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Mar 2019

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Preparation of HeLa Total Membranes and Assay of Lipid-inhibition of Serine Palmitoyltransferase Activity
HeLa全膜的制备及其对丝氨酸-棕榈酰转移酶活性的抑制作用   

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Abstract

Serine palmitoyltranferase (SPT) is a pyridoxal 5′ phosphate (PLP)-dependent enzyme that catalyzes the first and rate-limiting step of de novo synthesis of sphingolipids. SPT activity is homeostatically regulated in response to increased levels of sphingolipids. This homeostatic regulation of SPT is mediated through small ER membrane proteins termed the ORMDLs. Here we describe a procedure to assay ORMDL dependent lipid inhibition of SPT activity. The assay of SPT activity using radiolabeled L-serine was developed from the procedure established by the Hornemann laboratory. The activity of SPT can also be measured using deuterated L-serine but it requires mass spectrometry, which consumes money, time and instrumentation. The ORMDL dependent lipid inhibition of SPT activity can be studied in both cells and in a cell free system. This assay procedure is applicable to any type of mammalian cell. Here we provide the detailed protocol to measure SPT activity in the presence of either short chain (C8-ceramide) or long chain ceramide (C24-ceramide). One of the greatest advantages of this protocol is the ability to test insoluble long chain ceramides. We accomplished this by generating long chain ceramide through endogenous ceramide synthase by providing exogenous sphingosine and 24:1 acyl CoA in HeLa cell membranes. This SPT assay procedure is simple and easy to perform and does not require sophisticated instruments.

Keywords: Serine palmitoyltranferase (丝氨酸棕榈酰转移酶), Ceramide synthases (神经酰胺合酶), Sphingolipids (鞘脂), ORMDL (ORMDL), Ceramide (神经酰胺), Endoplasmic reticulum (内质网), Homeostasis (稳态)

Background

Serine palmitoyltranferase (SPT) is a multi-subunit enzyme that is widely expressed in eukaryotes and some prokaryotes (Hanada et al., 1997; Ikushiro et al., 2001; Hornemann et al., 2007). The first and rate-determining step of the sphingolipid biosynthetic pathway is catalyzed by SPT, producing 3-keto dihydrosphingosine from the condensation of serine and palmitoyl-CoA (Williams et al., 1984; Hanada, 2003). The mammalian SPT complex is composed of two large subunits and one small subunit. The large subunits are termed SPTLC1 and SPTLC2 and the small subunits are termed ssSPT (Nagiec et al., 1994; Lowther et al., 2012). SPT requires all three subunits to form a functional protein complex (Han et al., 2009). In some circumstances mammalian SPT is also composed of another large subunit termed SPTLC3 (Hornemann et al., 2009), which substitutes for SPTLC2. The functional SPT complex is formed by pairing SPTLC1 with either SPTCL2 or SPTLC3 and ssSPT. The active site of SPT resides in the SPTLC2 or SPTLC3 subunit. The substrate preference between the complex incorporating SPTLC2 and SPTLC3 differs. SPTLC2 prefers palmitoyl CoA. SPTLC3 prefers myristoyl-CoA (Hornemann et al., 2009). In certain situations SPT also incorporates alanine or glycine rather than serine into the precursor sphingolipid to generate a deoxysphingolipid base (Gable et al., 2010; Ferreira et al., 2018). SPT forms a stable complex with another ER resident protein termed ORMDL. This SPT-ORMDL interaction is crucial to maintain the homeostatic regulation of de novo biosynthesis of sphingolipids (Siow and Wattenberg, 2012; Davis et al., 2018). There are three closely related ORMDL family members found in mammals (Breslow et al., 2010; Siow and Wattenberg, 2012). We previously showed that increased levels of cellular ceramide inhibit SPT activity in an ORMDL-dependent manner in HeLa cells (Davis et al., 2019). The detailed mechanism behind the homeostatic regulation of SPT activity by the ORMDLs is largely unknown. SPT forms a stable complex with ORMDL, irrespective of cellular levels of ceramide. We propose that ceramides bind to either the ORMDLs or to the SPT-ORMDL complex to trigger the inhibition of SPT activity. We recently developed a cell free system in which membranes were isolated from HeLa cells and used to test the response of SPT activity to elevated sphingolipid. This system measures SPT activity in the presence or absence of ceramides with artificially short and natural chain lengths (Davis et al., 2019). We established the assay of ORMDL dependent lipid inhibition of SPT activity as a platform to test the functional interaction of SPT and ORMDL. This SPT assay procedure was developed from the procedure of Rutti et al. (2009). The cell free in vitro system has many advantageous over in vivo systems and both systems yield similar results (Davis et al., 2018). Using this cell free reconstitution system, we have determined that ORMDL dependent regulation of SPT activity is not mediated by post transcriptional or post translational mechanisms (Davis et al., 2018). This cell free system is a powerful tool to explore the detailed mechanism behind the homeostatic regulation of SPT by ORMDLs.

Materials and Reagents

  1. Vacuum filtration unit (0.2 μm) (VWR, catalog number: 10040-436 )
  2. T150 flask (Cyto One, catalog number: CC7682-4815 )
  3. 24-well plate (Cyto One, catalog number: CC7682-7524 )
  4. 26 gauge needle (BD, catalog number: 305110-269 )
  5. 1 ml syringe (BD, catalog number: 309659 )
  6. Thickwall Polycarbonate Tubes for Ultracentrifuge Rotors (Beckman Coulter, catalog number: 343776 )
  7. Screw cap tubes-2 ml (Axygen, catalog number: SCT-200-C-S )
  8. Scintillation tubes (PerkinElmer, catalog number: 6000292 )
  9. Disposable cell lifters-sterile (Fisher Scientific, catalog number: 08-100-240 )
  10. HeLa cells (ATCC, catalog number: CCL-2 )
  11. Dulbecco's minimal essential medium (DMEM Media) (Gibco, catalog number: 11960-044 )
  12. Fetal Bovine Serum (Gemini, catalog number: 900-108 )
  13. HEPES (Fisher Scientific, catalog number: BP310-1 )
  14. Penicillin-streptomycin, 10,000 U/ml (Gibco, catalog number: 15140-122 )
  15. Glutamine (Gibco, catalog number: 25030-081 )
  16. Trypsin-EDTA (Gibco, catalog number: 25200-056 )
  17. Collagen (Sigma, catalog number: C9791 )
  18. Sucrose (Fisher Scientific, catalog number: S5-3 )
  19. Myriocin (Cayman, catalog number: 63150 )
  20. Fumonisin B1 (Cayman, catalog number: 62580 )
  21. Tris (Fisher Scientific, catalog number: BP 152-5 )
  22. Complete mini EDTA-free Protease Inhibitor Cocktail (Roche, catalog number: 0 4693159001 )
  23. OptiMEM (Gibco, catalog number: 31985-070 )
  24. FA-Free BSA (Fisher, catalog number: BP9704-100 )
  25. Chloroform (Fisher, catalog number: C297-4 )
  26. Methanol (Fisher, catalog number: A452-4 )
  27. Liquid nitrogen
  28. C8 ceramide (Avanti, catalog number:860508)
  29. Sphingosine (Avanti, catalog number: 860490 )
  30. Palmitoyl CoA (Sigma, catalog number: P9716-10MG )
  31. 24:1 Coenzyme A (Avanti, catalog number: 870725 )
  32. Potassium hydroxide (Fisher, catalog number: P250-500 )
  33. DTT (Sigma, catalog number: D0632 )
  34. EDTA (Fisher, catalog number: S311-500 )
  35. Pyridoxal 5’ phosphate (Sigma, catalog number: P9255 )
  36. L-Serine (Sigma, catalog number:S4500)
  37. L-[3H(G)]-Serine (PerkinElmer, catalog number: 2477301 )
  38. Ecolite-Scintillation fluid (MP Biomedicals, catalog number: 882475 )
  39. Beta max-Scintillation fluid (MP Biomedicals, catalog number: 880020 )
  40. Serine free MEM (Gibco, catalog number: 11095-080 )
  41. Magnesium chloride (Fisher Scientific, catalog number: M87-100 )
  42. DMEM complete medium (see Recipes)
  43. C8-ceramide (see Recipes)
  44. Sphingosine (see Recipes)
  45. Palmitoyl CoA (see Recipes)
  46. 24:1 CoenzymeA (see Recipes)
  47. Fatty acid free BSA (see Recipes)
  48. 1 mM C8-ceramide (see Recipes)
  49. Methanol (see Recipes)
  50. Myriocin (see Recipes)
  51. Fumonisin B1 (see Recipes)
  52. Chloroform:methanol (2:1) (see Recipes)
  53. 10x Phosphate buffered saline (PBS) pH 7.4 (see Recipes)
  54. Alkaline methanol (see Recipes)
  55. Alkaline water (see Recipes)
  56. Digitonin (see Recipes)
  57. Pyridoxal 5’ phosphate (see Recipes)
  58. L-serine stock (see Recipes)
  59. 0.05% Trypsin (see Recipes)
  60. Scintillation fluid (see Recipes)
  61. Collagen (see Recipes)
  62. Collagen coating (see Recipes)

