Elsevier S & T

Lipidomics and Bioactive Lipids: Lipids and Cell Signaling: Lipids and Cell Signaling

Brown, H. Alex

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Table of contents
  • Contentsv
  • Contributorsxi
  • Prefacexvii
  • Volumes in Seriesxix
  • Chapter 1: Phospholipase A1 Assays Using a Radiolabeled Substrate and Mass Spectrometry1
  • 1. Introduction2
  • 2. Types of PLA12
  • 3. Conventional PLA1 Assay Using Radiolabeled Substrates3
  • 3.1. Materials3
  • 3.2. Preparation of radiolabeled phospholipid substrates3
  • 3.3. Assays5
  • 4. Novel PLA1 Assay Using ESI-MS6
  • 4.1. Materials6
  • 4.2. Preparation of recombinant PLA16
  • 4.3. Phospholipase A1 assay8
  • 4.4. PLA1 activity of intracellular PLA1s10
  • 5. Perspective11
  • Acknowledgments11
  • References11
  • Chapter 2: Real-Time Cell Assays of Phospholipase A2s Using Fluorogenic Phospholipids15
  • 1. Introduction16
  • 2. Fluorogenic PLA2 Substrates18
  • 2.1. Substrate specificity of PLA2s18
  • 2.2. Selection of fluorophores and design of fluorogenic PLA2 substrates18
  • 2.3. Real-Time cellular PLA2 assay using fluorogenic phospholipids22
  • 3. Measuring Cellular sPLA2 Activity Using PED6 and Red-PED623
  • 3.1. Materials23
  • 3.2. Labeling of cell membranes with PED6 and monitoring cellular sPLA2 activities23
  • 4. Measuring Cellular cPLA2alpha Activity Using DAPC24
  • 4.1. Synthesis of DAPC24
  • 4.2. Labeling of cell membranes with DAPC and monitoring cellular cPLA2alpha activity25
  • References26
  • Chapter 3: Analysis and Pharmacological Targeting of Phospholipase C beta Interactions with G Protei29
  • 1. Introduction30
  • 2. Methods31
  • 2.1. Protein expression and purification31
  • 2.2. PLCbeta assay37
  • 2.3. PLC assay optimization41
  • 2.4. Application of the PLC assay to evaluate peptide/small molecule modulation of Gbetagamma-depend42
  • 2.5. Evaluation of Gbetagamma–PLC binding43
  • 3. Concluding Remarks46
  • Acknowledgment47
  • References47
  • Chapter 4: Biochemical Analysis of Phospholipase D49
  • 1. Introduction50
  • 2. Assay of Recombinant PLD In Vitro52
  • 2.1. Expression of recombinant PLD152
  • 2.2. Purification of PLD152
  • 2.3. Chromatography (6His)52
  • 2.4. Size-exclusion chromatography (Superdex 200)53
  • 2.5. Anion-exchange chromatography (Q-Sepharose)55
  • 2.6. Characteristics of purified, full-length PLD156
  • 2.7. Purification of N-terminally truncated PLD156
  • 2.8. Assay of phospholipase D activity in vitro57
  • 3. Regulated PLD1 Activity59
  • 4. Preparation of Activators of PLD160
  • 4.1. Protein kinase Calpha (PKCalpha)60
  • 4.2. Myristoyl-ADP ribosylation factor 160
  • 4.3. Purification of geranylgeranylated RhoA, Rac1, and Cdc4260
  • 5. Effects of Activators on PLD1 Activity61
  • 6. Synergy between PLD1 Activators62
  • 7. Binding of PLD1 to Phospholipid Vesicles63
  • 8. Kinetic Parameters of PLD1 Catalytic Activity65
  • 9. Kinetic Analyses of Synergistic Responses69
  • 10. Phosphatidylinositol 4,5-Bisphosphate is an Essential PLD1 Activator69
  • 11. In Vivo PLD Assay Using Radioisotopes74
  • 12. In Vivo PLD Assay Using Deuterated 1-Butanol74
  • 12.1. Materials and methods75
  • 12.2. Application76
  • 13. Fluorescent In Vitro PLD Assay77
  • 13.1. Preparation of lipid substrates78
  • 13.2. Phospholipase D assay79
  • 14. Real-Time Diacylglycerol Lipase Assay80
  • 14.1. Synthesis of BD2-DAG80
  • 14.2. Protocol for BD2DAG preparation82
  • 14.3. Fluorescent assay for DAG-lipase activity83
  • Acknowledgments84
  • References85
  • Chapter 5: Measurement of Autotaxin/Lysophospholipase D Activity89
  • 1. Introduction90
