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Sphingolipid Metabolism and Cell Signaling, Part B
1st Edition - October 16, 2000
Editors: Alfred H. Merrill Jr., John N. Abelson, Yusuf A. Hannun, Melvin I. Simon Hardback ISBN:
9 7 8 - 0 - 1 2 - 1 8 2 2 1 3 - 2
9 7 8 - 0 - 0 8 - 0 4 9 6 6 9 - 6
This volume contains information on analyzing sphingolipids, sphingolipid transport and trafficking, and sphingolipid-protein interactions and cellular targets. Its companion…
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This volume contains information on analyzing sphingolipids, sphingolipid transport and trafficking, and sphingolipid-protein interactions and cellular targets. Its companion Volume 311 presents methods used in studying enzymes of sphingolipid biosynthesis and turnover, including inhibitors of some of these enzymes, genetic approaches, and organic and enzymatic syntheses of sphingolipids and analogs. The critically acclaimed laboratory standard for more than forty years, Methods in Enzymology is one of the most highly respected publications in the field of biochemistry. Since 1955, each volume has been eagerly awaited, frequently consulted, and praised by researchers and reviewers alike. Now with more than 300 volumes (all of them still in print), the series contains much material still relevant today--truly an essential publication for researchers in all fields of life sciences. Biochemists, molecular biologists, cell biologists, biomedical researchers, microbiologists, and developmental biologists. Contributors to Volume 312 Preface Methods in Enzymology Section I: Methods for Analyzing Sphingolipids : Analysis of Sphingoid Bases and Sphingoid Base 1-Phosphates by High-Performance Liquid Chromatography Introduction Measurement of Free Sphingoid Bases Measurement of Sphingoid Base 1-Phosphates Application of This Method for Assay of Sphingosine Kinase : Enzymatic Method for Measurement of Sphingosine 1-Phosphate Introduction Principle Materials and Methods Discussion Acknowledgments : Ceramide Mass Analysis by Normal-Phase High-Performance Liquid Chromatography Introduction Lipid Extraction Benzoylation Separation of Ceramide Benzoates by HPLC General Comments : Quantitative Determination of Ceramide Using Diglyceride Kinase Introduction History Procedure Data Interpretation Validation of the Assay Acknowledgment : Analysis of Sphingomyelin, Glucosylceramide, Ceramide, Sphingosine, and Sphingosine 1-Phosphate by Tandem Mass Spectrometry Introduction Mass Spectrometric Scanning Methods Free Sphingoid Bases: So, Sa, So-1-P, Sa-1-P Ceramides Glucosylceramides Sphingomyelin Summary Acknowledgments : Analyses of Glycosphingollpids by High-Performance Liquid Chromatography Introduction Chemical Structure and Supramolecular Organization Biologic Functions Principles of Isolation and Structural Characterization High-Performance Liquid Chromatography of Glycosphingolipids Materials and Methods Anion-Exchange HPLC of Gangliosides Acknowledgments : Sphingolipid Extraction and Analysis by Thin-Layer Chromatography Introduction Extraction Removal of Lipid Contaminants Removal of Salts and Other Small Hydrophilic Contaminants Separation of Anionic and Neutral Sphingolipids Thin-Layer Chromatography Visualization and Quantitative Determination of Sphingolipids on TLC Acknowledgments : Extraction and Analysis of Multiple Sphingolipids from a Single Sample Introduction Principle of DE MALDI-TOF MS Identification of Individual Molecular Species of Sphingolipid by DE MALDI-TOF MS Preparation of Lysosphingolipids Analysis of Lysosphingolipids and Sphingolipids by DE MALDI-TOF MS Gala-Series Glycosphingolipids Globo-Series Glycosphingolipids Ganglio-Series Glycosphingolipids: Gangliosides and Asialogangliosides Sphingomyelin Extraction and DE MALDI-TOF MS Analysis of Multiple Sphingolipids from a Single Sample : Purification of Sphingolipid Classes by Solid-Phase Extraction with Aminopropyl and Weak Cation Exchanger Cartridges Introduction Materials Chromatography Conditions and Procedures Comments Isolation and Purification of Sphingoid Bases from