Equipment

  1. Cell culture Hood (LABCONCO, catalog number: 36213043726 )
  2. -80 °C freezer (Thermo Scientific, model: 5815 )
  3. CO2 incubator (Thermo Scientific, model: 3110 )
  4. KONTES Dounce glass homogenizer (VWR, catalog number: KT885300-0007 )
  5. Table top refrigerated centrifuge with swingout rotor (Eppendorf, model: 5810 )
  6. Table top Ultracentrifuge (Beckman Coulter, model: Optima MAX-XP )
  7. TLA 120.2 rotor ( Backman Coulter, S/N: 16U1971)
  8. Scintillation counter (Backman Coulter, model: LS6500 )
  9. Water bath (Thermo Scientific, model: 2354 )
  10. Nitrogen gas (Airgas, catalog number: NI UHP300 )
  11. CO2 gas (Airgas, catalog number: CD USP50 )
  12. Vortexer (Vortex-Genie 2, model: SI-0236 )
  13. Table top high speed centrifuge (Thermo Scientific, model: accuSpin Micro 17 )

Procedure

  1. Extraction of L-[3H(G)]-serine
    1. Handle and store L-[3H(G)]-Serine only in designated and authorized locations with proper precautions.
    2. Dispose radioactive waste in specially designated waste containers.
    3. Take 2 µl of serine before and after extraction and measure radioactivity to calculate the recovery of L-[3H(G)]-Serine.
    4. Dispense 0.2 ml of the [3H]-serine into a 2 ml screw cap tube, then add 0.5 ml of chloroform:methanol (2:1 ratio) to the tube.
    5. Vortex the tube for 1 min.
    6. Centrifuge the tube at 16,200 x g for 2 min at room temperature in a high speed centrifuge.
    7. Transfer the upper aqueous phase into fresh 2 ml screw cap tubes.
    8. Place the tubes into the house vacuum flask for 30 min to remove the traces of organic solvents.
    9. Measure the radioactivity after extraction to calculate specific activity of L-[3H(G)]-serine.
    10. Use this extracted [3H]-serine to measure SPT activity in cells and membranes.
    11. Extracted [3H]-serine will give less background, i.e., low counts with myriocin treatment in SPT assay.

  2. Assay of lipid inhibition of serine palmitoyltransferase (SPT) activity in intact HeLa cells
    1. Grow HeLa cells in DMEM complete medium and maintain cells in a CO2 incubator at 37 °C and 5% CO2.
    2. On day-1, plate HeLa cells (7 x 104 cells/well) in a collagen coated 24-well plate with 1 ml of DMEM complete medium.
    3. See Recipes for the procedure of collagen coating in 24-well plates.
    4. Divide cells into 4 groups for control, C8-ceramide, myriocin treatment and protein analysis.
    5. Prepare 4 wells for each group and use average value for calculations.
    6. Myriocin is a specific inhibitor for SPT and is a negative control for this assay.
    7. On day-2, add 10 µM C8-ceramide or 1 µM myriocin to the cells and incubate for 1 h in a CO2 incubator, see recipe for C8-ceramide and myriocin preparation.
    8. Incubate control cells with methanol-BSA complex.
    9. After 1 h remove C8-ceramide or myriocin and gently wash cells with 0.25 ml of PBS at room temperature.
    10. Measure SPT activity by adding 0.25 ml of extracted 3H-serine to the cells in serine free MEM media (5 µCi/ml of serine free media) and incubate cells in a CO2 incubator for 1 h at 37 °C.
    11. After 1 h, remove radioactive serine and gently wash cells with 0.25 ml of PBS at room temperature.
    12. Stop the reaction by adding 400 µl of alkaline methanol and scrape the cells using plastic cell lifters.
    13. Transfer cells into 2 ml screw cap tubes.
    14. Extract total sphingolipids under alkaline conditions as described below in Section H.
    15. Refer to Figure 1A in Davis et al. (2019) and Figure 2A in Siow et al. (2015) for results.

  3. Assay of SPT activity in Permeabilized HeLa cells
    1. Plate HeLa cells (7 x 104 cells/well) in a collagen coated 24-well plate and incubate in a CO2 incubator for 24 h.
    2. Divide cells into 4 groups for control, C8-ceramide, myriocin treatment and protein analysis.
    3. Prepare 4 wells for each group and use average value for calculations.
    4. After 24 h, permeablize HeLa cells with 0.02% digitonin prepared in Opti-MEM.
    5. Add 250 µl of 0.02% digitonin and incubate cells for 3 min at 37 °C.
    6. After 3 min, remove digitonin and gently wash cells with 0.25 ml of PBS at room temperature.
    7. Prepare preincubation medium, 50 mM HEPES pH8.0, 1 mM EDTA and 20 µM pyridoxyl 5′-phosphate.
    8. Dilute C8-ceramide or myriocin with preincubation media and pre-warmed to 37 °C.
    9. Add 200 µl of preincubation medium with or without 10 µM C8-ceramide or 1 µM myriocin to permeabilized cells.
    10. Incubate cells at 37 °C for 30 min in a CO2 incubator.
    11. After 30 min add 200 µl of labelling media to each well, contains 2 μCi of extracted [3H]-serine, 1 mM L-serine, and 50 μM palmitoyl-CoA prepared in preincubation medium.
    12. Incubate cells for 60 min in a CO2 incubator.
    13. After 60 min remove the labelling media.
    14. Stop the reaction by adding 400 µl of alkaline methanol and scrape the cells using plastic cell lifters.
    15. Transfer cells into 2 ml screw cap tubes for lipid extraction.
    16. Extract total sphingolipids under alkaline condition as described below in Section H.
    17. Refer to Figure 1A in Davis et al. (2019) and Figure 2A-B in Siow and Wattenberg (2012) for results.