  • 2. Overview of Methods for Determination of Autotaxin/LysoPLD Activity93
  • 3 .Expression of V5-Tagged Autotaxin/LysoPLD in HEK293 Cells94
  • 4. Measurement of Autotaxin/LysoPLD Activity Using Radiolabeled Substrates95
  • 4.1. Source of reagents95
  • 4.2. Preparation of substrate96
  • 4.3. Assay buffer96
  • 4.4. Assay composition and incubation96
  • 4.5. Assay termination and product analysis96
  • 4.6. Kinetic analysis of recombinant autotaxin/lysoPLD using [14C]lysoPC substrate97
  • 5. Measurement of Autotaxin/LysoPLD Activity Using Fluorogenic Substrates98
  • 5.1. Source of reagents and supplies99
  • 5.2. Preparation of substrate100
  • 5.3. Assay composition and incubation100
  • 5.4. Kinetic analysis of autotaxin/lysoPLD using FS-3100
  • 6. Concluding Comments100
  • Acknowledgment102
  • References102
  • Chapter 6: Platelet-Activating Factor105
  • 1. Introduction105
  • 2. Procedure107
  • 2.1. Reagents107
  • 2.2. Standard curves108
  • 2.3. Lipid extraction109
  • 2.4. Sample cleanup112
  • 2.5. Platelet-activating factor quantitation113
  • Acknowledgments115
  • References115
  • Chapter 7: Quantitative Measurement of Phosphatidylinositol 3,4,5-trisphosphate117
  • 1. Introduction118
  • 2. Measuring Levels of Radioactively Labeled Phosphoinositides in Isolated Cells120
  • 2.1. Preparation of monomethylamine reagent121
  • 2.2. Radiolabeling of cells with [32P]Pi and stimulation with agonists121
  • 2.3. Extraction of cellular lipids121
  • 2.4. Deacylation of extracted lipids122
  • 2.5. High-performance liquid chromatography (HPLC) separation of deacylated lipids122
  • 3. Measuring PtdIns(3,4,5)P3 by Protein–Lipid Overlay122
  • 3.1. Preparation of recombinant GRP1 PH domain123
  • 3.2. Stimulation of neutrophils and extraction of cellular lipids125
  • 3.3. Neomycin bead-based purification of total PIs126
  • 3.4. Protein–lipid overlay126
  • 4. Conclusions126
  • Acknowledgments128
  • References128
  • Chapter 8: Measuring Phosphorylated Akt and Other Phosphoinositide 3-kinase-Regulated Phosphoprotein131
  • 1. Overview132
  • 1.1. Phosphoinositide 3-kinase (PI3K) introduction132
  • 1.2. PI3K signaling in B lymphocytes133
  • 2. Choosing a Downstream Readout: General Considerations134
  • 2.1. Downstream readout: Akt phosphorylation134
  • 2.2. Downstream readout: mTOR activation135
  • 2.3. Downstream readout: Phosphorylation of Erk136
  • 3. Protocols for Detection of PI3K-Regulated Phosphoproteins by Immunoblot137
  • 3.1. Choice of inhibitors137
  • 3.2. Stimulation of primary B lymphocytes138
  • 3.3. Harvest, lysis, and SDS-PAGE139
  • 3.4. Immunoblotting139
  • 3.5. Interpretation141
  • 4. Protocols for Detection of Phosphoproteins by Flow Cytometry142
  • 4.1. Cell type discrimination by surface marker staining143
  • 4.2. Inhibitor treatment, stimulation, and harvest of primary B lymphocytes144
  • 4.3. Phosflow detection of pAkt and pErk144
  • 4.4. Phosflow detection of pS6145
  • 4.5. Data analysis and interpretation145
  • 5. Discussion147
  • Acknowledgments150
  • References150
  • Chapter 9: Regulation of Phosphatidylinositol 4-Phosphate 5-kinase Activity by Partner Proteins155
  • 1. Introduction156
  • 2. Protocols158
  • 2.1. Preparation of PIP5K for in vitro activity and interaction assays158
  • 2.2. Preparation of PIP5K activators159
  • 2.3. In vitro assay of PIP5K activity161
  • 2.4. Assay for in vitro interactions of PIP5Kgamma661 with beta2 adaptin and talin head164
  • 2.5. Assay for in vivo interaction of endogenous PIP5Kgamma661 and beta2 adaptin165
  • Acknowledgments166
  • References166
  • Chapter 10: Biochemical Analysis of Inositol Phosphate Kinases171
  • 1. Introduction172
