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The basic principle for probing the transverse distribution of membrane lipids is simple. Lipids that are available for modification by enzymes or for covalent labeling in impermeable membrane vesicles are considered to be in the outer leaflet of the membrane bilayer, and those that are also accessible in permeable vesicles or only accessible in inside-out vesicles are considered to be in the inner leaflet of the membrane bilayer. However, the characteristics of the membrane preparation under investigation and the enzyme probe are critical in these experiments. The following criteria are important. [ABSTRACT FROM AUTHOR]

Interactions between lipids and proteins have been studied using a variety of spectroscopic methods. Nuclear magnetic resonance (usually using deuterated phospholipid) can give information about conformational changes of lipid molecules on binding to membrane proteins, but information about relative binding affinities and numbers of binding sites is more usually obtained using electron spin resonance (ESR) (1). In the ESR method, spin-labeled phospholipids or fatty acids are incorporated into biological membranes and give two component ESR spectra, with one component (the immobile component) representing phospholipid at the lipid-protein interface of the protein (annular sites) and the other (the mobile component) representing "free" phospholipid in the bulk bilayer phase away from the protein. An automated procedure for finding the relative amounts of the two components in the composite spectra has been presented (2,3). From such data, straight-line plots of the mobile: immobile lipid ratio against lipid:protein ratio are usually constructed, with the intercept on the lipid:protein ratio axis being taken to be the number of lipid-binding sites on the protein and the slope giving the relative binding constant; potential problems with this approach have been described (4). [ABSTRACT FROM AUTHOR]

The study of proteins and glycoproteins as biologically active ligands is an expanding area of research, stimulated in part by the identification of growth factors and cytokines and, in particular, those involved in the regulation of hemopoiesis and the inmiune response. The exquisite sensitivity and specificity of cellular responses to minute extracellular concentrations of these macromolecular ligands has demanded the design of equally sensitive assays in order to study their physiological properties. Additionally, abnormal responses owing to receptor or ligand mutation cannot be fully characterized without reliable analysis of the normal state. This chapter discusses some of the practical considerations for studying protein-protein binding. This can best be illustrated by describing the binding of serum-derived asialoglycoproteins to the asialoglycoprotein receptor, which is expressed almost exclusively on the sinusoidal plasma membrane of hepatocytes. This is a particularly definitive assay that identifies equimolar binding between the ligand and a single class of binding sites. However, it must be emphasized that this should not be regarded as a paradigm for ligandreceptor binding assays, but merely a guideline from which other assays appropriate for a particular ligand and its receptor may be fashioned. [ABSTRACT FROM AUTHOR]

Receptor-mediated endocytosis is the process used by all eukaryotic cells to internalize a variety of biologically important macromolecules, e.g., transport proteins that deliver nutrients to cells (1,2), plasma proteins (3), hormones and growth factors (4,5), and lysosomal enzymes (6). The process is initiated when cell surface proteins (receptors) bind specific macromolecules (ligands) with high affinity at the plasma membrane. Following ligand binding, receptor-ligand complexes are rapidly internalized through clathrin-coated pits and delivered to endosomes. In endosomes, sorting and segregation of ligands and receptors into one of several pathways occur (7). For example, some receptors recycle to the cell surface while their ligands are degraded to amino acids in lysosomes, whereas in other cases, both are degraded or recycled. In hepatocytes, receptors and ligand may also be transported across the cell via a process called transcytosis. [ABSTRACT FROM AUTHOR]

Movement of ions across cell membranes is associated with processes such as cell signaling, regulation of cell volume, maintenance of cell composition and pH, and energy transduction. Even in the erythrocyte, a very specialized cell with limited biological function, 11 different transport systems for Na+ and K+ have been identified (1). The different fluxes are characterized by their sensitivity to specific inhibitors, and can be monitored either by measuring the uptake and/or release of isotopes, such as 42K or 86Rb, which usually acts as K-congener in cation transport systems, or by measuring the net changes in ionic composition (intra- or extracellularly) caused by the continued action of the transport system of interest. In practice, the isotope and bulk ion determination techniques often require similar experimental protocols, and they will be considered together here. Two approaches will be described: intermittent determination of isotope/cation composition of intra- and extracellular fluid (Section 2.1.. and 13-3), and continuous recording of extracellular ionic composition (Section 2.2. and 3.2.). [ABSTRACT FROM AUTHOR]