  4. Preparation of HeLa cell lysate
    1. Seed 3 x 106 HeLa cells into one T-150 flask with DMEM complete medium.
    2. Grow HeLa cells for 48 h in a CO2 incubator to achieve 90-95% confluency.
    3. Remove the DMEM complete media and wash cells with 4 ml of PBS at room temperature.
    4. Add 4 ml of 0.05% trypsin to cells and incubate for 2 min at room temperature.
    5. After 2 min, remove trypsin and incubate cells at 37 °C for 5 min.
    6. Harvest cells with 10 ml of ice cold DMEM complete media.
    7. Collect cells by centrifugation for 10 min at 72 x g.
    8. Wash cells with 10 ml of ice cold PBS.
    9. Resuspend cells in 1.6 ml of swelling buffer (10 mM Tris pH 7.5, 15 mM KCl and 1 mM MgCl2) and incubate on ice for 15 min.
    10. To this, add 534 µl of 1 M sucrose, 7 µl of 200 mM EDTA and 80 µl of protease inhibitors cocktail from 25x stock.
      Note: 25x stock made by dissolving 1 protease inhibitor tablet in 2 ml of water.
    11. Homogenize the cells on ice with a 7 ml Dounce homogenizer using pestle-B with 30 to 40 strokes.
    12. Centrifuge the homogenate to remove unbroken cells and nuclei at 72 x g for 10 min at 4 °C with a swinging bucket rotor and collect the supernatant.
    13. Pass the cell free lysate through a 26 gauge needle with 1 ml syringe for 10 times.
    14. Aliquot 100 µl lysate into 1.5 ml tubes, snap freeze with liquid nitrogen and store at -80 °C until further analysis.

  5. Preparation of total membrane
    1. Prepare cell free lysate as described above in Section D.
    2. Transfer 1 ml of cell free lysate to each clean thick wall ultra-centrifuge tubes then centrifuge at 434,513 x g for 20 min at 4 °C in a TLA 120.2 rotor.
    3. Discard the supernatant.
    4. Resuspend the membrane pellet with 500 µl of membrane resuspension buffer (250 mM Sucrose, 25 mM Tris, pH 7.4 and 20 µl of protease inhibitors cocktail from 25x stock) and homogenize with the Dounce homogenizer using pestle-B with 10 strokes.
    5. Pass the membrane through a 26 gauge needle with 1 ml of syringe for 10 times.
    6. Aliquot 100 µl of membranes into 1.5 ml tubes and snap freeze with liquid nitrogen and store at -80 °C until further analysis.

  6. Assay of lipid inhibition of serine palmitoyltransferase activity in cell free lysate or total membranes
    1. Perform in vitro SPT activity in 2 ml screw cap tubes and total reaction volume of 200 µl.
    2. For assay of SPT activity with short chain ceramide, incubate cell free lysate (100 µg total protein) or membrane (50 µg total protein) in a pre-incubation buffer with or without 10 µM C8-ceramide or 1 µM myriocin on ice for 40 min.
    3. Pre-incubation buffer contains, 50 mM HEPES pH 8.0, 25 mM DTT, 2 mM EDTA and 20 µM PLP.
    4. After 40 min, add 100 µl of labelling mix then mix well.
    5. Labelling mix contains, 2 mM Serine, 100 µM palmitoyl CoA, 2 µCi extracted [3H]-serine.
    6. Incubate tubes for 60 min at 37 °C.
    7. Stop reaction by adding 400 µl of alkaline methanol.
    8. Extract total sphingolipid under alkaline conditions as described below in Section H.
    9. Refer to Figures 1A, B, 2A and 3A in Davis et al. (2019) for results.

  7. Assay of SPT activity with long chain ceramide
    1. For generation of long chain ceramide in membranes, incubate 50 µg of membrane with 20 μM sphingosine and 50 μM 24:1 CoA or 1 µM myriocin or 50 µM Fumonisin-B1 in a final volume of 100 µl in a buffer containing 20 mM HEPES pH 7.4, 25 mM KCl, 2 mM MgCl2 and 0.1% fatty acid free BSA.
    2. Fumonisin-B1 is an inhibitor of ceramide synthases.
    3. Incubate this reaction mix at 37 °C for 60 min to generate ceramide with 24:1 acyl chain by endogenous ceramide synthases.
    4. After 60 min, add 100 µl of labelling mix and incubate the tubes for additional 60 min at 37 °C.
    5. Labelling mix contains 50 mM HEPES pH 8.0, 1 mM DTT, 10 mM EDTA, 20 µM PLP, 2 mM serine,100 µM palmitoyl CoA and 2 µCi extracted [3H] serine.
    6. Stop the reaction by adding 400 µl of alkaline methanol and extract lipids under alkaline conditions as described below in Section H.
    7. Refer to Figure 2B-C in Davis et al. (2019) for results.

  8. Extraction of total sphingolipids under alkaline condition
    1. Add 100 µl of chloroform to the tube contains cells or membrane or lysate with 400 µl alkaline methanol.
    2. Vortex the tubes and centrifuge for 1 min at 16,200 x g in a high speed centrifuge.
    3. Add 500 µl of chloroform followed by 300 µl alkaline water and 100 µl of 2 N NH4OH to separate aqueous and organic phases.
    4. Vortex the tube for 1 min and spin at 16,200 x g for 1 min in a high speed centrifuge.
    5. Aspirate the upper aqueous phase.
    6. Add 1 ml of alkaline water to the lower organic phase, vortex well then centrifuge for 1 min at 16,200 x g.
    7. Aspirate the upper aqueous phase and repeat the above step 6 again.
    8. Transfer 350 µl of the lower organic phase into scintillation vials and dry under nitrogen gas.
    9. Add 5 ml of scintillation fluid, vortex well and count radioactivity using a scintillation counter.

Data analysis

  1. Expression of in vitro and in vitro SPT activity as [3H]-serine in total sphingolipids (CPM/mg/min).
    1. Here we provide the results obtained from previously performed experiments and detailed steps to calculate in vivo and in vitro SPT activity with results obtained from previously performed experiments.
    2. Table 1 shows results of in vitro SPT activity from HeLa cell membranes.

      Table 1. Serine palmitoyltransferase activity in total membranes of HeLa cells


    3. Normalize the CPM values to mg of total protein (50 µg of protein used for SPT assay)
      Control: 626.5/0.05 × 1 = 12,530 CPM/mg
      Myriocin: 47.6/0.05 × 1 = 952 CPM/mg
    4. Convert values to per min (SPT assay conducted for 60 min)
      Control: 12530/60 = 208.8 CPM/mg/min
      Myriocin: 952/60 = 15.8 CPM/mg/min
    5. In vivo and in vitro SPT activity is expressed as [3H] serine in total sphingolipids (CPM/mg/min).

  2. Expression of in vitro SPT activity as [3H]-serine in total sphingolipids (pMol/mg/min)
    1. Total radioactivity in substrate (labelling mix) is 1,280,980 CPM.
    2. SPT reaction mix contains 200 nMol of serine.
    3. Consider 200 nMol of serine is equivalent to 1,280,980 CPM.
    4. Calculate specific activity from this value by using this formula,
      nMol of serine/CPM in substrate × mean CPM from SPT assay = X nMol,
      e.g., 200/1,280,980 × 626 = 0.097 nMol of [3H] serine in total sphingolipids.
    5. Mean value of control SPT activity is 626 CPM.
    6. Convert the above value to per mg protein, 0.097/0.05 ×1 = 1.94 nMol/mg protein.
    7. Convert the above value to per min, 1.94/60 = 0.032 nMol/mg/min.
    8. Convert value from nmol to pmol by multiplying the above value by 1,000 (0.032 × 1,000 = 32 pMol/mg/min).
    9. In vitro SPT activity is expressed as [3H]-serine in total sphingolipids (pMol/mg/min).

