Methods in Membrane Biology

Examination of the tables of contents of journals - biochemical, molecular biological, ultrastructural, and physiological-provides convincing evidence that membrane biology will be in the 1970s what biochemical genetics was in the 1960s. And for good reason. If genetics is the mechanism for main taining and transmitting the essentials of life, membranes are in many ways the essence of life. The minimal requirement for independent existence is the individualism provided by the separation of "life" from the environment. The cell exists by virtue of its surface membran~. One might define the first living organism as that stage of evolution where macromolecular catalysts or self-reproducing polymers were first segregated from the surrounding milieu by a membrane. Whether that early membrane resembled present cell membranes is irrelevant. What matters is that a membrane would have provided a mechanism for maintaining a local concentration of molecules, facilitating chemical evolution and allowing it to evolve into biochemical evolution. That or yet more primitive membranes, such as a hydrocarbon monolayer at an air-water interface, could also have provided a surface that would facilitate the aggregation and specific orientation of molecules and catalyze their interactions. If primitive membranes were much more than mere passive barriers to free diffusion, how much more is this true of the membranes of contemporary forms of life. A major revolution in biological thought has been the recogni tion that the cell, and especially the eukaryotic cell, is a bewildering maze of membranes and membranous organelles.

Contenu
1 Preparation and Use of Liposomes as Models of Biological Membranes.- 1. Introduction and Historical Survey.- 2. Model Systems.- 2.1. Multilamellar Liposome.- 2.2. Microvesicle.- 2.3. Macrovesicles.- 3. Physical Properties of Membrane Molecules in Aqueous Media.- 3.1. Microscopy.- 3.2. X-Ray Diffraction.- 3.3. Light Scattering.- 3.4. Microcalorimetry.- 3.5. Centrifugation.- 3.6. Temperature Jump.- 3.7. Nuclear Magnetic Resonance.- 3.8. Spin Labels.- 3.9. Microelectrophoresis.- 4. Methods.- 4.1. Surface Area.- 4.2. Dialysis.- 4.3. Equilibrium Diffusion (Low Permeabilities).- 4.4. Nonequilibrium Fluxes (High Permeabilities).- 5. Materials.- 5.1. Egg Phosphatidylcholine.- 5.2. Phosphatidic Acid.- 5.3. Ox-Brain Phospholipids.- 5.4. Characterization and Purity.- 6. References.- 2 Thermodynamics and Experimental Methods for Equilibrium Studies with Lipid Monolayers.- 1. Introduction.- 2. Thermodynamics.- 2.1. The Monolayer System.- 2.2. Equilibrium and Supercompressed States.- 2.3. The Gibbs Phase Rule and Phase Separation in Monolayers.- 2.4. The Film Balance Experiment: A Thermodynamic Analysis.- 2.5. Molecular Thermodynamics of Lipid Monolayers.- 3. Experimental Methods.- 3.1. The Film Balance.- 3.2. Evaluation of ? Using Radiotracers.- 3.3. Preparation and Use of Materials for the Film Balance.- 3.4. Other Techniques.- 4. References.- 3 Circular Dichroism and Absorption Studies on Biomembranes.- 1. Introduction.- 1.1. On the Complexity of Biomembrane Spectra.- 2. Distortions in the Absorption and Circular Dichroism Spectra of Biomembranes.- 2.1. Absorption Effects.- 2.2. Circular Dichroism: Differential Absorption Effects.- 2.3. Instrumental Artifacts.- 3. On Calculations of the Particulate Poly-L-Glutamic Acid Model System.- 4. On the Presence or Absence of Artifact at 222 nm in the CD of Biomembranes.- 5. Approximate Corrections.- 5.1. Pseudo Reference State Approach.- 5.2. Consideration of Membrane Heterogeneity.- 5.3. On Alternate Sources of Absorption Data.- 6. Analysis of Corrected Membrane Data.- 6.1. Specialized Structures Proposed for Specialized Functions.- 6.2. Domains.- 6.3. Empirical Correlations.- 7. References.- 4 Isolation and Serological Evaluation of HL-A Antigens Solubilized from Cultured Human Lymphoid Cells.- 1. Introduction.- 2. Extraction of Soluble Histocompatibility Antigens.- 2.1. Cell Surface Location.- 2.2. Criteria of Solubility.- 2.3. Antigen Source Material.- 2.4. Extraction Techniques.- 2.5. Mechanism of Action of Chaotropic Ions.- 3. Purification of HL-A Antigens.- 3.1. Preparative PAGE.- 4. Physicochemical Characterization of HL-A Antigens.- 4.1. Physical Properties.- 4.2. Chemical Properties.- 5. Molecular Representation of HL-A Antigens on the Lymphoid Cell Surface.- 5.1. Membrane-Antigen Models.- 6. Serological Assays.- 6.1. The Cytotoxic Test.- 6.2. The Blocking Assay.- 6.3. The Quantitative Absorption Test.- 6.4. Comments and Conclusions.- 7. Perspectives.- 8. References.- 5 Dissociation and Reassembly of the Inner Mitochondrial Membrane.- 1. Components of the Inner Mitochondrial Membrane.- 1.1. Introduction.- 1.2. Electron Transport System.- 1.3. Energy Transfer System.- 1.4. Ion Transport and Membrane Lipids.- 1.5. Topology in the Membrane: Extrinsic and Intrinsic Proteins.- 1.6. The Scope of This Chapter.- 2. Methods of Dissociation of the Inner Membrane.- 2.1. Mechanical Disintegration.- 2.2. Chaotropic Agents.- 2.3. Detergents.- 2.4. Organic Solvents.- 2.5. Alkali, Salt, Chelating Agent, and Other Agents.- 3. Methods of Fractionation of Membrane Proteins.- 3.1. Centrifugation.- 3.2. Gel Filtration.- 3.3. Salting Out in the Presence of Detergents.- 3.4. Ion-Exchange Chromatography.- 3.5. Dialysis.- 3.6. Gel Adsorption.- 4. Analysis of the Membrane Components.- 4.1. Light Absorption.- 4.2. Gel Electrophoresis.- 4.3. Immunological Localization of Membrane Proteins.- 4.4. Radioimmunoassay of the Membrane Proteins.- 4.5. Enzyme Assay of the Membrane System.- 5. Purification of Extrinsic Proteins.- 5.1. Proteins of Energy Transfer.- 5.2. Proteins of Electron Transport.- 6. Purification of Intrinsic Proteins.- 6.1. Deficient Particles.- 6.2. Red-Green Separation.- 6.3. Proteins of Energy Transfer.- 7. Reassembly.- 7.1. Adsorption of Extrinsic Proteins on the Membrane.- 7.2. Formation of Vesicles with Intrinsic Proteins and Lipids.- 7.3. Ion Transport and Anisotropy of the Membrane.- 8. References.