Many cell functions are controlled by molecular signals (hormones, neurotransmitters, and so forth) that interact with cell-surface receptors and trigger specific intracellular responses. Intracellular signaling can be difficult to study in isolated, purified systems, because these events depend on cellular architecture to a large extent. In intact cells, access to intracellular systems is limited by the restricted permeability of the surface membrane (plasma membrane). Much useful information can be obtained when the macromolecular elements of intracellular signaling are made accessible through a plasma membrane that is permeable to small (<1000 dalton), but not large molecules. For example, the nucleotide requirements (ATP, GTP, and so on) of exocytosis were assessed in cells made permeable to such molecules (1-3). Such permeabilized cells are also useful for studying processes modulated by guanine-nucleotide-binding proteins (G-proteins) (1-4). [ABSTRACT FROM AUTHOR]

Despite a 10,000-fold gradient of Ca2+ across the cell membrane, the concentration of cytosolic free calcium, [Ca2+]i, is regulated with remarkable constancy. A combination of mechanisms precisely regulate [Ca2+]i at nanomolar levels. These include influx of Ca2+ via plasma membrane calcium channels, release and redistribution of Ca2+ from internal stores, and efflux of Ca2+ by the action of ATP-driven calcium pumps. [ABSTRACT FROM AUTHOR]

Part A. Sample and Standards Preparation In Chapter 18, the extraction procedures for recovery of the phosphoinositols and phosphoinositides from cells are described, together with simple separation procedures to resolve them into their general classes (InsP1; InsP2, and so on, and PtdIns, PtdInsP, and PtdInsP2). However the metabolism of phosphoinositols is complex (1,2), leading to the formation of several isomeric forms in each class (see Fig.1 of Chapter 18 and Note Al). [ABSTRACT FROM AUTHOR]

Part A. Protein Kinase-Mediated Phosphorylation Events Protein kinases (ATP:protein phosphotransferases) regulate a wide range of cellular events, including the transduction of signals leading to cell growth. Although the protein kinase superfamily encompasses a large and structurally diverse group of enzymes, protein and DNA sequencing data indicate that they share a common evolutionary origin with a particularly highly conserved catalytic core structure and function. At present, the well characterized protein kinases (PK) can be divided into four main subclasses [ABSTRACT FROM AUTHOR]

Part A. Biosynthesis and Extraction of Phosphoinositides and Phosphoinositols The minor inositol-containing membrane phospholipids, the phosphoinositides, play a central role in cell signal transduction. Activation of a hormone-sensitive phospholipase C (phosphoinositidase C) results in the rapid catabolism of the polyphosphoinositides to form the two second messengers inositol 1,4,5-trisphosphate (Ins(l,4,5)P3), a water soluble phosphoinositol that promotes the release of Ca2+ from intracellular stores, and diacylglycerol (DG), which remains in the plasma membrane and activates protein kinase C (1-3). See Fig. 1 for a summary of the pathways. [ABSTRACT FROM AUTHOR]

Part A. Identification of G-Proteins The past decade has seen the emergence of a rapidly expanding superfamily of regulatory proteins, the G-proteins, that transduce as diverse a range of biological functions as protein synthesis, transmembrane signaling, intracellular trafficking, and cell proliferation (reviewed in 1). The application of biochemical and molecular biological techniques has substantially increased our understanding of the structure and function of G-proteins and has revealed a highly conserved primary structure and molecular mechanism throughout evolution. The central mechanistic concept is that G-proteins can exist in two interconvertible conformational states, one inactive (GDP-bound) and one active (GTP-bound). This basic cycle of GTP binding and hydrolysis (by an intrinsic GTPase) can confer both directionality and amplification to G-protein-mediated events. Although novel individual species have recently been identified (e.g., 2), there are presently three major classes of G-proteins [ABSTRACT FROM AUTHOR]

Cyclic adenosine 3′,5′-monophosphate (cAMP) and cyclic guanosine 3′,5′-monophosphate (cGMP) have between them been implicated in the mediation of many physiological phenomena. Over the last 30 years, a number of techniques have been employed to assess changes in the amounts of these two molecules. Many, however, suffered from lack of sensitivity and/or ease of performance. Since the early 1970s, two methods have been developed that to a large extent overcome these problems. The first is based on competitive binding to endogenous proteins (1). The second method is a competitive radioimmunoassay that, with minor modifications, can be used to detect levels of cyclic nucleotides down to 1 fmol/assay tube; this is discussed in detail below (2,3). The method is equally applicable to the measurement of all cyclic nucleotides; however, for clarity only that for cAMP is described. [ABSTRACT FROM AUTHOR]