Hydrolysis Mixtures of Parent Sphingolipids Isolation of Free Sphingoid Bases from Serum and Urine : Ganglioside Analysis by High-Performance Thin-Layer Chromatography Introduction Sample Preparation: Extraction and Isolation of Glycosphingolipids Solvent Systems for Ganglioside Separation by HPTLC Chemical Detection of GSLs on TLC Plates HPTLC-Overlay of Glycosphingolipids Combined HPTLC and Mass Spectrometry (HPTLC-MS) Acknowledgment : Purification and Analysis of Gangliosides Introduction Three-Step Purification Method Metabolic Radiolabeling and Analysis of Cellular and Shed Gangliosides Purification of Gangliosides by HPLC Acknowledgments : Thin-Layer Chromatography Blotting Using Polyvinylidene Difluoride Membrane (Far-Eastern Blotting) and Its Applications Introduction Far-Eastern blotting Rapid Purification of Glycosphingolipids by Far-Eastern Blotting Far-Eastern Blotting/Mass Spectrometry (MS) Binding Study on PVDF Membrane Enzyme Reaction on Glycosphingolipid-Transferred PVDF Membrane : Thin-Layer Chromatography Immunostaining : Monoclonal Anti-Glycosphingolipid Antibodies Introduction Strategy for Generation of Anti-Glycolipid Antibodies Methods for Generation of Anti-Glycolipid Monoclonal Antibodies Properties of Monoclonal Antibodies Directed to Glycolipid Antigens : Immunolocalization of Gangliosides by Light Microscopy Using Anti-Ganglioside Antibodies Introduction Factors That Affect Binding of Anti-Ganglioside Antibodies to Gangliosides in Biologic Membranes Experimental Protocols for Using Anti-Ganglioside Antibodies : Cloud-Point Extraction of Gangliosides using Nonionic Detergent C14EO6 Introduction Principles of Cloud-Point Extraction Use of Nonionic Polyoxyethylene Detergent, C14EO6, for Cloud-Point Extraction Protocol for Cloud-Point Extraction of Gangliosides from Cultured Cells and Tissue Samples Acknowledgment : Analyses of Glycosphingolipids Using Clam, Mercenaria mercenaria, Ceramide Glycanase Introduction Preparation of Purified Clam Ceramide Glycanase Purification of Clam CGase by Ion Exchange, Hydrophobic, and Dye Matrices Enzyme Assay Properties of Purified Clam CGase Glycosphingolipid Substrate Specificity of CGase from Clam Isolation and Visualization of Oligosaccharides after Hydrolysis by Clam CGase Analysis of GSLs in Conjunction with Fluorophore-Assisted Carbohydrate Electrophoresis (FACE) : Quantitative Analyses of Binding Affinity and Specificity for Glycolipid Receptors by Surface Plasmon Resonance Introduction Optical Principle of Surface Plasmon Resonance Detection Glycolipid Immobilization on Sensor Chip Surfaces Binding Specificities Kinetic and Affinity Analyses Summary Acknowledgments : Use of Circular Dichroism for Assigning Stereochemistry of Sphingosine and Other Long-Chain Bases Introduction Circular Dichroic Exciton Chirality Method Application of Exciton Chirality Method to Sphingosine and Dihydrosphingosine Two-Step Chromophoric Derivatization Attempts to Lower the Scale of Stereochemical Analysis : Infrared Determination of Conformational Order and Phase Behavior in Ceramides and Stratum Corneum Models Introduction Experimental Techniques IR Spectral Regions Sensitive to Ceramide Structure and Organization New Methods in IR Spectroscopy of Biologic Molecules Summary Acknowledgments : Use of Nuclear Magnetic Resonance Spectroscopy in Evaluation of Ganglioside Structure, Conformation, and Dynamics Introduction Ganglioside Purity and Preparation of Samples Nuclear Magnetic Resonance Experimental Section Nuclear Magnetic Resonance Spectroscopy of Gangliosides: Choice of Solvent and Temperature Primary Structure Secondary Structure Acknowledgment : Fluorescence Quenching Assay of Sphingolipid/Phospholipid Phase Separation in Model Membranes Introduction Principles of Using Fluorescence Quenching to Detect Phase Separation: Behavior of Simple Binary Lipid Mixtures Effect of Composition on Fluorescence in Fully Miscible Lipid Mixtures Effect of Composition on Fluorescence In Partially Immiscible Lipid Mixtures Effect of Temperature on Fluorescence Relationship between Lipid Structure, Lipid