Recipes

  1. DMEM complete medium
    500 ml of DMEM media
    50 ml of FBS
    10 ml of HEPES pH 8.0
    5 ml of glutamine and 5 ml Penicillin-streptomycin
  2. C8-ceramide
    10 mg of C8-ceramide dissolved in 1.174 ml of methanol as a 20 mM stock and store at -20 °C
  3. Sphingosine
    10 mg of sphingosine dissolved in 3.34 ml of methanol as a 10 mM stock store at -20 °C
  4. Palmitoyl CoA
    10 mg of palmitoyl CoA dissolved in 1.99 ml of DMSO as a 5 mM stock and store at -20 °C
  5. 24:1 CoenzymeA
    Dissolve 5 mg of 24:1 Coenzyme A in 857.2 µl of DMSO to make 5 mM Stock and store at -20 °C
  6. Fatty acid free BSA
    Fatty acid free BSA prepared in PBS as a 2% stock and filter sterilize using 0.2 µm syringe filter and store at 4 °C
  7. 1 mM C8-ceramide
    BSA complex 2.5 µl of 20 mM C8-ceramide mixed with 47.5 µl of 2% fatty acid free BSA, made every time fresh
  8. Methanol:BSA complex
    2.5 µl of methanol mixed with 47.5 µl of 2% fatty acid free BSA, made every time fresh
  9. Myriocin
    Dissolve myriocin in methanol as a 1 mM stock and store at -20 °C
  10. Fumonisin B1
    1 mg of Fumonisin B1 dissolved in 92.4 µl of DMSO as a 15 mM stock and store at -20 °C
  11. Chloroform:methanol (2:1)
    Mix 2 volume of chloroform and 1 ml volume of methanol
  12. 10x Phosphate buffered saline (PBS) pH 7.4
    15.4 mM KH2PO4, 1.55 M NaCl and 28 mM Na2HPO4·7H2O in water, if required adjust pH to 7.4 with HCl
  13. Alkaline methanol
    Dissolve 0.7 g potassium hydroxide in 100 ml of methanol
  14. Alkaline water
    Add 100 µl of 2 N NH4OH in 100 ml water
  15. Digitonin
    Prepare 1% digitonin in DMSO as a stock and dilute to 0.02% in Opti-Mem
  16. Pyridoxal 5’ phosphate
    Prepared in water as a 20 mM stock and store at -20 °C
  17. L-serine stock
    Prepare serine in water as a 50 mM stock and store at -20 °C
  18. 0.05% Trypsin
    Dilute stock 0.25% trypsin into 0.05% with Phosphate buffered saline 7.4 and filter sterilize and store at 4 °C
  19. Scintillation fluid
    We use Ecolite scintillation fluid to measure radioactivity in aqueous solutions and Beta max to measure radioactivity in organic solvents
  20. Collagen
    Dissolve 10 mg of collagen in 10 ml of 0.1 N acetic acid and mix it for 3 h at room temperature, then dilute collagen to 50 µg/ ml in PBS and store at 4 °C
  21. Collagen coating
    Equilibrate collagen to room temperature and add 1 ml to each well of 24-well plate, incubate at room temperature for 5 min. After incubation completely remove all collagen by pipetting. Collagen is reusable at least for 10 times.

Acknowledgments

This assay protocol is developed from the method of Rutti et al. (2009). This work was supported by funding from National Institute of Health grant RO1HL131340 to B.W.

Competing interests

Authors have no conflicts of interest to disclose

References

  1. Breslow, D. K., Collins, S. R., Bodenmiller, B., Aebersold, R., Simons, K., Shevchenko, A., Ejsing, C. S. and Weissman, J. S. (2010). Orm family proteins mediate sphingolipid homeostasis. Nature 463(7284): 1048-1053. 
  2. Davis, D., Kannan, M. and Wattenberg, B. (2018). Orm/ORMDL proteins: Gate guardians and master regulators. Adv Biol Regul 70: 3-18. 
  3. Davis, D. L., Gable, K., Suemitsu, J., Dunn, T. M. and Wattenberg, B. W. (2019). The ORMDL/Orm-serine palmitoyltransferase (SPT) complex is directly regulated by ceramide: Reconstitution of SPT regulation in isolated membranes. J Biol Chem 294(13): 5146-5156.
  4. Ferreira, C. R., Goorden, S. M. I., Soldatos, A., Byers, H. M., Ghauharali-van der Vlugt, J. M. M., Beers-Stet, F. S., Groden, C., van Karnebeek, C. D., Gahl, W. A., Vaz, F. M., Jiang, X. and Vernon, H. J. (2018). Deoxysphingolipid precursors indicate abnormal sphingolipid metabolism in individuals with primary and secondary disturbances of serine availability. Mol Genet Metab 124(3): 204-209. 
  5. Gable, K., Gupta, S. D., Han, G., Niranjanakumari, S., Harmon, J. M. and Dunn, T. M. (2010). A disease-causing mutation in the active site of serine palmitoyltransferase causes catalytic promiscuity. J Biol Chem 285(30): 22846-22852. 
  6. Han, G., Gupta, S. D., Gable, K., Niranjanakumari, S., Moitra, P., Eichler, F., Brown, R. H., Jr., Harmon, J. M. and Dunn, T. M. (2009). Identification of small subunits of mammalian serine palmitoyltransferase that confer distinct acyl-CoA substrate specificities. Proc Natl Acad Sci U S A 106(20): 8186-8191. 
  7. Hanada, K. (2003). Serine palmitoyltransferase, a key enzyme of sphingolipid metabolism. Biochim Biophys Acta 1632(1-3): 16-30. 
  8. Hanada, K., Hara, T., Nishijima, M., Kuge, O., Dickson, R. C. and Nagiec, M. M. (1997). A mammalian homolog of the yeast LCB1 encodes a component of serine palmitoyltransferase, the enzyme catalyzing the first step in sphingolipid synthesis. J Biol Chem 272(51): 32108-32114.
  9. Hornemann, T., Penno, A., Rutti, M. F., Ernst, D., Kivrak-Pfiffner, F., Rohrer, L. and von Eckardstein, A. (2009). The SPTLC3 subunit of serine palmitoyltransferase generates short chain sphingoid bases. J Biol Chem 284(39): 26322-26330. 
  10. Hornemann, T., Wei, Y. and von Eckardstein, A. (2007). Is the mammalian serine palmitoyltransferase a high-molecular-mass complex? Biochem J 405(1): 157-164.
  11. Ikushiro, H., Hayashi, H. and Kagamiyama, H. (2001). A water-soluble homodimeric serine palmitoyltransferase from Sphingomonas paucimobilis EY2395T strain. Purification, characterization, cloning, and overproduction. J Biol Chem 276(21): 18249-18256. 
  12. Lowther, J., Naismith, J. H., Dunn, T. M. and Campopiano, D. J. (2012). Structural, mechanistic and regulatory studies of serine palmitoyltransferase. Biochem Soc Trans 40(3): 547-554.
  13. Nagiec, M. M., Baltisberger, J. A., Wells, G. B., Lester, R. L. and Dickson, R. C. (1994). The LCB2 gene of Saccharomyces and the related LCB1 gene encode subunits of serine palmitoyltransferase, the initial enzyme in sphingolipid synthesis. Proc Natl Acad Sci U S A 91(17): 7899-7902.
  14. Rutti, M. F., Richard, S., Penno, A., von Eckardstein, A. and Hornemann, T. (2009). An improved method to determine serine palmitoyltransferase activity. J Lipid Res 50(6): 1237-1244.
  15. Siow, D. L. and Wattenberg, B. W. (2012). Mammalian ORMDL proteins mediate the feedback response in ceramide biosynthesis. J Biol Chem 287(48): 40198-40204.
  16. Williams, R. D., Wang, E. and Merrill, A. H., Jr. (1984). Enzymology of long-chain base synthesis by liver: characterization of serine palmitoyltransferase in rat liver microsomes. Arch Biochem Biophys 228(1): 282-291.
  17. Siow, D., Sunkara, M., Dunn T.M., Morris, A.J., Wattenberg, B. (2015). ORMDL/serine palmitoyltransferase stoichiometry determines effects of ORMDL3 expression on sphingolipid biosynthesis. J Lipid Res. 56 (4): 898-908.

