Sphingolipid Metabolism and Cell Signaling, Part B (eBook)

Yusuf A. Hannun, Alfred H. Merrill Jr. (Herausgeber)

eBook Download: PDF | EPUB

2000 | 1. Auflage
688 Seiten
Elsevier Science (Verlag)
978-0-08-049669-6 (ISBN)

Infos

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.

Inhalt

Front Cover 1
Sphingolipid Metabolism and Cell Signaling 4
Copyright Page 5
Table of Contents 6
Contributors to Volume 312 12
Preface 18
Volumes in Series 20
Section I: Methods for Analyzing Sphingolipids 40
Chapter 1. Analysis of Sphingoid Bases and Sphingoid Base 1-Phosphates by High-Performance Liquid Chromatography 42
Chapter 2. Enzymatic Method for Measurement of Sphingosine 1-Phosphate 48
Chapter 3. Ceramide Mass Analysis by Normal-Phase High-Performance Liquid Chromatography 55
Chapter 4. Quantitative Determination of Ceramide Using Diglyceride Kinase 61
Chapter 5. Analysis of Sphingomyelin, Glucosylceramide, Ceramide, Sphingosine, and Sphingosine 1-Phosphate by Tandem Mass Spectrometry 71
Chapter 6. Analyses of Glycosphingolipids by High-Performance Liquid Chromatography 84
Chapter 7. Sphingolipid Extraction and Analysis by Thin-Layer Chromatography 103
Chapter 8. Extraction and Analysis of Multiple Sphingolipids from a Single Sample 119
Chapter 9. Purification of Sphingolipid Classes by Solid-Phase Extraction with Aminopropyl and Weak Cation Exchanger Cartridges 140
Chapter 10. Ganglioside Analysis by High-Performance Thin-Layer Chromatography 154
Chapter 11. Purification and Analysis of Gangliosides 174
Chapter 12. Thin-Layer Chromatography Blotting Using Polyvinylidene Difluoride Membrane (Far-Eastern Blotting) and Its Applications 184
Chapter 13. Thin-Layer Chromatography Immunostaining 196
Chapter 14. Monoclonal Anti-Glycosphingolipid Antibodies 199
Chapter 15. Immunolocalization of Gangliosides by Light Microscopy Using Anti-Ganglioside Antibodies 218
Chapter 16. Cloud-Point Extraction of Gangliosides Using Nonionic Detergent C14EO6 226
Chapter 17. Analyses of Glycosphingolipids Using Clam, Mercenaria mercenaria, Ceramide Glycanase 235
Chapter 18. Quantitative Analyses of Binding Affinity and Specificity for Glycolipid Receptors by Surface Plasmon Resonance 244
Chapter 19. Use of Circular Dichroism for Assigning Stereochemistry of Sphingosine and Other Long-Chain Bases 256
Chapter 20. Infrared Determination of Conformational Order and Phase Behavior in Ceramides and Stratum Corneum Models 267
Chapter 21. Use of Nuclear Magnetic Resonance Spectroscopy in Evaluation of Ganglioside Structure, Conformation, and Dynamics 286
Chapter 22. Fluorescence Quenching Assay of Sphingolipid/ Phospholipid Phase Separation in Model Membranes 311
Section II: Methods for Analyzing Aspects of Sphingolipid Metabolism in Intact Cells 330
Chapter 23. Synthesis of Fluorescent Substrates and Their Application to Study of Sphingolipid Metabolism in Vitro and in Intact Cells 332
Chapter 24. Selection of Mammalian Cell Mutants in Sphingolipid Biosynthesis 343
Chapter 25. Selection of Yeast Mutants in Sphingolipid Metabolism 356
Chapter 26. Fluorescence-Based Selection of Gene-Corrected Hematopoietic Stem and Progenitor Cells Based on Acid Sphingomyelinase Expression 369
Chapter 27. Mammalian Ganglioside Sialidases: Preparation and Activity Assays 378
Section III: Sphingolipid–Protein Interactions and Cellular Targets 398
Chapter 28. Effects of Sphingosine and Other Sphingolipids on Protein Kinase C 400
Chapter 29. Kinetic Analysis of Sphingoid Base Inhibition of Yeast Phosphatidate Phosphatase 412
Chapter 30. Assays of Sphingosine-Dependent Kinase for 14-3-3 Protein 420
Chapter 31. Synthesis and Use of Caged Sphingolipids 426
Chapter 32. Binding of Sphingosine 1-Phosphate to Cell Surface Receptors 440
Chapter 33. Use of Short-Chain Ceramides 446
Chapter 34. Analysis of Ceramide-Activated Protein Phosphatases 459
Chapter 35. Use of Affinity Chromatography and TID-Ceramide Photoaffinity Labeling for Detection of Ceramide-Binding Proteins 468
Chapter 36. Lectin-Mediated Cell Adhesion to Immobilized Glycosphingolipids 477
Chapter 37. Analysis of Glycolipid-Dependent Cell Adhesion Based on Carbohydrate–Carbohydrate Interaction 486
Chapter 38. Analysis of Interactions between Glycosphingolipids and Microbial Toxins 498
Chapter 39. Oxidation of Aglycone of Glycosphingolipids: Serine and Ceramide Acid Precursors for Soluble Glycoconjugates 512
Chapter 40. Separation of Glycosphingolipid-Enriched Microdomains from Caveolae Characterized by Presence of Caveolin 527
Chapter 41. Reconstitution of Sphingolipid–Cholesterol Plasma Membrane Microdomains for Studies of Virus–Glycolipid Interactions 534
Chapter 42. Analysis of Ceramides Present in Glycosylphosphatidylinositol Anchored Proteins of Saccharomyces cerevisiae 545
Chapter 43. Preparation of Functionalized Lipid Tubules for Electron Crystallography of Macromolecules 554
Section IV: Sphingolipid Transport and Trafficking 560
Chapter 44. Applications of BODIPY–Sphingolipid Analogs to Study Lipid Traffic and Metabolism in Cells 562
Chapter 45. Using Biotinylated Gangliosides to Study Their Distribution and Traffic in Cells by Immunoelectron Microscopy 573
Chapter 46. Assays for Transmembrane Movement of Sphingolipids 601
Section V: Other Methods 620
Chapter 47. Compilation of Methods Published in Previous Volumes of Methods in Enzymology 622
Author Index 632
Subject Index 670

