This volume is the third of a series on Membrane Proteins and, like the pre ceding manuals, is the result of an International Advanced Course entitled Isolation and Characterization of Membrane Proteins: Biochemical and Bio physical Aspects sponsored by the Federation of European Biochemical So cieties (FEBS) and the Italian Research Council (CNR). The success of the course and the continuous development in the field of membrane biology has prompted me to publish also in this case the protocols of the experiments which were carried out by the students. The students have been able not only to perform the experiments pub lished in this manual without help from the instructors, but also to suggest improvements, which have been incorporated in the published version. Care has been taken in making the planning and the execution of the ex periments as simple as possible, by listing in detail all the necessary pieces of equipment, test tubes, pipettes, chemicals, etc. At the same time the intro duction and the "philosophy" have been limited to the essential, as also the references, only those having been listed which may help in a better under standing of the principles and of the biological background of a given experi ment. Front Matter....Pages I-XII Front Matter....Pages 1-1 Purification of Cytochrome c Reductase and Oxidase by Affinity Chromatography....Pages 3-10 Molecular Weight Estimation of Membrane Proteins: Gel Permeation Chromatography and Sucrose Gradient Centrifugation....Pages 11-23 The Problem of Light Scattering in the Absorbance and Fluorescence Studies of Proteins in Membranes....Pages 24-31 Reproducible Preparation of Phosphatidylserine Vesicles for Fluorescence Studies of Protein Incorporation into Lipids....Pages 32-41 Purification and Reconstitution of the Proton Translocating Membrane Bound Inorganic Pyrophosphatase from Rhodospirillum rubrum ....Pages 42-48 The Function of Transmembrane Channels: Ion Transport Studies by 23 Na NMR....Pages 49-55 Membrane Signal Transduction via Protein Kinase C....Pages 56-66 Extraction, Partial Purification, and Reconstitution of a Mixture of Carriers from the Inner Mitochondrial Membrane....Pages 67-75 Front Matter....Pages 77-77 The Effect of Polypeptides on the Fluidity of Lipid Bilayers....Pages 79-85 Polypeptide-Lipid Interactions as Studied by 13 C NMR....Pages 86-94 Conformational Changes in Polypeptides and Proteins Brought About by Interactions with Lipids....Pages 95-106 Front Matter....Pages 107-107 Two Examples of Selective Fluorescent Labeling of SH-Groups with Eosin-5-Maleimide: The ADP/ATP Translocator and the Cytochrome c Oxidase Subunit III of Bovine Heart Mitochondria....Pages 109-118 Hydrophobic Photolabeling with 125 I-TID of Red Blood Cell Membranes....Pages 119-123 Use of Fluorescent Probes of the Adenine Nucleotide Carrier for Binding Studies and Analysis of Conformational Changes....Pages 124-135 Spin Labeling of Membranes and Membrane Proteins....Pages 136-148 Front Matter....Pages 149-149 Functional Reconstitution of the Mitochondrial Cytochrome b-c 1 Complex: Effect of Cholesterol....Pages 151-159 Changes of the Membrane Surface Potential Measured by Amphiphilic Fluorescent and ESR Probes....Pages 160-167 Reconstitution of Cytochrome c Oxidase....Pages 168-172 Measurement of Pyruvate Transport in Mitochondria....Pages 173-181 Membrane Proteins: Function, Purification, And Characterization -- Lipid-protein Interaction -- Protein Modification -- Protein Reconstitution. Edited By Angelo Azzi, Lanfranco Masotti, Arnaldo Vecli ; With Contributions By A. Azzi ... [et Al.]. Papers From A Course Sponsored By The Federation Of European Biochemical Societies And The Italian Research Council. Includes Bibliographies.
