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Über dieses Buch

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.



Analytical Separation Techniques


Electrophoretic Transfer of Chloroplast Membrane Proteins from SDS-Gels onto Nitrocellulose and Their Immunological Detection

The electrophoretic transfer of proteins out of a polyacrylamide slab gel onto a sheet of nitrocellulose was introduced by Towbin et al. (1979). Their work was based on the previously developed technique for the transfer of nucleic acids onto papers or membranes of derivatized cellulose and, together with the technical know-how of several manufacturers, opened a wide new area of experimental procedures in protein analysis (for a review see Technical Bulletins of Bio-Rad and of Schleicher and Schüll Inc.).
A. Boschetti, J. Karlen, H. P. Michel

Investigation of Amphiphilic Nature of Different Forms of Acetylcholinesterase from Torpedo Marmorata by Charge Shift Crossed Immunoelectrophoresis

The electric organ of Torpedo marmorata contains two different classes of acetylcholinesterase (AChE) (for review see Massoulie and Bon 1982) which can be distinguished by their solubilization and sedimentation behavior. One class comprises the high salt-soluble enzyme forms (HSS-AChE), the other the detergent-soluble enzyme (DS-AChE). Both classes can be purified by affinity chromatography. HSS-AChE is an asymmetric collagen tailed form sedimenting at 17S and 13S with a molecular weight of the catalytic subunit of 68,000. The 17S form consists of three, the 13 S of two catalytically active tetramers. These are attached to collagen-like tails forming a triple helix. DS-AChE is a dimer with a S-value of 6.5S in the presence of Triton X-100 and has a subunit molecular weight of 66,000. By limited digestion of DS-AChE with proteinase K a catalytically active enzyme sedimenting at 7.5S is formed.
S. Stieger, U. Brodbeck

Separation of Hydrophobic Membrane Proteins by Phase Partition: Characterization by Two-Dimensional Gel Electrophoresis and Silver-Staining

Most membrane glycoproteins are integral components of the lipid bilayer, and therefore contain a hydrophobic sequence. Several methods are available for characterization of these hydrophobic components: These include hydrophobic labelling, sequence determination, hydrophobic chromatography and phase partition. An elegant version of the latter technique was described by Bordier (1981). This consists of using a non-ionic detergent with a cloud-point somewhat higher than room temperature to partition proteins into hydrophobic and hydrophilic fractions.
K. J. Clemetson, M.-L. Zahno, B. Wyler

Quantitative Analysis of Plant Membrane Lipids by a Combined TLC-GLC Procedure

In the analysis of membrane acyl lipids two aspects are very often of major interest: The quantitative determination of the particular lipid components and their fatty acid composition. The scope of the experiment is the separation of a lipid mixture by two-dimensional TLC and the subsequent separation and quantitative determination by GLC of the fatty acids. From the total amount of fatty acids, the amount of particular lipid components is calculated.
W. Eichenberger

Localization of Phosphatidylglycerol in the Membrane of Acholeplasma laidlawii

The two layers of a biological membrane can have different phospholipid compositions. A good example of this is the erythrocyte membrane, where the outer layer consists mainly of choline-containing phospholipids, whereas the aminophospholipids are found preferentially in the inner layer. Phospholipid asymmetry is furthermore detectable in plasma membranes of eukaryotic cells, in virus membranes, and in various bacterial membrane systems.
J. Op Den Kamp

Protein Structure and Interactions


Crystallization of Two Membrane Proteins: Bacteriorhodopsin and Photosynthetic Reaction Centres

The only method to determine the spatial structure of proteins at a resolution sufficient to trace the peptide chain and to position the amino acids is X-ray crystallography. Crystallography depends on the availability of large, well-ordered crystals. Membrane proteins could not be crystallized until recently. The main reason for the lack of success in the crystallization of membrane proteins lies in the amphiphilic nature of the membrane proteins surface: Those surface domains exposed to the aqueous phases on both sides of the membrane are hydrophilic, like the surface of globular proteins, whereas the surface domains in contact with the alkane chains of the lipids must be highly hydrophobic. As a consequence, typical membrane proteins can only be solubilized with the help of detergents: Detergent micelles replace the lipids of the membrane and shield the hydrophobic surface domains of the membrane proteins against water. Mild detergents have to be used to solubilize membrane proteins in a functionally active state.
H. Michel

Investigation by Crossed Immunoelectrophoresis of Membrane-Cytoskeleton Interactions in Human Erythrocyte Membranes