简介

[摘要] 丝氨酸Palmitoyltranferase (SPT)是吡哆醛5 ' 磷酸(PLP)依赖酶催化第一和限速步骤中从头合成鞘脂。SPT活动是Homeostatically调控响应水平的提高鞘脂。这SPT的稳态调节是通过小ER膜蛋白介导称为ORMDLs。在这里,我们描述了一种方法用放射性标记的L-丝氨酸以测定SPT活性的SPT活性。测定的ORMDL依赖性抑制脂质从由规定的程序被开发Hornemann 实验室。 SPT的活性也可以使用氘化的L-丝氨酸进行测定,但需要进行质谱分析,这会耗费金钱,时间和仪器。可以在细胞和无细胞系统中研究ORMDL依赖性脂质对SPT活性的抑制作用。在这里,我们提供了详细的协议来测量存在短链(C8-神经酰胺)或长链神经酰胺(C24-神经酰胺)时SPT活性。该协议的最大优势之一我们通过在HeLa细胞膜中提供外源鞘氨醇和24:1酰基辅酶A通过内源性神经酰胺合酶生成长链神经酰胺来实现这一目标。需要精密的仪器。

[背景 ] 丝氨酸palmit oyltranferase (SPT)是一种多亚基酶是在真核生物和原核生物一些广泛表达(花田等人,1997; Ikushiro 。等人,2001; Hornemann 等人,2007).The 第一和速率SPT催化鞘脂生物合成途径的确定步骤,由丝氨酸和棕榈酰-CoA的缩合反应生成3-酮基二氢鞘氨醇(Williams 等,1984; Hanada,2003)。哺乳动物的SPT复合物由两个大的亚基组成。大的亚基称为SPTLC1和SPTLC2,小的亚基称为ssSPT (Nagiec 等,1994; Lowther 等,2012).SPT 需要全部三个亚基形成功能性蛋白复合物(Han 等) Al。,2009)。在某些情况下,哺乳动物SPT还由另一个称为SPTLC3的大亚基组成(Hornemann Et Al。,2009),它取代SPTLC2。功能性SPT复合物是通过将SPTLC1与SPTCL2或S PTLC3配对而形成的。ssSPT。活动 SPT驻留在复杂结合之间的SPTLC2或SPTLC3 subunit.The底物偏好的现场SPTLC2和SPTLC3 differs.SPTLC2喜欢棕榈CoA.SPTLC3喜欢肉豆蔻- 辅酶A (Hornemann 。等人,2009) 。在某些情况下,SPT还结合丙氨酸或甘氨酸救父丝氨酸到前体鞘脂生成一个Deoxysphingolipid 基地(山墙等,2010;费雷拉等人,2018) 。SPT形成稳定的复合物与其他ER驻地蛋白质称为ORMDL这SPT-ORMDL互动是至关重要的。维持鞘脂从头生物合成的体内稳态调节(Siow和Wattenberg ,2012; Davis 等,2018)。在哺乳动物中发现了三个密切相关的ORMDL家族成员(Breslow 等,2010; Siow和Wattenberg,2012)我们以前表明,增加的水平在HeLa细胞中的ORMDL依赖性方式的细胞的神经酰胺抑制SPT活性号第(戴维斯等人,2019).The 稳态regulat后面详细的机理 ORMDLs对SPT活性的影响尚不清楚.SPT与ORMDL形成稳定的复合物,无论神经酰胺的细胞水平如何。我们建议神经酰胺与ORMDLs或SPT-ORMDL复合物结合以触发SPT活性的抑制。我们最近开发了一种无细胞系统,该系统从HeLa细胞中分离出膜并用于测试SPT活性对鞘脂升高的反应。该系统可在存在或不存在具有人工短链和天然链长的神经酰胺的情况下测量SPT活性(Davis 等,2019) 。我们建立了测定ORMDL相关脂质抑制SPT活动为平台,以测试SPT与ORMDL。这SPT试验过程中的功能相互作用开发板的等静压从程序的Rutti 等人(2009)。无细胞体外系统有许多优于体内系统而这两个系统产生类似的结果(戴维斯等人,2018) 。使用本无细胞重构体系,我们已经确定 ORMDL依赖调节这SPT活动不是介导的转录后或者翻译后机制(中-戴维斯等人。,2018) 。这种细胞系统是一个强大的工具来了解详细机制的稳态调节SPT通过ORMDLs后面。

关键字:丝氨酸棕榈酰转移酶, 神经酰胺合酶, 鞘脂, ORMDL, 神经酰胺, 内质网, 稳态

材料和试剂


 


真空过滤单元(0.2 μ米)(VWR,目录号:10040-436)
T150烧瓶(Cyto One,目录号:CC7682-4815)
24 - 孔板(的Cyto 一,目录号:CC7682-7524)
26号针(BD,目录号305110-269)
1 ml注射器(BD,货号:309659)
用于超速离心机转子的厚壁聚碳酸酯管(贝克曼库尔特,目录号:343776)
螺帽管2 ml(Axygen ,目录号:SCT-200-CS)
闪烁管(Perkin Elmer,目录号:6000292)
一次性无菌细胞提升器(Fisher Scientific,目录号:08-100-240)
HeLa细胞(ATCC,目录号:CCL-2)
Dulbecco的基本必需培养基(DMEM 培养基)(Gibco ,目录号:11960-044)
胎牛血清(双子,目录号:900-108)
HEPES(Fisher S Cientific,目录号:BP310-1)
青霉素链霉素,10,000 U / ml(Gibco,目录号:15140-122)
谷氨酰胺(Gibco,目录号:25030-081)
胰蛋白酶-EDTA(Gibco,目录号:25200-056)
胶原蛋白(Sigma,目录号:C9791)
蔗糖(Fisher Scientific,目录号:S5-3)
Myriocin(开曼群岛,货号:63150)
伏马菌素B1(Cayman,货号:62580)
Tris(Fisher S Cientific,目录号:BP 152-5)
完整的不含EDTA的迷你蛋白酶抑制剂鸡尾酒(罗氏,目录号:04693159001)
OptiMEM (Gibco,目录号:31985-070)
无FA BSA(Fisher,目录号:BP9704-100)
氯仿(Fisher,目录号:C297-4)
甲醇(Fisher,目录号:A452-4)
液氮
C8神经酰胺(Avanti,目录号:860508)
鞘氨醇(Avanti,目录号:860490)
蓟马toyl辅酶A(Sigma,目录号:P9716-10MG )
24:1辅酶A(Avanti,目录号:870725)
氢氧化钾(Fisher,目录号:P250-500)
DTT(Sigma,目录号:D0632)
EDTA(Fisher,目录号:S311-500)
5'磷酸吡rid醛(Sigma,目录号:P9255)
L-丝氨酸(Sigma,目录号:S4500)
L- [ 3 H(G)]-丝氨酸(Perkin Elmer,目录号:2477301)
Ecolite -Scintillation流体(MP Biomedicals公司,目录号:882475)
Beta max闪烁液(MP Biomedicals,目录号:880020)
无丝氨酸MEM(Gibco,目录号:11095-080)
氯化镁(Fisher S Cientific,目录号:M87-100)
DMEM完全培养基(请参阅食谱)
C8神经酰胺(请参阅食谱)
鞘氨醇(请参见食谱)
棕榈酰CoA(请参阅食谱)
24:1 辅酶A (请参阅食谱)
不含脂肪酸的BSA(请参阅食谱)
1 mM C8-神经酰胺(请参阅食谱)
甲醇(请参阅食谱)
肉豆蔻素(请参阅食谱)
伏马菌素B1(请参阅食谱)
氯仿:甲醇(2:1)(请参阅食谱)
10x磷酸盐缓冲液(PBS)pH 7.4 (请参阅食谱)
碱性甲醇(请参阅食谱)
碱性水(请参阅食谱)
地高辛(请参阅食谱)
磷酸吡rid醛5'(请参阅食谱)
L-丝氨酸库存(请参阅食谱)
0.05%胰蛋白酶(请参阅食谱)
闪烁液(请参阅配方)
胶原蛋白(请参阅食谱)
胶原蛋白涂层(请参阅食谱)
 