  • 2. Experimental Methods174
  • 2.1. IP kinase expression constructs174
  • 2.2. Expression and purification of IP kinases175
  • 2.3. Enzymatic generation of IPs for use as high-performance liquid chromatography (HPLC) standards175
  • 2.4. Analysis of inositol phosphates by thin-layer chromatography (TLC)177
  • 2.5. Kinetic analysis of human IHPK1178
  • 2.6. Purification of inositol phosphates179
  • 2.7. Analysis of PP-IP5 by proton-decoupled 31P NMR180
  • 3. Conclusions182
  • Acknowledgments183
  • References183
  • Chapter 11: Analysis of Phosphoinositides and Their Aqueous Metabolites187
  • 1. Introduction188
  • 2. Cell Sample Extraction191
  • 2.1. Acidified ‘‘Bligh & Dyer’’194
  • 2.2. Neutral extraction196
  • 3. Lipid Phase: TLC, HPLC Separation, and Desalting197
  • 3.1. TLC197
  • 3.2. Indirect Partisil 10 SAX HPLC-deacylated PIs analysis199
  • 3.3. Direct Econosphere NH2 HPLC201
  • 3.4. Post-Econosphere desalting202
  • 4. Aqueous Phase: HPLC Separation, Desalting, and Scintillant Extraction203
  • 4.1. Partisil 10 SAX HPLC–phosphate203
  • 4.2. Partisil 10 SAX HPLC–formate206
  • 4.3. Nucleodex beta-OH HPLC206
  • 4.4. Further Partisil HPLC systems207
  • 4.5. Desalting209
  • 4.6. Scintillant extraction211
  • 5. Chemical Identification212
  • 5.1. Periodate oxidation212
  • 5.2. Acidified butanol214
  • 6. ESI-MS/MS Identification214
  • 6.1. GPIs in general215
  • 6.2. GroPIns4P versus MePIns4P216
  • 7. Standards219
  • 7.1. InsP(n-1)219
  • 7.2. LysoPtdIns4P221
  • 7.3. GroPIns5P222
  • 7.4. Cyclic IPs224
  • 7.5. MePIns4P225
  • 8. Final Considerations226
  • Acknowledgments227
  • References227
  • Chapter 12: Combination of C17 Sphingoid Base Homologues and Mass Spectrometry Analysis as a New App233
  • 1. Introduction234
  • 2. Mass Spectrometry Analysis235
  • 3. Ceramide Synthase236
  • 4. In Vitro Ceramide Synthase Method236
  • 5. Sphingosine Kinase237
  • 6. In Vitro Sphingosine Kinase Method238
  • 7. In Cells Labeling with C17 Sphingoid Base239
  • Acknowledgments240
  • References240
  • Chapter 13: Measurement of Mammalian Sphingosine-1-Phosphate Phosphohydrolase Activity In Vitro and243
  • 1. Introduction244
  • 2. Principle249
  • 3. Measurement of SPP Activity in Cell Lysates249
  • 3.1. Preparation of cell lysates249
  • 3.2. Preparation of labeled S1P250
  • 3.3. In vitro SPP assay251
  • 4. Measurement of SPP Activity in Live Cells252
  • 4.1. Measurement of S1P uptake and hydrolysis in nonpermeabilized cells252
  • 4.2. TLC of sphingoid base phosphates253
  • Acknowledgments253
  • References253
  • Chapter 14: A Rapid and Sensitive Method to Measure Secretion of Sphingosine-1-Phosphate257
  • 1. Introduction258
  • 2. Measurement of S1P259
  • 2.1. Principle259
  • 2.2. Materials259
  • 2.3. Preparation of labeled S1P260
  • 2.4. Determination of recovery of [3H]sphingosine and [3H]S1P by differential extraction260
  • 2.5. Labeling of adherent cells with [3H]sphingosine260
  • 2.6. Labeling of nonadherent cells with [3H]sphingosine261
  • 2.7. Calculations261
  • 3. Conclusions and Perspectives262
  • Acknowledgments263
  • References263
  • Chapter 15: Ceramide Kinase and Ceramide-1-Phosphate265
  • 1. Introduction266
  • 2. Recombinant Expression and Kinetic Analysis of CERK269
  • 2.1. Principle269
  • 2.2. Reagents270
  • 2.3. Buffers270
  • 2.4. Procedures270
  • 3. In Vitro Kinetic Analysis of CERK Activity Using Mixed Micellar Assays272
  • 3.1. Principle272
  • 3.2. Reagents275
  • 3.3. Buffers275
  • 3.4. Procedures275
  • 4. Effective Delivery of C1P to Cells in Tissue Culture to Study Biological Effects278
  • 4.1. Principle278
  • 4.2. Reagents280
  • 4.3. Procedures280
