Methods in Membrane Biology: Volume 2

ISBN-13: 9781461589624 / Angielski / Miękka / 2012 / 363 str.

Edward D. Korn

Opis

The purposes of this senes were discussed in the preface to Volume I: to present "a range of methods . . . from the physical to the physiological . . . in sufficient detail for the reader to use them in his laboratory" and also to describe "the theoretical backgrounds of the methods and their limita tions in membrane biology" so that the reader will be enabled "to evaluate more critically and to understand more fully data obtained by methods foreign to his] usual experiences. " The chapter by Lee, Birdsall, and Metcalfe with which Volume 2 begins accomplishes these twin goals with a thorough description of the application of nuclear magnetic relaxation measurements to membrane biology together with a lucid and succinct integration of the results of such studies into present concepts of the organi zation of membrane lipids. This then permits speculation on the physical basis of membrane permeability. The powerful tool of NMR spectroscopy will have even fuller application with the development of techniques, al ready partially exploited, for l3C-Iabeling of specific carbon atoms in lipid molecules and with extension of the observations to membrane proteins. The following two chapters, by Glick and by Laine, Stellner, and Hako mori, describe the isolation and characterization of membrane glycoproteins and membrane glycolipids, respectively."

Spis treści

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 Oxidoreductase Accessibility.- 5. Conclusion.- 6. References.- 5Kinetic Studies of Transport Across Red Blood Cell Membranes.- 1. Introduction.- 2. General Considerations.- 2.1. Kinetic Analysis of the Carrier Model.- 2.2. Integrated Rate Equation Treatment.- 2.3. On Errors and Error Analysis in Kinetic Measurements.- 2.4. Some General Technical Considerations.- 3. Experimental Methods—Mediated Transport.- 3.1. Sugar Transport.- 3.2. Amino Acid Transport.- 3.3. Transport of Nucleosides.- 3.4. Monovalent Cation Transport.- 3.5. Calcium Transport.- 3.6. Anion Transport.- 4. Experimental Methods—Nonmediated Transport.- 4.1. Phenomenological Equations.- 4.2. Measurement of for the Membrane of Red Blood Cells.- 4.3. Diffusion of Water and Nonelectrolytes.- 4.4. Determination of the Reflection Coefficient ?.- 5. References.