The red blood cell needs a high stability as well as a high deformability and elasticity to be able to pass through the capillaries of the circulation system without being disrupted. These properties are supported by the cytoskeleton of the cell membrane and by its dynamic interactions with membrane spanning proteins and the lipid bilayer. A model for these postulated interactions is shown in Fig. 1: Spectrin (Band 1 and Band 2; nomenclature according to Fairbanks et al.) is linked with the syndeins (Bands 2.1, 2.2., 2.3 and 2.6) which are in turn associated with Band 3, the major integral membrane protein. These interactions are considered as the major forces which anchor the membrane skeleton to the bilayer core. Within the membrane cytoskeleton spectrin (Bands 1 and 2) is believed to form a tetramer which is built up of two heterodimers. The proteins called Band 4.1a and Band 4.1b bind to the ends of these tetramers and connect spectrin to Band 5 (the red cell actin). It has further been postulated that Bands 4.1a and 4.1b are attached to the lipid bilayer, a suggestion which has also been made for spectrin. However, the association sites have not yet been identified.
P. Ott, P. Bütikofer

Labelling 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

Labeling of membranes from within the lipid core aims at identifying those polypeptide segments of membrane proteins that are buried within the lipid bilayer (Brunner 1981, Bayley 1982). This method, therefore, represents a useful complement to the more established surface labeling techniques of membranes.
J. Brunner, R. Aggeler, B. Reber

Selective Labeling of the ADP/ATP Translocator with Eosin-5-Maleimide

The ADP/ATP translocator is located in the inner membrane of mitochondria, where it catalyzes the vectorial exchange between cytosolic ADP and matrix ATP, a key process in the cellular energy supply of aerobic organism (for review see Klingenberg 1980; Vignais 1976; Vignais et al. 1982). In beef heart mitochondria the translocator is the most abundant integral protein, being about 10% of the total mitochondrial protein. The Mr of the monomeric translocator is ~ 32,500. The translocator has been isolated as a complex with carboxyatractylate (CAT) or bongkrekic acid using extraction procedures with Triton X-100 and it was found that the complex is a dimer. A complete amino acid sequence of the translocator revealed that the monomeric protein contains 4 cysteins over 297 amino acids. Masking or modification of sulfydryl groups, which are essential for the nucleotide translocation, causes the inhibition of the translocation activity. At least one sulfydryl group is maked when CAT is bound to the translocator.
M. Müller

Site-Directed Hydrophobic Labeling of Membrane Proteins: NBD-Modification of Bacteriorhodopsin

Membrane proteins which are involved in the signal transmittance through a biological membrane or participate in the transfer of solutes across cell membranes are presumed to span the lipid bilayer. The proteins’ transmembrane disposition can be assayed by various techniques including labeling procedures with hydrophilic chemical reagents, enzyme-mediated modifications, protease accessibility and antibody interaction. Convincing evidence for the intramembraneous disposition of a protein arises from hydrophobic labeling studies. Those segments of intrinsic membrane proteins that are in close association with the hydrocarbon core of a lipid bilayer may be identified by using hydrophobic photogenerated reagents (Gitler and Bercovici 1980, Brunner and Semenza 1981, Ross et al. 1982).
H. Sigrist, E. Kislig, P. Allegrini



Reconstitution of Na, K-ATPase

The Na, K-ATPase (EC or sodium pump is a membrane-spanning protein-lipid complex composed of a catalytic α subunit (about 100,000 mol wt), of a β subunit (35–65,000 mol wt) and of phospholipid and cholesterol (for a recent review, see Jørgensen 1982).
B. M. Anner, M. M. Marcus, M. Moosmayer

Import of Proteins by Isolated Mitochondria

Proteins of a mitochondrion have two origins. Only about one-tenth (by mass) are derived from the mitochondrial genome; the information to produce the bulk of mitochondrial protein is encoded on nuclear genes. Nuclear gene products are synthesized in the extramitochondrial cytoplasm and then incorporated into mitochondria by a process called import. This process consists of a few distinct steps: translation of the proteins as precursors, recognition and binding of precursors to the mitochondrial surface, translocation of bound precursors into or across one or both mitochondrial membranes, covalent modification or “maturation” of translocated precursors, and assembly of the mature proteins into functional complexes. Important features of this process include the following:
many precursors — but not all — are larger than their mature counter-parts, due to a transient amino terminal “signal sequence” on the primary translation product; (2) binding of precursors to mitochondria is a post-translation event; (3) translation of many precursors — but not all — requires an electrochemical protential across the mitochondrial inner membrane; (4) the most common covalent modification of a precursor is proteolytic removal of its signal sequence by a specific protease located in the mitochondrial matrix.
R. Hay, W. Oppliger

Isolation and Functional Reconstitution of Rat Liver Cytochrome Oxidase

Cytochrome oxidase is the terminal component of the respiratory chain of mitochondria. It functions as an electron carrier, between cytochrome c and oxygen, and as a proton pump, thus participating as a redox pump in the conversion of metabolic energy during the synthesis of ATP (Azzi 1980; Wainio 1983). Cytochrome oxidase has been purified from mitochondria of different sources. The most common technique used to purify beef heart cytochrome oxidase is based on the solubilization of mitochondria by cholate, followed by salt fractionation to separate the different cytochromes (Hartzell et al. 1978). Other techniques are based on hydrophobic chromatography (Nagasawa et al. 1979), and affinity chromatography (Bill et al. 1982; Rascati and Parsen 1979). An additional experimental approach, which has been often used to purify rat liver cytochrome oxidase, is based on differential solubilization using nonionic detergents of the Triton series (Ades and Cascarano 1977). Triton X-114 solubilize the b—c1 complex but not the cytochrome oxidase which can be subsequently solubilized using Triton X-l00 (Slinde and Flatmark 1976).
P. Gazzotti

