Methods in Membrane Biology

1 Immunoelectron Microscopy and Immunofluorescence in Membrane Biology.- 1. Introduction.- 2. Preparation of Antibodies and Other Ligands.- 2.1. The Antibody Molecule.- 2.2. Antisera.- 2.3. Purification of Immunoglobulin and Antibody.- 2.4. Monovalent Antibodies.- 2.5. Hybrid Antibodies.- 2.6. Other Ligands.- 3. Immunoelectron Microscopy (IEM).- 3.1. IEM Techniques.- 3.2. IEM Markers.- 3.3. General Membrane Labeling Methods.- 3.4. Preparation of Antibody-Marker Conjugates.- 3.5. Procedures for Labeling Cells and Cellular Membranes.- 3.6. Problems of Interpretation of the Pattern of Labeling on Biological Membranes.- 4. Immunofluorescence.- 4.1. Introduction.- 4.2. Outline of the Technique. Fluorescent markers.- 4.3 Applications and Interpretation. Comparison with IEM.- 5. Concluding Remarks.- 6. References.- 2 Synaptic Membranes and Junctions from Brain.- 1. Introduction.- 1.1. Criteria for Identifying Synaptic Components in Subcellular Fractions.- 2. Subcellular Fractionation Methods.- 2.1. Isolation of Synaptosomal Plasma Membrane.- 2.2. Isolation of Synaptic Junction-Enriched Subfractions.- 2.3. Purity of the Isolated Fractions.- 3. Properties of Isolated Membranes and Junctions.- 3.1. Ultrastructure.- 3.2. Protein Components.- 3.3. Prospects for Further Neurochemical Study.- 4. References.- 3 High-Sensitivity Differential Scanning Calorimetry in the Study of Biomembranes and Related Model Systems.- 1. Introduction.- 2. Differential Scanning Calorimetry.- 2.1. General Description.- 2.2. The Privalov Calorimeter.- 2.3. Fundamental Principles of DSC.- 2.4. Calorimetric Detectability of Equilibrium Processes.- 3. Applications of High-Sensitivity DSC.- 3.1. Pure Lipids.- 3.2. Lipid Mixtures.- 3.3. Biological Membranes.- 4. Conclusion.- 5. References.- 4 Paramagnetic Hydrophilic Probes in NMR Investigations of Membrane Systems.- 1. Introduction.- 2. Interaction of Paramagnetic Ions with Phospholipid Bilayers.- 2.1. Nature of the Interaction.- 2.2. Identification of Binding Sites.- 2.3. Number of Binding Sites.- 2.4. Lifetime of the Complex.- 3. Changes in the NMR Spectra of Phospholipid Vesicles Caused by Paramagnetic Ions. Separation of Signals from Inward- and Outward-Facing Molecules.- 3.1. Relaxation Probes.- 3.2. Shift Probes.- 4. Environmental Factors Affecting the Paramagnetic Changes of the N(CH3)3 Proton Resonance.- 4.1. Metal-to-Phospholipid Ratio.- 4.2. Temperature.- 4.3. Acidity of the Medium.- 4.4. Foreign Ions.- 4.5. Lipid Environment.- 5. Applications.- 5.1. Stability and Ion Permeability of Sonicated Liposomes.- 5.2. Size Determination of Sonicated Liposomes.- 5.3. Packing Differences of Phospholipid Molecules on the Inner and Outer Surfaces of the Vesicle Bilayer.- 5.4. Outside-Inside Distribution of Lipids in Sonicated Liposomes of Mixed Composition.- 5.5. Intermembrane Phospholipid Exchange.- 5.6. Structure of Serum Lipoproteins.- 5.7. Miscellaneous.- 6. References.- 5 Membrane Mutants of Mammalian Cells in Culture.- 1. Introduction.- 2. Altered Permeability.- 2.1. Nucleoside Transport.- 2.2. Amino Acid Transport.- 2.3. Methotrexate Resistance.- 2.4. Colchicine Resistance.- 2.5. Resistance to Actinomycin D and Puromycin.- 3. Altered Response to Membrane-Specific Agents.- 3.1. Ouabain Resistance.- 3.2. Lectin Resistance.- 3.3. Resistance to Diphtheria Toxin.- 3.4. Resistance to Antisera.- 3.5. Altered Adhesive Properties.- 4. Remarks Concerning Experimental Strategies.- 4.1. Cell Type.- 4.2. Cloning and Culturing.- 4.3. Single versus Complex Selections.- 4.4. Revertants.- 4.5. Hybrids.- 5. Concluding Remarks.- 6. References.