The physicochemical and mechanical properties of phospholipids dispersed in an aqueous medium provide the molecular framework for many of the dynamic properties of cell membranes (1,2). The ability of protein molecules embedded in the plasma membrane to move laterally from one location to another, for example, in lymphocyte capping, depends on the presence of a low-viscosity (fluid) lipid domain in the membrane. Traffic of membrane materials to and from the cell surface depends on the ability of membrane vesicles to fuse with and to bud from the plasma membrane. Such processes are essential for the release of neurotransmitters into the synaptic cleft and play a role in a variety of diseases involving infection of cells by enveloped viruses, such as influenza virus (3). [ABSTRACT FROM AUTHOR]

The first measurement of the transmembrane diffusion of phospholipids in membranes was carried out by Kornberg and McConnell in 1971 (1). These authors sonicated an aqueous suspension of egg lecithin mixed with a small percentage of a spin-labeled phosphatidylcholine. The spin-labeled lipid had a nitroxide group on its polar head group that was directly exposed to the aqueous phase. Sodium ascorbate at 0°C, a nonpermeant reducing agent, very rapidly and selectively abolished the paramagnetism of the spin-labeled molecules present on the external monolayer of the vesicles. By subsequent exposure to ascorbate, it was possible to infer a half-time of randomization of approx 6.5 h at 30°C (1). This pioneer work was at least qualitatively confirmed by several laboratories using different experimental approaches in model systems as well as in biological membranes (for a review, see ref. 2). Thus, the nitroxide probe, in spite of possible steric hindrance and polarity characteristics, does not seem to modify profoundly the rate of phospholipid transverse diffusion in a lipid bilayer. Yet, when the transverse movement is governed by a specific carrier protein (2), it is necessary to move the probe away from the lipid moiety recognized by the carrier. In such a case, the selective accessibility to ascorbate is more difficult to achieve. Nevertheless, nitroxides can [ABSTRACT FROM AUTHOR]

The function of many glycoproteins is directly related to the structure of their oligosaccharide chains (1-5). Subtle changes in oligosaccharide structure involving the linkage of or loss of single monosaccharide units are known to occur in many biological events. Knowledge of this fine structure is necessary if the function of glycoproteins is to be understood. [ABSTRACT FROM AUTHOR]

The synthesis, characterization, and use of fluorescently labeled lipid probes to follow transport in living cells at a microscopic level have greatly expanded our knowledge of intracellular lipid trafficking. Although lipids containing a number of covalently attached fluorophores have been synthesized, most transport studies have used lipids labeled with 4-nitrobenzo-2-oxa-l,3-diazole (NBD).** Several lines of evidence suggest that NBD-labeled lipids faithfully mimic their native counterparts (see ref. 1 for review). For example: [ABSTRACT FROM AUTHOR]

An important topic in membrane biochemistry is the determination of the sidedness of the components comprising a biological membrane. Neither phospholipids nor membrane proteins are distributed symmetrically over inner and outer aspects of cellular membranes, and knowledge of the topological distribution is a prerequisite for understanding the functional properties of membranes. This chapter will deal with a recently developed, noninvasive technique to assess the distribution of phospholipids over the two leaflets of biological membranes with emphasis on the distribution of negatively charged phospholipids (1,2). [ABSTRACT FROM AUTHOR]

Phospholipid-transfer proteins (PL-TP) have been isolated from a number of tissues, including heart, liver, and brain (1). These proteins catalyze the one-for-one exchange in vitro of phospholipid molecules between membranes, lipoproteins, or liposomes. The functions of PL-TP in vivo have not been established; however, it is likely that they are involved in the transfer of phospholipid between organelle membranes during membrane biogenesis and membrane remodeling. Since PL-TP only exchange the phospholipid of the outer leaflet of membrane bilayers and do not appear to alter the structure of membranes, they provide a unique tool for investigating the transverse distribution of membrane phospholipids. [ABSTRACT FROM AUTHOR]