Composition, and Domain Formation Sample Composition in Experiments in Which Lipid Composition Is Varied Sample Composition in Experiments in Which Temperature Is Varied Sample Preparation: Lipid Mixing Dispersing Lipid Samples in Buffer at Ambient Temperatures Dispersing Lipid Samples in Buffer at Elevated Temperatures Fluorescence Measurements Data Analysis Factors Limiting Sensitivity and Interpretation of Quenching Experiments Section II: Methods for Analyzing Aspects of Sphingolipid Metabolism in Intact Cells : Synthesis of Fluorescent Substrates and Their Application to Study of Sphingolipid Metabolism in Vitro and in Intact Cells Introduction Synthesis of Fluorescent Sphingolipids : Selection of Mammalian Cell Mutants in Sphingolipid Biosynthesis Introduction CHO Cells as Somatic Cell Genetic Tools Isolation of CHO Mutants Resistant to SM-Directed Cytolysin Metabolic Labeling of Lipids with Radioactive Serine Analyses of the Contents of SM and GM3 In CHO Cells Identification of Defective Steps in LY-A and LY-B Strains Acknowledgments : Selection of Yeast Mutants in Sphingolipid Metabolism Introduction Principle of Genetic Selection Suppressors of Ca 2+-Sensitive Phenotype of csg2 Null Mutants Isolation and Characterization of the Suppressor Mutants : Fluorescence-Based Selection of Gene-Corrected Hematopoietic Stem and Progenitor Cells Based on Acid Sphingomyelinase Expression Introduction General Methods Typical Results from Pulse-Chase Labeling of Normal, ASMKO, and Transduced Cells Comments Acknowledgments : Mammalian Ganglioside Sialidases: Preparation and Activity Assays Introduction Methods Acknowledgments Section III: Sphingolipid–Protein Interactions and Cellular Targets : Effects of Sphingosine and Other Sphingolipids on Protein Kinase C Introduction Effectiveness and Specificity of Interaction Use of Sphingolipids in Cell Systems Manipulation of Experimental Systems Solutions Monitoring PKC and PKC-Mediated Responses to Sphingolipids : Kinetic Analysis of Sphingoid Base Inhibition of Yeast Phosphatidate Phosphatase Introduction Preparation of Enzymes Preparation of Radioactive Phosphatidate Preparation of Sphingoid Bases Preparation of Triton X-100/Lipid-Mixed Micelles Phosphatidate Phosphatase Assay Analysis of Kinetic Data Inhibition of Phosphatidate Phosphatase Activity by Sphingoid Bases and Structural Requirements for Inhibition Kinetic Analysis of Sphingoid Base Inhibition Acknowledgments : Assays of Sphingosine-Dependent Kinase for 14-3-3 Protein Introduction Cell Culture and Stimulation with TPA Separation of SDKl-Containing Fraction by Chromatography on Q-Sepharose Standard SDK1 Activity Assay Simplified SDK1 Activity Assay : Synthesis and Use of Caged Sphingolipids Introduction Materials and Methods Summary of Findings Conclusions and Future Goals Acknowledgments : Binding of Sphingosine 1-Phosphate to Cell Surface Receptors Introduction Preparation of [ 32P]SPP Binding Assay Factors Affecting Binding of SPP to SPPR Expressing Cells Conclusions Acknowledgments : Use of Short-Chain Ceramides Introduction Why Use Short-Chain Ceramides? When Is the Use of Short-Chain Ceramides Informative? How Should Short-Chain Ceramides Be Used? Examples: Use of Short-Chain Ceramides in Cell Biology Supporting and Corroborating Lines of Investigation Conclusions Acknowledgments : Analysis of Ceramide-Activated Protein Phosphatases Introduction Assay Methods Acknowledgments : Use of Affinity Chromatography and TID-Ceramide Photoaffinity Labeling for Detection of Ceramide-Binding Proteins Introduction Ceramide-Affinity Chromatography [ 125I]TID-Ceramide Photoaffinity Labeling Acknowledgments : Lectin-Mediated Cell Adhesion to Immobilized Glycosphingolipids Introduction Lectin-Transfected Cells Glycosphingolipid-Coated Microwells Quantification of Cell Adhesion General Considerations Acknowledgments : Analysis of Glycolipid-Dependent Cell Adhesion Based on Carbohydrate–Carbohydrate Interaction I Introduction II GM3-Dependent Adhesion of Mouse B16 Melanoma Cells to Mouse or Human Endothelial Cells Adhesion of B16 Melanoma Cells to Nonactivated