设备


 


细胞培养罩(LABCONCO,目录号:36213043726)
-80 °C冰柜(Thermo Scientific,型号:8155)
CO 2 培养箱(Thermo S Cientific,型号:3110)
康特斯Dounce玻璃均质机(VWR,目录号:KT885300-0007)
带有台式转子的台式冷冻离心机(Eppendorf,型号:5810)
台式超速离心机(贝克曼库尔特公司,型号:Optima MAX-XP)
TLA 120.2转子(Backman Coulter,S / N:16U1971)
反闪烁(Backman C Oulter,M Odel:LS6500)
水浴(Thermo Scientific,型号:2354)
氮气(Airgas,目录号:NI UHP300)
CO 2 气(Airgas,目录号:CD USP50 )
Vortexer (Vortex-Genie 2,型号:SI-0236)
台式高速离心机(Thermo Scientific,型号:accuSpin Micro 17)
 


程序


 


L- [ 3 H(G)]-丝氨酸的提取
L- [ 3 H(G)]-Serine只能在适当的预防措施下在指定和授权的地方使用和存放。
将放射性废物处置在专门指定的废物容器中。
在提取前后取2 µl丝氨酸,并测量放射性以计算L- [ 3 H(G)]-丝氨酸的回收率。
将0.2 ml的[ 3 H]-丝氨酸分配到2 ml螺帽管中,然后将0.5 ml 氯仿:甲醇(比例为2:1)加入管中。
将试管涡旋1分钟。
在室温下,以高速离心机将试管以16,200 x g 离心2分钟。
将上层水相转移到新鲜的2 ml螺帽管中。
将试管放入家用真空瓶中30分钟,以去除痕量有机溶剂。
提取后测量放射性以计算L- [ 3 H(G)]-丝氨酸的比活度。
使用提取的[ 3 H]丝氨酸测量细胞和膜中的SPT活性。
提取的[ 3 H]-丝氨酸将产生较少的背景,即在SPT分析中用myriocin处理时计数较低。
 


完整HeLa细胞中脂质抑制丝氨酸棕榈酰转移酶(SPT)活性的测定
在DMEM完全培养基中培养HeLa细胞,并将细胞维持在37°C和5%CO 2 的CO 2 培养箱中。
在第1天,板HeLa细胞(7×10 4 细胞/孔)在胶原包被的24 - 孔平板用1ml的DMEM完全培养基中。
见食谱胶原涂层的过程在24 - 孔板中。
将细胞分为4组,分别用于对照,C8-神经酰胺,myriocin处理和蛋白质分析。
每组准备4口井,并使用平均值进行计算。
十四烷是SPT的特异性抑制剂,是该测定的阴性对照。
在第2天,向细胞中加入10 µM C8-神经酰胺或1 µM桃红霉素,并在CO 2 培养箱中孵育1小时,请参见C8-神经酰胺和桃红霉素制备方法。
用甲醇-BSA复合物孵育对照细胞。
1小时后,取出C8-神经酰胺或myriocin,并在室温下用0.25 ml PBS轻轻洗涤细胞。
通过向无丝氨酸MEM培养基(5 µCi / ml无丝氨酸的培养基)中的细胞中加入0.25 ml提取的3 H-丝氨酸,并将细胞在CO 2 培养箱中于37 °C 孵育1 h,来测量SPT活性。
1小时后,取出放射性丝氨酸,并在室温下用0.25 ml PBS轻轻洗涤细胞。
加入400 µl碱性甲醇终止反应,并使用塑料细胞提升器刮擦细胞。
将细胞转移到2 ml螺帽管中。
总鞘脂类在提取碱性条件。如下文在小号挠度H.
参阅˚F igure 1A中戴维斯等人。(2019 )和˚F igure 2A中{人萧,2015#20} 人萧等人。(2015 )用于结果。
 


透化HeLa细胞中SPT活性的测定
板HeLa细胞(7×10 4 细胞/孔)在包被的24胶原- 孔板,孵育在CO 2 培养箱中培养24小时。
将细胞分为4组,分别用于对照,C8-神经酰胺,myriocin处理和蛋白质分析。
每组准备4口井,并使用平均值进行计算。
24小时后,用Opti-MEM中制备的0.02%洋地黄皂苷透化HeLa细胞。
加入250μl0.02%洋地黄皂苷并在37 °C 下孵育细胞3分钟。
3分钟后,取出洋地黄皂苷并在室温下用0.25 ml PBS轻轻洗涤细胞。
预温育培养基中的制备,50mM的HEPES pH 8.0中,1mM EDTA和20微摩尔吡哆5 ' 磷酸。
用预温育培养基稀释C8-神经酰胺或myriocin,并预热至37 °C 。
向透化的细胞中加入200 µl带有或不带有10 µM C8-神经酰胺或1 µM十四烷的预培养培养基。
在CO 2 培养箱中于37 °C 孵育细胞30分钟。
30分钟后,添加标签介质到每个孔的200μl的,包含2 微居里萃取[的3 H] -丝氨酸,1米中号L-丝氨酸,和50 μ 中号棕榈酰-辅酶A 在预温育培养基中制备。
将细胞在CO 2 培养箱中孵育60分钟。
60分钟后,取出标签纸。
加入400 µl碱性甲醇终止反应,并使用塑料细胞提升器刮擦细胞。
将细胞转移到2 ml螺帽管中进行脂质提取。
总鞘脂类在提取碱性条件。如下文在小号挠度H.
参阅˚F igure 1A中戴维斯等人。(2019 )和˚F igure在2A-B 人萧和瓦滕伯格(2012 )为结果。
 


HeLa细胞裂解液的制备
将3 x 10 6 HeLa细胞接种到装有DMEM完全培养基的T-150烧瓶中。
在CO 2 培养箱中培养HeLa细胞48小时,以达到90-95%的融合度。
除去DMEM完全培养基,并在室温下用4 ml PBS洗涤细胞。
向细胞中加入4 ml 0.05%的胰蛋白酶,并在室温下孵育2分钟。
2分钟后,取出胰蛋白酶,将细胞在37 °C 下孵育5分钟。
用10 ml冰冷的DMEM完全培养基收获细胞。
通过以72 x g 离心10分钟收集细胞。
用10 ml冰冷的PBS洗涤细胞。
将细胞重悬于1.6 ml溶胀缓冲液(10 mM Tris pH 7.5、15 mM KCl 和1 mM MgCl 2 )中,并在冰上孵育15分钟。
为此,从25x储备液中加入534 µl 1 M蔗糖,7 µl 200 mM EDTA和80 µl蛋白酶抑制剂混合物。
注意:通过将1个蛋白酶抑制剂片剂溶解在2 ml 水中制成2 5x原料。