The proteoglycans (PGs) are a large and varied family of complex macromolecules whose physical properties are dominated by their large sulfated polysaccharide chains (for a recent review, see ref. 1). They are widely distributed in the animal kingdom, but are not associated with any particular anatomical or cellular sites, being instead almost ubiquitously present both on cells and within extracellular matrices. On cells they are usually constituents of the plasma membranes, though in the case of various cells of the hemopoietic lineage they can also be found in high concentrations within secretory granules. [ABSTRACT FROM AUTHOR]

Purification of membrane proteins of necessity requires the use of detergents to solubilize the proteins prior to their isolation. Although reconstitution of membrane proteins has been achieved without the use of detergents (e.g., by sonication of membrane proteins with lipids), detergent-based methods are more widely used and appear to be more reproducible (1). If a particular protein is to be purified in an active form, then particular attention must be paid to the choice of detergent. If the protein has not been purified previously, the first step is to solubilize the membrane preparation with a range of detergents and to perform assays of function on the solubilized material (see alsoChapter 22 of Biomembrane Protocols: I. Isolation and Analysis). It may be necessary to include exogenous lipid during the solubilization step to stabilize the protein (2). Ideally, the chosen detergent should have a high critical micelle concentration to enable the detergent to be removed following purification. Ionic detergents, such as cholate, usually fall into this category, although the nonionic detergent octylglucoside also has a high critical micelle concentration. Nonionic detergents, such as Triton X-100 and C12E8, cannot be removed by conventional dialysis, but it is reported that Extracti-Gel D™ (Pierce, Rockford, IL), when used in the form of a column, will remove such detergents from protein samples (3). [ABSTRACT FROM AUTHOR]

Protein-protein interactions in biological membranes can be studied using conventional chemical crosslinking methods, crosslinked products being detected either by altered molecular weights on SDS-polyacrylamide gels or using specific antibodies. Unfortunately, such studies can give crosslinked products too large to enter the gels, and it can be difficult to eliminate the possibility that crosslinked products form as a result of diffusion of the components in the membrane rather than because a complex of the components is present in the membrane (1). An alternative is to use spectroscopic techniques, and both electron spin resonance (ESR), especially saturation transfer ESR, and fluorescence techniques have been used to study protein-protein interactions in membranes. The ESR technique is sensitive to rates of motion of membrane proteins and can detect aggregation of membrane proteins from the resulting reduction in the rate of rotation of the proteins (2,3). The fluorescence technique operates on too fast a time scale (ns) to be sensitive to protein rotation, but can measure distances between protein molecules in the membrane, which would be expected to be smaller for molecules present in an aggregated form than for those distributed randomly in the plane of the membrane. [ABSTRACT FROM AUTHOR]

Part A. Production of Hybridoma Cell Lines A strategy for raising monoclonal antibodies involves immunization of mice; fusion of the mouse spleen cells with a myeloma cell line; selection, cloning, and freezing of hybridoma cell lines; and screening for monoclonal antibody production. Part A of this chapter will be concerned with the production of hybridoma cell lines, and Part B with the screening procedures. [ABSTRACT FROM AUTHOR]

Integral membrane proteins of physiological importance, such as ion channels, transporters, receptors, and enzymes, are usually minor components of the membrane. This low abundance, coupled with their hydrophobicity and frequent instability in detergent solution, renders them very difficult to purify for detailed investigation. As a consequence, most of our knowledge of these proteins has come from gene cloning, which has yielded the amino acid sequences of a large number of membrane proteins. This information allows the study of the tissue and subcellular distribution of the proteins, their topology in the membrane, and their isolation, using antipeptide antibodies, since antibodies raised against short peptides (10-20 amino acid residues) frequently recognize the corresponding sequence in intact proteins (1). [ABSTRACT FROM AUTHOR]

This method selectively labels glycoproteins on the cell surface. Tritiated borohydride reduction may follow chemical oxidation of vicinal hydroxyl groups by sodium periodate or enzymatic oxidation by galactose oxidase (GAO).** The periodate oxidation is more effi cient. It generates aldehydes on the terminal sialic acid residues of glycoproteins. The enzymatic oxidation is milder, and by virtue of the molecular size of the enzymes, more likely to be limited to the cell surface. Since most mammalian glycoproteins have a penultimate galac tose or N-acetylgalactosamine residue followed by sialic acid, the latter is removed by simultaneous incubation in neuraminidase, rendering the galactose and galactosamine residues available to the enzymatic activity of GAO that oxidizes these residues at the carbon-6 position. The method is generally applicable and may be used in conjunction with methods described in Part B of this chapter and in Chapter 2. See alsoChapter 4, Part D in this volume. [ABSTRACT FROM AUTHOR]