Spectral Techniques


Fluorescent Labeling of Band 3 Protein from Erythrocytes

Fluorescence spectroscopy has become a valuable tool in the study of membrane proteins. Measurements of the fluorescence of extrinsic probes as well as of that intrinsic to the proteins have provided a considerable insight into the structure and mobility of these systems. The use of fluorescent probes to study membrane proteins is illustrated in the practical exercise described here, where the interaction of the mercurial reagent fluorescein mercuric acetate with Band 3 protein of erythrocytes is examined.
R. P. Casey, P. S. O’Shea, A. Azzi

Spin Labeling of Membranes and Membrane Proteins

Electron spin resonance (ESR) or electron paramagnetic resonance (EPR) has improved during the last 20 years to a powerful technique to derive structural and dynamic information about membranes and membrane components. The source of an ESR signal is an unpaired electron, which may exist intrinsically in a metalloprotein (e.g., cytochrome c oxidase) or be introduced as a stable free radical (spin label). The most commonly used spin labels are nitroxides of the general formula shown in Fig. 1, where the unpaired electron is located in the pz-orbital of the nitrogen atom. R1 and R2 are groups which give the nitroxide its specific reactivity and/or physicochemical properties. The shape of the ESR spectrum of a spin label depends on the concentration and on the motion of the molecule and on the polarity of the environment. The sensitivity of spin labels to these parameters are used to study the motion, the lateral distribution and the interaction of membrane components as well as the fluidity and the polarity of biological membranes.
R. Bolli, S. Feuerstein-Thelen, A. Azzi

Spectroscopic Measurements in the Subsecond to Second Time Domains: Use of an Optical Multichannel Analyzer for Spectral and Temporal Data Acquisition

A new generation of spectrophotometers is becoming available that use photodiode arrays instead of the traditional photomultiplier tube to detect a signal of interest. While conventional instruments are restricted to one or at most two wavelengths at a time and are often relatively slow, the new instruments can record an entire spectrum in as little as 10 ms and can repeat this process at rates of up to 100 spectra s-1. The information is presented in digitized form and is thus suitable for on-line display, handling and analysis with modern computers. Here we give a brief overview of the new instrumentation, with special emphasis on the fluorescence apparatus constructed at the Theodor Kocher Institute.
D. A. Deranleau, V. Von Tscharner

Use of a Potential Sensitive Dye in Studies on Phospholipid and Sarcoplasmic Reticulum Vesicles

A wide variety of dyes, when applied to biological membranes, change their optical properties along with a change in membrane potential. Experiments performed on neurons, where the membrane potential was measured simultaneously by an intracellular microelectrode, showed good agreement between the electrical and optical determinations (see Cohen and Salzberg 1978 for review).
H. Oetliker, H. Lüdi



Beta-Adrenoceptor Regulation in Organs of Rats Chronically Treated with Reserpine or Isoproterenol. Radioligand-Receptor Binding Studies

There is increasing evidence that target cells are able to adjust their response to the action of neurotransmitters. Recently, adaptive changes in the number of β-adrenoceptors have been identified as a feedback regulatory mechanism in tissues of several organs and in cell culture systems. This phenomenon, known as “receptor up- or down- regulation” or “receptor hypo- or hypersensitization” is assumed to maintain a balanced response to autonomic innervation, thus protecting the target cell or organ from the effects of abnormal neurotransmitter levels.
U. Honegger, S. Probst

Functional Changes in the Beta-Adrenoceptor Adenylate Cyclase System During Reticulocyte Maturation in vitro

The development of reticulocytes into circulating erythrocytes represents the final step in red cell maturation and is associated with major changes in cell metabolism (Yoshikawa and Rapoport 1974), as well as in membrane structure and function. Membrane alterations include modification of protein and lipid composition (see Light and Tanner 1978) changes in various membrane transport mechanisms for amino acids, sugars and ions (see Tucker and Young 1982) and loss of sensitivity toward certain hormones. Reticulocytes thus constitute a useful model system for studying regulatory processes that are involved in cell differentiation. The present experiment is designed to investigate specifically maturation-associated inactivation of the adrenergic receptor effector system.
J.-B. Montandon, H. Porzig

Acetylcholine Receptor-Enriched Membrane Fragments from the Electric Organ of Torpedo Marmorata

The nicotinic acetylcholine receptor (AcChR) is without doubt the best known of all receptors under investigation. This is due to the following features. Large amounts can be extracted from the electric ray, Torpedo species. It is the target for the snake venom neurotoxins, a property that permits a precise assay for the receptor and has allowed the design of a powerful affinity chromatography resin.
B. Schwendimann


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