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.

I. Membrane Proteins: Function, Purification, and Characterization

Purification of Cytochrome c Reductase and Oxidase by Affinity Chromatography
Molecular Weight Estimation of Membrane Proteins: Gel Permeation Chromatography and Sucrose Gradient Centrifugation
The Problem of Light Scattering in the Absorbance and Fluorescence Studies of Proteins in Membranes
Reproducible Preparation of Phosphatidylserine Vesicles for Fluorescence Studies of Protein Incorporation into Lipids
Purification and Reconstitution of the Proton Translocating Membrane Bound Inorganic Pyrophosphatase from Rhodospirillum rubrum
The Function of Transmembrane Channels: Ion Transport Studies by 23Na NMR
Membrane Signal Transduction via Protein Kinase C
Extraction, Partial Purification, and Reconstitution of a Mixture of Carriers from the Inner Mitochondrial Membrane
II. Lipid-Protein Interaction
The Effect of Polypeptides on the Fluidity of Lipid Bilayers
Polypeptide-Lipid Interactions as Studied by 13C NMR
Conformational Changes in Polypeptides and Proteins Brought About by Interactions with Lipids
III. Protein Modification
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
Hydrophobic Photolabeling with 125I-TID of Red Blood Cell Membranes
Use of Fluorescent Probes of the Adenine Nucleotide Carrier for Binding Studies and Analysis of Conformational Changes
Spin Labeling of Membranes and Membrane Proteins
IV. Protein Reconstitution
Functional Reconstitution of the Mitochondrial Cytochrome b-c1 Complex: Effect of Cholesterol
Changes of the Membrane Surface Potential Measured by Amphiphilic Fluorescent and ESR Probes
Reconstitution ofCytochrome c Oxidase
Measurement of Pyruvate Transport in Mitochondria.