  • 5. Analysis of Levels of Kinase-Derived C1P in Cells281
  • 5.1. Principle281
  • 5.2. Reagents282
  • 5.3. Procedure283
  • 6. Analysis of CERK Localization in Cells284
  • 6.1. Principle284
  • 6.2. Reagents284
  • 6.3. Procedure284
  • 7. Analysis of CERK Function by siRNA-Mediated Manipulation of CERK Expression286
  • 7.1. Principle286
  • 7.2. Reagents287
  • 7.3. Procedure287
  • 8. Analysis of CERK mRNA Levels by Q-PCR288
  • 8.1. Principle288
  • 8.2. Reagents288
  • 8.3. Procedure289
  • Acknowledgments289
  • References290
  • Chapter 16: Measurement of Mammalian Diacylglycerol Kinase Activity In Vitro and in Cells293
  • 1. Introduction294
  • 2. In Vitro Assay of DGK295
  • 2.1. Detergent micelles295
  • 2.2. Liposomes296
  • 2.3. Substrate296
  • 2.4. Enzyme297
  • 2.5. ATP298
  • 2.6. Assay conditions298
  • 2.7. Product isolation299
  • 2.8. Enzyme kinetics299
  • 3. Measuring DGK Activity in Subcellular Compartments300
  • 3.1. Nuclear isolation300
  • 3.2. Sucrose gradient centrifugation for isolation of nuclei300
  • 3.3. Membrane-depleted nuclei301
  • 4. Measuring DGK Activity in Cultured Cells301
  • 4.1. Label and harvest cells302
  • 4.2. Extract lipids302
  • 4.3. Separate lipids302
  • 5. Summary303
  • References303
  • Chapter 17: Lipid Phosphate Phosphatases from Saccharomyces cerevisiae305
  • 1. Introduction306
  • 2. Preparation of Radiolabeled Substrates307
  • 3. Assay Methods307
  • 4. Growth of Yeast308
  • 5. Purification Procedure308
  • 5.1. Preparation of cell extract308
  • 5.2. Preparation of microsomal membranes308
  • 5.3. Preparation of Triton X-100 extract308
  • 5.4. DE53 (DEAE-cellulose) chromatography309
  • 5.5. Affi-Gel blue chromatography309
  • 5.6. Hydroxylapatite chromatography309
  • 5.7. Mono Q I chromatography309
  • 5.8. Mono Q II chromatography310
  • 5.9. Enzyme purity310
  • 5.10. Identification of DPP1 and LPP1 genes310
  • 6. Properties of DPP1- and LPP1-Encoded Lipid Phosphate Phosphatases311
  • Acknowledgment313
  • References313
  • Author Index317
  • Subject Index335
Book details
  • Vendor Elsevier S & T
  • SKU 9780123739650
  • ISBN-13 9780080554099
  • Author Brown, H. Alex
  • Category Science
  • Subject Organic

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This volume in the well-established Methods in Enzymology series features methods for the study of lipids using mass spectrometry techniques. Articles in this volume cover topics such as Phospholipase A1 assays using a radio-labeled substrate and mass spectrometry; Real-time Cell Assays of Phospholipases A2 Using Fluorogenic Phospholipids; Analysis and Pharmacological Targeting of Phospholipase C â interactions with G proteins; Biochemical Analysis of Phospholipase D.; Measurement of Autotaxin/Lysophospholipase D Activity; Platelet-Activating Factor; Quantitative measurement of PtdIns(3,4,5)P3; Measuring Phosphorylated Akt And Other Phosphoinositide 3-Kinase-Regulated Phosphoproteins In Primary Lymphocytes; Regulation of Phosphatidylinositol 4-Phosphate 5-Kinase activity by partner proteins; Biochemical Analysis of Inositol Phosphate Kinases; Analysis of the phosphoinositides and their aqueous metabolites; Combination of C17-sphingoid base homologues and mass spectrometry analysis as a new approach to study sphingolipid metabolism; Measurement of mammalian sphingosine-1-phosphate phosphohydrolase activity in vitro and in vivo; A rapid and sensitive method to measure secretion of sphingosine-1-phosphate; Ceramide Kinase and Ceramide-1-Phosphate; Measurement of Mammalian Diacylglycerol Kinase Activity in vitro and in Cells; Lipid Phosphate Phosphatases from Saccharomyces cerevisiae.