Sphingolipid Metabolism and Cell Signaling, Part B - 1st Edition - ISBN: 9780121822132, 9780080496696

Sphingolipid Metabolism and Cell Signaling, Part B, Volume 312

1st Edition

Serial Volume Editors: Alfred Merrill, Jr. Yusuf Hannun
Editor-in-Chiefs: John Abelson Melvin Simon
eBook ISBN: 9780080496696
Hardcover ISBN: 9780121822132
Imprint: Academic Press
Published Date: 16th October 2000
Page Count: 688
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Table of Contents

  • Contributors to Volume 312
  • Preface
  • Methods in Enzymology
  • Section I: Methods for Analyzing Sphingolipids
    • [1]: 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
    • [2]: Enzymatic Method for Measurement of Sphingosine 1-Phosphate
      • Introduction
      • Principle
      • Materials and Methods
      • Discussion
      • Acknowledgments
    • [3]: Ceramide Mass Analysis by Normal-Phase High-Performance Liquid Chromatography
      • Introduction
      • Lipid Extraction
      • Benzoylation
      • Separation of Ceramide Benzoates by HPLC
      • General Comments
    • [4]: Quantitative Determination of Ceramide Using Diglyceride Kinase
      • Introduction
      • History
      • Procedure
      • Data Interpretation
      • Validation of the Assay
      • Acknowledgment
    • [5]: 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
    • [6]: 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
    • [7]: 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
    • [8]: 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
    • [9]: 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
    • [10]: 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
    • [11]: 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
    • [12]: 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
    • [13]: Thin-Layer Chromatography Immunostaining
      • Introduction
    • [14]: 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
    • [15]: 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
    • [16]: 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
    • [17]: 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)
    • [18]: 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
    • [19]: 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
    • [20]: 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
    • [21]: 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
    • [22]: 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
    • [23]: Synthesis of Fluorescent Substrates and Their Application to Study of Sphingolipid Metabolism in Vitro and in Intact Cells
      • Introduction
      • Synthesis of Fluorescent Sphingolipids
    • [24]: 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
    • [25]: Selection of Yeast Mutants in Sphingolipid Metabolism
      • Introduction
      • Principle of Genetic Selection
      • Suppressors of Ca2+-Sensitive Phenotype of csg2 Null Mutants
      • Isolation and Characterization of the Suppressor Mutants
    • [26]: 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
    • [27]: Mammalian Ganglioside Sialidases: Preparation and Activity Assays
      • Introduction
      • Methods
      • Acknowledgments
  • Section III: Sphingolipid–Protein Interactions and Cellular Targets
    • [28]: 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
    • [29]: 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
    • [30]: 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
    • [31]: Synthesis and Use of Caged Sphingolipids
      • Introduction
      • Materials and Methods
      • Summary of Findings
      • Conclusions and Future Goals
      • Acknowledgments
    • [32]: 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
    • [33]: 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
    • [34]: Analysis of Ceramide-Activated Protein Phosphatases
      • Introduction
      • Assay Methods
      • Acknowledgments
    • [35]: 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
    • [36]: Lectin-Mediated Cell Adhesion to Immobilized Glycosphingolipids
      • Introduction
      • Lectin-Transfected Cells
      • Glycosphingolipid-Coated Microwells
      • Quantification of Cell Adhesion
      • General Considerations
      • Acknowledgments
    • [37]: 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, Gg3 or GM3 Liposomes
      • III Gb4-Dependent Adhesion of Human Embryonal Carcinoma 2102 Cells
      • Comments
      • Acknowledgment
    • [38]: 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
    • [39]: 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
    • [40]: 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
    • [41]: 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
    • [42]: Analysis of Ceramides Present in Glycosylphosphatidylinositol Anchored Proteins of Saccharomyces cerevisiae
      • Principle
      • Overall Approach
      • Experimental Procedures
      • Acknowledgments
    • [43]: Preparation of Functionalized Lipid Tubules for Electron Crystallography of Macromolecules
      • Introduction
      • Lipids
      • Procedure
      • Buffers
      • Crystallization Trials
      • Acknowledgments
  • Section IV: Sphingolipid Transport and Trafficking
    • [44]: 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
    • [45]: 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-C18-GM1 by Immunoelectron Microscopy
      • Localization of Biotin-C18-GM1 by Preembedding Labeling
      • Localization of Biotin-C18-GM1 in LR Gold Embedded Samples
      • Localization of Biotin-C18-GM1 on Ultrathin Cryosections
      • Use of Biotin-C8-GM1 for Studies of Ganglioside Transport in Cells: Applications and Limitations
      • Acknowledgments
    • [46]: 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
    • [47]: 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


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.


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Ratings and Reviews

About the Serial Volume Editors

Alfred Merrill, Jr. Serial Volume Editor

Affiliations and Expertise

Emory University, Atlanta, Georgia, U.S.A.

Yusuf Hannun Serial Volume Editor

Affiliations and Expertise

Medical University of South Carolina, Charleston, U.S.A.

About the Editor-in-Chiefs

John Abelson Editor-in-Chief

Affiliations and Expertise

California Institute of Technology, Division of Biology, Pasadena, U.S.A.

Melvin Simon Editor-in-Chief

Affiliations and Expertise

The Salk Institute, La Jolla, CA, USA