Human or Mouse Endothelial Cells, and Its Inhibition by LacCer, Gg 3 or GM3 Liposomes III Gb 4-Dependent Adhesion of Human Embryonal Carcinoma 2102 Cells Comments Acknowledgment : Analysis of Interactions between Glycosphingolipids and Microbial Toxins Introduction Assay of Glycolipid Binding Retrograde Intracellular Glycolipid Traffic Use of Toxin Conjugates to Monitor Glycolipid Retrograde Transport within Cells Acknowledgments : Oxidation of Aglycone of Glycosphingolipids: Serine and Ceramide Acid Precursors for Soluble Glycoconjugates Introduction Oxidation of Sphingosine Double Bond of Glycolipids Synthesis of Glycoconjugates Summary Acknowledgment : Separation of Glycosphingolipid-Enriched Microdomains from Caveolar Membrane Characterized by Presence of Caveolin Introduction Preparation of Low-Density Detergent-Insoluble Membrane Fraction Separation of DIM into GM3 /Src-Contalning and Caveolin-Containing Subfractions by Antibodies Lipid Composition of DIM Subfractions of Mouse Melanoma B16 Cells: GSL Signaling Domain and Caveolin-Containing Fraction Comments Acknowledgment : Reconstitution of Sphingolipid–Cholesterol Plasma Membrane Mlcrodomalns for Studies of Virus-Glycolipid Interactions Introduction Surface Pressure Measurements Kinetics Studies Reconstitution of Cholesterol-GalCer Microdomains Mapping of GalCer-Binding Site on gp120 Interaction of CD4 with Glycosphingolipid Microdomains Reconstitution of HIV-1 Fusion Complex in Glycosphingolipid Microdomain Glycosphingolipid Microdomains as Preferential Sites of Formation of HIV-1 Fusion Complex Acknowledgments : Analysis of Ceramides Present in Glycosylphosphatidylinositol Anchored Proteins of Saccharomyces cerevisiae Principle Overall Approach Experimental Procedures Acknowledgments : Preparation of Functionalized Lipid Tubules for Electron Crystallography of Macromolecules Introduction Lipids Procedure Buffers Crystallization Trials Acknowledgments Section IV: Sphingolipid Transport and Trafficking : Applications of BODIPY–Sphingolipid Analogs to Study Lipid Traffic and Metabolism in Cells Introduction Measurements of Concentration-Dependent Spectral Shift of BODIPY–Lipids in Living Cells Conclusions : Using Biotinylated Gangliosides to Study Their Distribution and Traffic in Cells by Immunoelectron Microscopy Introduction Materials and General Methods Biotinylated Ganglioside GM1 Incorporation of Biotinylated GM1 into Cultured Fibroblasts Localization of Biotin-C 18-GM1 by Immunoelectron Microscopy Localization of Biotin-C 18-GM1 by Preembedding Labeling Localization of Biotin-C 18-GM1 in LR Gold Embedded Samples Localization of Biotin-C 18-GM1 on Ultrathin Cryosections Use of Biotin-C 8-GM1 for Studies of Ganglioside Transport in Cells: Applications and Limitations Acknowledgments : Assays for Transmembrane Movement of Sphingolipids Introduction General Aspects of Assays for Translocation of Sphingolipids across Membranes Calculating Translocation of Sphingolipids across Membranes Chemical Modification (Table I) Enzymatic Modification Noncovalent Protein Binding Lipid Exchange and Transfer Proteins Application of Analogs of Lipids of Interest Acknowledgments Section V: Other Methods : Compilation of Methods Published in Previous Volumes of Methods in Enzymology Introduction Sphingolipid Metabolism Inhibitors of Sphingolipid Metabolism Chemical and Enzymatic Syntheses Methods for Isolating and Analyzing Sphingolipids Methods for Analyzing Aspects of Sphingolipid Metabolism in Intact Cells Sphingolipid-Protein Interactions and Cellular Targets Sphingolipid Transport and Trafficking Other Methods Author Index Subject Index
Published: October 16, 2000
Hardback ISBN: 9780121822132
eBook ISBN: 9780080496696
Alfred H. Merrill Jr.
Affiliations and expertise
Emory University, Atlanta, Georgia, U.S.A. John N. Abelson
Affiliations and expertise
California Institute of Technology, Division of Biology, Pasadena, U.S.A. Yusuf A. Hannun
Affiliations and expertise
Medical University of South Carolina, Charleston, U.S.A. Melvin I. Simon
Affiliations and expertise
The Salk Institute, La Jolla, CA, USA