%

Inactivation of Ceramide Synthase 6 in Mice Results in an Altered Sphingolipid Metabolism and Behavioral Abnormalities

Ebel¹,

Dorp²,

Petrasch‐Parwez³

et al. 2013

Journal of Biological Chemistry

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Background: Ceramide synthases N-acylate (dihydro-)sphingosine to (dihydro-)ceramide in mammals. Results: Enzymatically inactive ceramide synthase 6 in mice (CerS6KO) results in an altered sphingolipid metabolism and behavioral abnormalities. Conclusion: Catalytically active CerS6 is necessary to maintain sphingolipid homeostasis in mice. Significance: The CerS6KO mouse reveals for the first time the metabolic and physiological consequences of CerS6 inactivation.

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A fluorescent assay for ceramide synthase activity

Cited by 32 publications

References 22 publications

A rapid ceramide synthase activity using NBD-sphinganine and solid phase extraction

A rapid ceramide synthase activity using NBD-sphinganine and solid phase extraction

Following the flux of long-chain bases through the sphingolipid pathway in vivo using mass spectrometry

Inactivation of Ceramide Synthase 6 in Mice Results in an Altered Sphingolipid Metabolism and Behavioral Abnormalities

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