Methods in Membrane Biology: Volume 2

1Nuclear Magnetic Relaxation and the Biological Membrane.- 1. Introduction.- 2. Theory of Nuclear Magnetic Resonance.- 2.1. Nuclear Magnetic Moments.- 2.2. Effects of Nuclear Dipolar Interactions.- 2.3. Molecular Motion.- 2.4. Calculation of Relaxation Times.- 2.5. Spin Diffusion.- 2.6. Chemical Shifts and Coupling Constants.- 3. Practice of Nuclear Magnetic Resonance.- 3.1. Continuous-Wave Techniques.- 3.2. Pulse Techniques.- 3.3. Fourier Transform NMR Spectroscopy.- 3.4. Spin Decoupling.- 3.5. Measurement of Nuclear Magnetic Relaxation Times.- 4. NMR Studies of Lipids.- 4.1. Physical Properties of Phospholipids.- 4.2. 13C Spectra of Lipids.- 4.3. 1H Spectra of Lipids.- 4.4. 31P Spectra of Lipids.- 4.5. Deuterium Spectra of Labeled Lipids.- 4.6. 19F Spectra of Labeled Lipids.- 5. NMR Studies of Biological Membranes.- 5.1. Studies of Membranes without Probe Molecules.- 5.2. Studies with Isotopically Labeled Phospholipids.- 5.3. Small Molecule-Membrane Interactions.- 6. The Biological Membrane.- 6.1. Dynamic Processes in Membranes.- 6.2. Lipid Bilayers.- 6.3. Permeability of Phospholipid Bilayers.- 6.4. Diffusion in Biological Membranes.- 6.5. The Membrane as a Fluid Mosaic.- 7. Appendix.- 7.1. Properties of Vectors.- 7.2. Nuclear Precession.- 7.3. Rotating Frame.- 7.4. Continuous Wave: Rapid Adiabatic Passage.- 7.5. Pulse Experiments.- 8. References.- 2Isolation and Characterization of Surface Membrane Glycoproteins from Mammalian Cells.- 1. Introduction.- 1.1. Relevance to Biological Phenomena.- 1.2. Definition of Glycoproteins and Microheterogeneity.- 1.3. Problems of Obtaining Glycoproteins from Surface Membranes.- 2. Starting Material.- 2.1. Direct.- 2.2. Indirect.- 3. Markers for Glycoproteins.- 3.1. Biological Activity.- 3.2. Fucose.- 3.3. Sialic Acids and Hexosamines.- 3.4. Total Hexoses.- 3.5. Galactose.- 4. Separation of Glycoproteins.- 4.1. Column Chromatography.- 4.2. Electrophoresis.- 4.3. Affinity Chromatography.- 5. Identification of Components.- 5.1. Carbohydrate Units.- 5.2. Glycopeptide Bond.- 5.3. Protein Units.- 6. General Comments.- 7. References.- 3Isolation and Characterization of Membrane Glycosphingolipids.- 1. Introduction.- 1.1. Definition, Classification, and Nomenclature of Glycosphingolipids.- 1.2. Aspects of Glycolipids and Possible Functions.- 2. Isolation of Glycolipids.- 2.1. Preparation of Ganglioside from Various Organs and Erythrocyte Stroma.- 2.2. Separation of Ganglioside Fraction into Hematoside, Mono-, Di-, and Trisialoganglioside.- 2.3. Separation of Ceramide Megalosaccharide and Ganglioside.- 2.4. Separation of Glycolipids from the Lower Phase of the Folch Partition.- 2.5. Separation of Neutral Glycolipids by Column Chromatography.- 2.6. Separation and Preparation of Glycolipids by Thin-Layer Chromatography.- 2.7. Separation of Glycolipids with Positional and Anomeric Isomers.- 2.8. Quantitative Isolation of Total Glycosphingolipid.- 3. Characterization of Glycolipids.- 3.1. Determination of Carbohydrate Components.- 3.2. Determination of Carbohydrate Sequence and Anomeric Linkages.- 3.3. Determination of Anomeric Configuration in Carbohydrate Chains by Proton Magnetic Resonance.- 3.4. Position of Glycosyl Linkages.- 4. References.- 4Preparation of Impermeable Inside-Out and Right-Side-Out Vesicles from Erythrocyte Membranes.- 1. Introduction.- 2. Preparation of Sealed Inside-Out and Right-Side-Out Vesicles.- 2.1. Erythrocytes.- 2.2. Unsealed Ghosts.- 2.3. Modes of Vesicle Formation.- 2.4. Generation of Inside-Out Vesicles.- 2.5. Generation of Right-Side-Out Vesicles.- 2.6. Density Gradient Purification of Sealed Vesicles.- 2.7. Purification by Aqueous Partition.- 2.8. Properties of Vesicle Preparations.- 3. Preparation of Sealed Ghosts.- 3.1. Rationale.- 3.2. Procedures.- 4. Assay of Sidedness and Sealing.- 4.1. Rationale.- 4.2. Acetylcholinesterase Accessibility.- 4.3. Sialic Acid Accessibility.- 4.4. Glyceraldehyde-3-Phosphate Dehydrogenase Accessibility.- 4.5. NADH-Cytochrome c