Enzymes, Receptors, and Carriers of Biological Membranes: A Laboratory Manual

ISBN-13: 9783540137511 / Angielski / Miękka / 1984 / 168 str.

A. Azzi; U. Brodbeck; P. Zahler

Opis

This manual follows at a distance of 3 years the previous one entitled Mem brane Proteins, and, like its predecessor, it is the result of an International Advanced Course sponsored by FEBS, SKMB and SNG, which was held in Bern in September 1983. The experiments offered to the students in the course had to be largely up. dated or chosen from new areas of membrane research, because of the sub stantial and rapid development of the field. Using the protocols of the course, the participants (graduate students, postdoctoral fellows and also senior scientists), in most cases not at all ex pert in biomembrane research, were able to repeat all the experiments suc cessfully. Those few protocols which for some reason did not fulfill the role we expected were modified. These protocols have now been collected in this manual, which we are able to offer to a number of biology, biochemistry and biophysics laborato ries, hoping that the selected number of methods which have been success fully used during the Advanced Course may be useful to them. This manual is also intented for teachers of practical classes, who may use it as a text book and as source of selected references, collected not in the library, but in the laboratory, from the notebooks of the young researchers who have contributed so much to the success of the Course."

Spis treści

I. Analytical Separation Techniques.- Electrophoretic Transfer of Chloroplast Membrane Proteins from SDS-Gels onto Nitrocellulose and Their Immunological Detection.- Investigation of Amphiphilic Nature of Different Forms of Acetylcholinesterase from Torpedo Marmorata by Charge Shift Crossed Immunoelectrophoresis.- Separation of Hydrophobic Membrane Proteins by Phase Partition: Characterization by Two-Dimensional Gel Electrophoresis and Silver-Staining.- Quantitative Analysis of Plant Membrane Lipids by a Combined TLC-GLC Procedure.- Localization of Phosphatidylglycerol in the Membrane of Acholeplasma laidlawii.- II. Protein Structure and Interactions.- Crystallization of Two Membrane Proteins: Bacteriorhodopsin and Photosynthetic Reaction Centres.- Investigation by Crossed Immunoelectrophoresis of Membrane-Cytoskeleton Interactions in Human Erythrocyte Membranes.- III. Labeling and Crosslinking.- Labeling of the Hydrophobic Core of Membranes with 3-trifluoromethyl-3-(m-[125I] iodophenyl) diazirine: Measurement of the Time-Course of the Photolabeling Process.- Selective Labeling of the ADP/ATP Translocator with Eosin-5-Maleimide.- Site-Directed Hydrophobic Labeling of Membrane Proteins: NBD-Modification of Bacteriorhodopsin.- IV. Reconstitution.- Reconstitution of Na, K-ATPase.- Import of Proteins by Isolated Mitochondria.- Isolation and Functional Reconstitution of Rat Liver Cytochrome Oxidase.- V. Spectral Techniques.- Fluorescent Labeling of Band 3 Protein from Erythrocytes.- Spin Labeling of Membranes and Membrane Proteins.- Spectroscopic Measurements in the Subsecond to Second Time Domains: Use of an Optical Multichannel Analyzer for Spectral and Temporal Data Acquisition.- Use of a Potential Sensitive Dye in Studies on Phospholipid and Sarcoplasmic Reticulum Vesicles.- VI. Receptors.- Beta-Adrenoceptor Regulation in Organs of Rats Chronically Treated with Reserpine or Isoproterenol. Radioligand-Receptor Binding Studies.- Functional Changes in the Beta-Adrenoceptor Adenylate Cyclase System During Reticulocyte Maturation in vitro.- Acetylcholine Receptor-Enriched Membrane Fragments from the Electric Organ of Torpedo Marmorata.