使用7毫升Dounce匀浆器使用杵B将其在冰上匀浆30到40次。
离心匀浆以在4 °C下用转桶式转子以72 x g的速度除去未破碎的细胞和细胞核10分钟,并收集上清液。
用1 ml注射器将无细胞裂解液通过26号针头10次。
等分试样100 微升裂解物至1.5mL管中,搭扣用液氮,并储存在冷冻- 80 ℃,直至进一步分析。
 


总膜的制备
无细胞裂解物,准备将如上所述在小号挠度D.
将1 ml无细胞裂解液转移到每个干净的厚壁超离心管中,然后在TLA 120.2转子中于4 °C 以434,513 x g 离心20分钟。
丢弃目标。
用500 µl膜重悬缓冲液(250 mM蔗糖,25 mM Tris,pH 7.4和20 µl蛋白酶抑制剂混合物(来自25x储备液))重悬膜沉淀,并用Dounce匀浆器使用杵笔B匀浆10次。
用1毫升注射器将膜通过26号针头10次。
将100 µl 膜分装到1.5 ml试管中,并用液氮速冻并保存在-80 °C 直至进一步分析。
 


脂质抑制无细胞裂解物或总膜中丝氨酸棕榈酰转移酶活性的测定
在2 ml螺帽管中进行体外SPT活性,反应总体积为200 µl。
为了用短链神经酰胺测定SPT活性,在有或没有10 µM C8-神经酰胺或1 µM阿霉素的预孵育缓冲液中于冰上孵育40分钟的无细胞裂解物(总蛋白100 µg)或膜(总蛋白50 µg)。分钟
预孵育缓冲液包含50 mM HEPES pH 8.0、25 mM DTT,2 mM EDTA和20 µM PLP。
40分钟后,添加100 µl标记混合物,然后充分混合。
标记混合物包含2 mM丝氨酸,100 µM棕榈酰CoA,2 µCi 提取的[ 3 H]-丝氨酸。
将试管在37 °C下孵育60分钟。
加入400 µl碱性甲醇终止反应。
总鞘脂在提取碱性条件。如下文在小号挠度H.
参阅˚F igures 1A,1B,2A和3A中戴维斯等人。(2019 )用于结果。
 


用长链神经酰胺测定SPT活性
在膜中产生长链神经酰胺,用20 Myu M 鞘氨醇和50 Myu M 24 孵育50 Myug膜:1 CoA或1 Myu M Myriocin或50 Myu M Fumonisin -B1,最终体积为100 Myu L在缓冲液中含有20 mM HEPES pH 7.4、25 mM KCl ,2 mM MgCl 2 和0.1%不含脂肪酸的BSA。
伏马菌素-B1 是神经酰胺合酶的抑制剂。
将该反应混合物在37 °C下孵育60分钟,以通过内源性神经酰胺合酶生成具有24:1酰基链的神经酰胺。
60分钟后,加入100 μ 标记混合物的升,并在37温育额外60分钟管℃下。
标记混合物包含50mM的HEPES pH 8.0中,1mM的DTT,10mM的EDTA,20 μM PLP,2mM的丝氨酸,100 μ 中号棕榈酰辅酶A和2 μ 次萃取[ 3 H]丝氨酸。
该反应通过停止增加400 MYU 模式L碱性甲醇和脂类提取物在碱性条件下所描述的下面小号挠度H.
参阅˚F igure 2B-C在戴维斯等人。(2019 )用于结果。
 


碱性条件下总鞘脂的提取
向装有细胞或膜或含有400 µl碱性甲醇的裂解液的试管中加入100 µl氯仿。
将试管涡旋并在高速离心机中以16,200 xg 的速度离心1分钟。
加入500 µl氯仿,然后加入300 µl碱性水和100 µl 2 N NH 4 OH分离水相和有机相。
将试管涡旋1分钟,然后在高速离心机中以16,200 xg 旋转1分钟。
吸出上层水相。
向下部有机相中加入1 ml碱性水,充分涡旋,然后以16,200 xg 离心1分钟。
吸出上层水相,并再次重复上述步骤6。
将350 µl下层有机相转移至闪烁瓶中,并在氮气下干燥。
加入5 ml闪烁液,充分涡旋并使用闪烁计数器对放射性进行计数。
 


数据分析


 


I N的体外表达和体外SPT活性为总鞘脂中的[ 3 H]-丝氨酸(CPM / Mg / Min)。
在这里,我们提供了从先前进行的实验获得的结果和详细步骤来计算我Ñ体内和i的体外SPT活性与来自先前进行的实验获得的结果。
Ť 能够1 示出的结果体外SPT活动从HeLa细胞的膜。
 


表1. HeLa细胞总膜中丝氨酸棕榈酰转移酶的活性


管号


控制(CPM)


肌球蛋白(CPM)


管1


659


55


管2


649


43


管3


571


45


意思


626


47


标清


48





 


将CPM值归一化为总蛋白mg(用于SPT分析的50 µg蛋白)
对照:626.5 / 0.05 × 1 = 12 ,,530 CPM / mg             


十四霉素:47.6 / 0.05 × 1 = 952 CPM /毫克             


将值转换为每分钟(进行60分钟的SPT分析)
对照:12530/60 = 208.8 CPM / mg / min                                         


肌球蛋白:9 52/60 = 15.8 CPM / mg / min                                         


在体内和i的体外SPT活性表示为[ 3 H] SER 总共鞘脂INE(CPM /毫克/分钟)。
的表达我在体外SPT活性[ 3 H] -丝氨酸到TAL鞘脂(皮摩尔/毫克/分)
在底物(标记混合物)总放射性为1 ,280 ,980 CPM。
SPT反应混合物包含200 nMol 的丝氨酸。
考虑20 0 纳摩尔丝氨酸的相当于1 ,280 ,980 CPM。
使用此公式从该值计算比活,
丝氨酸的nMol / 底物中的CPM × SPT分析得出的平均CPM = X nMol ,


例如1,200 / 1 ,280 ,980 × 626 = 0.097 纳摩尔[的3 总共鞘脂H]丝氨酸。


对照SPT活性的平均值为626 CPM。
将上述值转换为每毫克蛋白质0.097 / 0.05 × 1 = 1.94 nMol / 毫克蛋白质。
将上述值转换为每分钟1.94 / 60 = 0.032 nMol / mg / min。
从纳摩尔到转换值皮摩尔由MULT iplying上述值由1000 (0.032 × 1 ,000 = 32 皮摩尔/毫克/分钟)。
体外SPT活性表示为[ 3 中总的鞘脂H] -丝氨酸(皮摩尔/毫克/分钟)。
 


菜谱


 


DMEM完全培养基
500 ml DMEM培养基
50 ml FBS
10 ml HEPES pH 8.0 5 ml谷氨酰胺和5 ml青霉素-链霉素
C8-神经酰胺将
10 mg C8-神经酰胺溶解在1.174 ml甲醇中,作为20 mM储备液,并储存在-20 °C下
鞘氨醇
10 mg鞘氨醇溶解于3.34 ml甲醇中,作为-20 °C下10 mM的储备液
Palmitoyl CoA将
10 mg的Palmitoyl CoA溶解在1.99 ml DMSO中,作为5 mM储备液,并储存在-20 °C
24:1 辅酶
A将5 mg的24:1辅酶A溶于857.2 µl的DMSO中,制成5 mM储备液,并储存在-20 °C下
不含
脂肪酸的BSA在PBS中以2%的储备液制备的不含脂肪酸的BSA,并使用0.2 µm注射器式过滤器过滤并灭菌,并储存在4 °C下
1毫米C8-神经酰胺
每次新鲜时,将2.5 µl的BSA复合物2.5 µl的20 mM C8-神经酰胺与47.5 µl的不含2%脂肪酸的BSA混合