Textauszug

[1]

Analysis of Sphingoid Bases and Sphingoid Base 1-Phosphates by High-Performance Liquid Chromatography

Alfred H. Merrill, Jr.; Thomas B. Caligan; Elaine Wang; Katherine Peters; JoyceOu

Introduction

Long-chain bases (sphingosine, sphinganine, phytosphingosine, and homologs of these compounds) and their derivatives (sphingoid base 1-phosphates, psychosines, etc.) are highly bioactive compounds that appear as intermediates of sphingolipid metabolism and cell signaling, and are elevated on disruption of sphingolipid metabolism in disease. Among the methods that are available for quantitation of sphingoid bases, reversed-phase high-performance liquid chromatography (HPLC) of the fluorescent, o-phthalaldehyde derivatives remains one of the most sensitive and informative since it is sensitive and able to resolve individual molecular species. 1,2 This chapter describes these procedures as well as their application for analysis of sphingoid bases1 and sphingoid base 1-phosphates2 as substrates and products of sphingosine kinase.

Measurement of Free Sphingoid Bases

Free sphingoid bases are relatively easy to analyze because they can be extracted in high yield using standard organic solvents and, once glycero-lipids are removed by base treatment, there are relatively few contaminants that elute on reversed-phase HPLC in the vicinity of the o-phthalaldehyde derivatives of most naturally occurring sphingoid bases.1 Because there is considerable interest in analyzing the amounts of sphinganine in blood, tissues, and urine from animals exposed to naturally occurring inhibitors of ceramide synthase (fumonisins), many methods have been developed for sphingoid base extraction and analysis,3–7 most of which are modifications of the method described here.

Lipid Extraction

Sphingoid bases can be extracted from most mammalian samples (tissues, cells in culture, serum, etc.) as follows. Tissues are usually homogenized in 4 volumes (w/v) of ice-cold 0.05 M potassium phosphate buffer (pH 7.0) and 0.1 ml of the homogenate is used. Cells in culture are chilled, washed with cold buffered saline, and scraped from the dishes (106 cells are usually used). Blood, serum, or plasma (0.1–0.5 ml) can be used directly. The samples are placed in 13 × 100-mm screw-cap test tubes (standard borosilicate tubes with Teflon caps); 1.5 ml of CHCl3: methanol (1:2, v/v) is added (more of this solvent can be added if phase separation occurs); an internal standard of 50–400 pmol of eicosasphinganine (C20-sphinganine, which is available commercially from a number of suppliers, such as Matreya, Pleasant Gap, PA) is added; and the extract components are mixed vigorously (for example, by sonication in a bath-type sonicator). Next, 2 ml each of CHCl3 and water are added, and the two phases are separated by centrifugation using a tabletop centrifuge. The upper phase is discarded, and the CHCl3 phase is washed 2–3 times with water, drained through a small column (Pasteur pipette) containing anhydrous sodium sulfate (granular), and dried in vacuo.

The extracts are resuspended in 1 ml of 0.1 M KOH in methanol and CHC13 (4:1, v/v), incubated at 37° for 1 hr, and the sphingoid bases are extracted by adding 2 ml each of CHCl3 and water and the CHCl3 phase is washed with water, dried over sodium sulfate, and the solvent removed in vacuo.

Formation and Analyses of o-Phthalaldehyde Derivatives of Sphingoid Bases

The o-phthalaldehyde (OPA) derivatives are prepared by a dissolving the base-treated extracts in 50 μl of methanol, and adding (with rapid mixing) 50 μl of OPA reagent. The OPA reagent is prepared by mixing (a) 99 ml of 3% (w/v) boric acid in water (pH adjusted to 10.5 with KOH), (b) 1 ml of ethanol containing 50 mg of OPA, and (c) 50 μ1 of 2-mercaptoethanol. The OPA reagent is stable for approximately 1 week when stored in the refrigerator.

After incubation for approximately 15 min at room temperature, 250–500 μ1 of the mobile phase solvent for HPLC (methanol: 5 mM potassium phosphate buffer, pH 7.0, usually in a ratio of 90:10, v/v) is added. After a few minutes, the samples are centrifuged briefly in a microcentrifuge to clarify, and aliquots are analyzed by HPLC. Until injection, the OPA derivatives are kept at ≤8° the derivatives are usually stable for up to 48 hr when kept cold.

HPLC analyses are conducted using a C18 reversed-phase column (Waters, Milford, MA, Radial Pak C18 column: 5 μm, type 8NVC18) with a small Cig guard column (2.5 cm; Universal Scientific, Atlanta, GA), and an isocratic solvent system (methanol: 5 mM potassium phosphate, pH 7.0, at 90:10, v/v) at a flow rate of 2 ml/min. The fluorescence of the OPA derivatives is measured with an excitation wavelength of 340 nm and an emission wavelength of 455 nm (or a 418-nm cutoff filter).

Under these conditions, the retention times of sphingosine, sphinganine, and 4-hydroxysphinganine (phytosphinganine) are approximately 11, 15, and 7 min, respectively. The C20-sphinganine internal standard elutes at approximately 20 min. The sensitivity of the method will depend on the fluorescence detector; with a Shimadzu (Kyoto, Japan) RF-535 spectroflu-orometer, we have found that fluorescence is proportional to the amount of sphingosine from at least 10–1000 pmol of injected sphingoid base.