甲醇:BSA 复合物
每次新鲜时,将2.5 µl甲醇与47.5 µl 2%不含脂肪酸的BSA混合


Myriocin将Myriocin
溶解为1 mM的甲醇溶液,并储存在-20 °C
伏马菌素B1将
1 mg 伏马菌素B1溶解在92.4 µl DMSO中,作为15 mM储备液,并储存在-20 °C下
氯仿:甲醇(2:1)
混合2体积的氯仿和1体积的甲醇
10×磷酸盐缓冲盐水(PBS)pH 7.4的
15.4毫KH 2 PO 4 ,1.55 M氯化钠和28 mM的钠2 HPO 4·水中7H 2 O,如果需要,用HC L将PH调节至7.4
碱性甲醇将
0.7克氢氧化钾溶于100毫升甲醇
碱性水在100毫升水中
添加100微升2 N NH 4 OH
毛地黄皂苷
在DMSO制备1%毛地黄皂苷作为库存,并稀释至0.02%中的Opti-MEM
在水中以20 mM的储备液
制备5' 磷酸吡stock醛,并在-20 °C下储存
L-丝氨酸原液
以50 mM的储备液在水中制备丝氨酸并储存在-20 °C


0.05%胰蛋白酶将
0.25%胰蛋白酶用磷酸盐缓冲液7.4稀释至0.05%,然后过滤灭菌并在4 °C下储存
闪烁液
我们使用Ecolite 闪烁液来测量水溶液中的放射性,使用Beta max来测量有机溶剂中的放射性
胶原蛋白将
10 mg胶原蛋白溶于10 ml 0.1 N乙酸中,在室温下混合3 h,然后在PBS中将胶原蛋白稀释至50 µg / ml,并在4 °C下保存
胶原涂覆
下平衡胶原至室温和1ml添加到每个孔中的24 - ,在室温下孔板,孵育5 min.After孵育,通过移液完全除去所有胶原胶原是可重复使用至少10次。
 


Acknowledg 发言:


 


该测定方案是根据Rutti 等人(2009)的方法开发的,这项工作得到了美国国立卫生研究院(NIH)向BW提供的RO1HL131340资助


 


 


完成利益


 


作者没有利益冲突要披露


 


参考文献


 


Breslow,DK,Collins,SR,Bodenmiller,B.,Aebersold,R.,Simons,K.,Shevchenko,A.,Ejsing,CS和Weissman,JS(2010)。Orm家族蛋白介导鞘脂稳态。Nature 463(7284) ):1048-1053。              
Davis,D.,Kannan,M. and Wattenberg,B.(2018)。Orm / ORMDL蛋白:Gate Guardians和Master Regulators。Adv Biol Regul 70:3-18。              
戴维斯(DL),盖布尔(Kable ),Suemitsu ,J.,邓恩(TM)和瓦滕贝格(BW)(2019)。生物化学杂志294(13):5146-5156。
Ferreira,CR,Goorden,SMI,Soldatos,A.,Byers,HM,Ghauharali-van der Vlugt,JMM,Beers-Stet,FS,Groden,C.,van Karnebeek,CD,Gahl,WA,Vaz,FM,江,X. and Vernon,HJ(2018)。脱氧鞘脂前体表明在患有丝氨酸可及性的原发性和继发性紊乱的个体中鞘脂代谢异常。Mol Genet Metab 124(3):204-209。              
Gable,K.,Gupta,SD,Han,G.,Niranjanakumari,S.,Harmon,JM和Dunn,TM(2010)。丝氨酸棕榈酰转移酶活性位点的致病突变引起催化混杂.J Biol Chem 285 (30):22846-22852。             
Han,G.,Gupta,SD,Gable,K.,Niranjanakumari,S.,Moitra,P.,Eichler,F.,Brown,RH,Jr.,Harmon,JM和Dunn,TM(2009)。赋予丝氨酸-CoA底物特异性的哺乳动物丝氨酸棕榈酰转移酶的亚基 .Proc Natl Acad Sci USA 106(20):8186-8191。              
Hanada,K.(2003)。丝氨酸棕榈酰转移酶,鞘脂代谢的关键酶。Biochim Biophys Acta 1632(1-3):16-30。              
Hanada,K.,Hara,T.,Nishijima,M.,Kuge,O.,Dickson,RC和Nagiec,MM(1997)。酵母LCB1的哺乳动物同源物编码丝氨酸棕榈酰转移酶的一种成分,该酶催化第一个步骤在神经鞘脂类合成。生物化学杂志272(51):32108-32114。
Hornemann,T.,Penno,A.,Rutti,MF,Ernst,D.,Kivrak-Pfiffner,F.,Rohrer,L.和von Eckardstein,A.(2009)。丝氨酸棕榈酰转移酶的SPTLC3亚基产生短链类鞘氨醇。 。碱生物化学杂志284(39):26322-26330。              
Hornemann,T.,Wei,Y.和von Eckardstein,A.(2007)。哺乳动物的丝氨酸棕榈酰转移酶是一种高分子质量的复合物吗? Biochem J 405(1):157-164。
Ikushiro,H.,Hayashi,H.和Kagamiyama,H.(2001)。鞘脂单胞菌EY2395T菌株的水溶性同型二聚丝氨酸棕榈酰转移酶。纯化,鉴定,克隆和过量生产 .J Biol Chem 276(21):18249- 18256。              
Lowther,J.,Naismith,JH,Dunn,TM和Campopiano,DJ(2012)。丝氨酸棕榈酰转移酶的结构,机理和调控研究。Biochem Soc Trans 40(3):547-554。
Nagiec,MM,Baltisberger,JA,Wells,GB,Lester,RL和Dickson,RC(1994)。酿酒酵母的LCB2基因和相关的LCB1基因编码丝氨酸棕榈酰转移酶的亚基,这是鞘脂合成中的初始酶.Proc Natl Acad Sci美国91(17):7899-7902。
Rutti,MF,Richard,S.,Penno,A.,von Eckardstein,A.和Hornemann,T.(2009)。一种确定丝氨酸棕榈酰转移酶活性的改进方法。J Lipid Res 50(6):1237-1244。
Siow,DL和Wattenberg,BW(2012)。哺乳动物ORMDL蛋白介导神经酰胺生物合成中的反馈响应。J Biol Chem 287(48):40198-40204。
Williams,RD,Wang,E.和Merrill,AH,Jr.(1984)。肝脏长链碱基合成的酶学:大鼠肝微粒体中丝氨酸棕榈酰转移酶的表征。Arch Biochem Biophys 228(1):282-291。
Siow,D.,Sunkara,M.,Dunn TM,Morris,AJ,Wattenberg,B.(2015)。ORMDL /丝氨酸棕榈酰转移酶的化学计量确定ORMDL3表达对鞘脂生物合成的影响。J Lipid Res .56(4):898- 908。
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Copyright: © 2020 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Kannan, M., Davis, D. L., Suemitsu, J., Oltorik, C. D. and Wattenberg, B. (2020). Preparation of HeLa Total Membranes and Assay of Lipid-inhibition of Serine Palmitoyltransferase Activity. Bio-protocol 10(12): e3656. DOI: 10.21769/BioProtoc.3656.
  2. Davis, D. L., Gable, K., Suemitsu, J., Dunn, T. M. and Wattenberg, B. W. (2019). The ORMDL/Orm-serine palmitoyltransferase (SPT) complex is directly regulated by ceramide: Reconstitution of SPT regulation in isolated membranes. J Biol Chem 294(13): 5146-5156.
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