Comments

Some samples also contain fluorescent contaminants that may be mistaken for sphingoid bases, but this can be ascertained by examining the extract on HPLC without OPA derivatization.

Sphingoid bases of 18-carbon atoms predominate in most mammalian samples; however, other homologs (including 20-carbon species) are found in milk sphingolipids and brain gangliosides, for example, and are common in other organisms, such as yeast, fungi, and plants. Hence, the HPLC profile for a given sample should be examined without adding the C20-sphinganine internal standard to ensure that it does not interfere with the analysis. When other sphingoid bases are present, the HPLC solvent can be varied (including the use of a gradient system) to achieve the desired separation. Other alkylamines can also be used as internal standards, but the fluorescence yield versus that of sphingoid bases must be determined.

This procedure is also able to analyze psychosine and presumably other lysosphingolipids, although these will probably not be extracted efficiently by this procedure. For more polar sphingoid bases and derivatives, the solid-phase extraction described below can be substituted.

Other Modifications to This Method

A number of simplified versions of this method have been published.3–8 One that we have used omits the first extraction step by first mixing the sample with 1 ml of CHCl3: methanol (1:4, v/v) containing 0.1 M KOH, incubating it for 1 hr at 37°, then continuing the two-phase extraction described above to obtain a dry extract for OPA derivatization and HPLC. In most cases, this gives a slightly higher yield of the sphingoid bases.

Other methods for analyzing sphingoid bases have been summarized in Ref. 1. There is also a method that uses sphingosine kinase to quantify sphingosine.8

Measurement of Sphingoid Base 1-Phosphates

The analysis of sphingoid base 1-phosphates presents two additional challenges: their high water solubility results in partial extraction by organic solvents, and the OPA derivatives of sphingoid base 1-phosphates are more unstable. These problems were solved by the following modifications.2

Solid-Phase Extraction of Sphingoid Base 1-Phosphates

Each sample is mixed with an equal volume of methanol, then gravity loaded onto a small RP-18 column. The column is prepared by suspending LiChroprep RP-18 (40- to 63-µm particle size, EM Science, Gibbston, NJ) in methanol :H20 (1:1, v/v), allowing the matrix to settle, then aspirating off the fines. The column (approximately 5 mm × 4 cm) is prepared by placing a small amount of coarse glass wool in the neck of a Pasteur pipette, adding the RP-18 slurry, then washing the resin (the RP-18 is sometimes washed first with hexane to remove fluorescent contaminants) with at least 3 ml of methanol: H2O (1:1, v/v) before addition of the sample. If necessary, precipitated material in the sample-methanol mixture can be removed by centrifugation in a tabletop centrifuge.

As soon as the last portion of the sample is loaded, the column is washed with 2 ml methanol :H2O (1:1, v/v) followed by 4 ml of methanol :H2O (3:1, v/v) containing 0.1% acetic acid. Sphingoid base 1-phosphates are next eluted with 4 ml of methanol: H2O (9:1) containing 10 mM potassium phosphate buffer (pH 7.2), and the solvent is removed under vacuum, with care to analyze the samples as soon as possible after drying to minimize decomposition. Using this procedure, recoveries are typically ∼85%.

Preparation and Analysis of o-Phthalaldehyde Derivatives

The OPA derivatives are prepared by a minor modification of the method described above for free sphingosine. This modification is the addition of 100 μ1...

Produkt-Details

• Erscheint lt. Verlag: 25.10.2000
• Mitarbeit: Chef-Herausgeber: John N. Abelson, Melvin I. Simon
• Sprache: englisch
• Themenwelt: Naturwissenschaften ► Biologie ► Biochemie
• Themenwelt: Naturwissenschaften ► Biologie ► Genetik / Molekularbiologie
• Themenwelt: Naturwissenschaften ► Biologie ► Zellbiologie
• Themenwelt: Technik
• ISBN-10: 0-08-049669-5 / 0080496695
• ISBN-13: 978-0-08-049669-6 / 9780080496696