The ability to compartmentalize function has enabled multicellular organisms to evolve sophisticated physiological systems. At the cellular level, compartmentalization of function is established by the maintenance of asymmetric structure and function between the apical and basolateral surfaces of the cell. This asymmetry (polarity) extends on a biochemical level to the membrane proteins of the apical and basolateral plasma membrane. Indeed, many of the polarized functions of epithelia are mediated by membrane proteins enriched or restricted to one particular cellular domain (1,2). [ABSTRACT FROM AUTHOR]

In order to determine the structure of a protein by X-ray crystallography, well ordered three-dimensional crystals are required. However, despite the wealth of experience accumulated in the course of the crystallization and structural analyses of several hundred soluble globular proteins and their complexes, the process of crystallization still remains something of an art, and is often the rate limiting step of any analysis. For membrane proteins that present an additional challenge by virtue of their amphipathic nature, experience is considerably more limited, and the first three-dimensional crystals suitable for X-ray analysis were only reported in 1980 (1,2). Many membrane proteins form two-dimensional arrays in situ, and these may be studied by electron microscopy and electron diffraction of tilted specimens to determine their three-dimensional structure, but only in the pioneering study of bacteriorhodopsin has the resolution of the structural analysis approached that obtainable by X-ray crystallography (3). The formation of two-dimensional crystalline arrays will not be considered in this chapter. The first membrane protein crystal structure, the bacterial photosynthetic reaction center complex, was solved in 1985 (4); this was followed by the second reaction center structure in 1986 (5,6), and more recently porin in 1991 (7). Since these pioneering studies, increasing numbers of membrane protein crystallizations have been reported. For recent reviews, see refs. 8-11. [ABSTRACT FROM AUTHOR]

Glycolipids are an important group of diverse molecules present in the plasma membrane and to a more limited extent in the intracellular membranes of the Golgi and lysosomal systems of eukaryotic cells. They are prominent as antigenic determinants and as the ligands for specific lectin binding, and there are many examples in which they have been used as cell specific markers. The cellular glycolipid pattern frequently shows changes with the state of differentiation and may be altered following viral transformation. In animal cells the most common and most varied glycolipids are glycosphingolipids, and the methods of isolation and analysis given are mainly for this class of molecule. Methods are given for the extraction of total glycolipid from small volumes of tissue and for the isolation of the glycolipids from such extracts. Methods of fractionating and quantitating the acidic and neutral glycolipids are described, which do not require the use of specialized equipment such as high-performance liquid chromatography (HPLC), gas chromatography (GC), or mass spectroscopy and which can be performed easily in the general laboratory. [ABSTRACT FROM AUTHOR]

A strategy for determining the structure of a glycosaminoglycan (GAG) can be divided into three areas. The first is to establish its saccharide composition. This requires complete depolymerization of the parent chains to disaccharide products that can be purified, separated, and identified either by comparison with reference standards or by further structural analysis (1-6). [ABSTRACT FROM AUTHOR]

Membrane enzymes may be peripheral or integral proteins. The attachment of peripheral enzymes is mediated mainly by association with other membrane proteins and consists primarily of hydrophilic, electrostatic interactions. Integral membrane proteins are primarily bound by hydrophobic interactions with the lipid bilayer, either via one or more transmembrane peptide domains (1) or by possession of a glycosyl-phosphatidylinositol (GPI) membrane anchor at the carboxy-terminus (2). The purification of a membrane enzyme thus requires two additional considerations compared to the isolation of a cytosolic enzyme: the isolation of a membrane fraction containing the enzyme and the solubilization of the enzyme from this fraction. Once solubilization has been achieved and maintained, the enzyme can be treated essentially as a soluble enzyme and subjected to a wide range of purification techniques. [ABSTRACT FROM AUTHOR]

Two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) using isoelectric focusing (IEF) in the first dimension, followed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) in the second as described by O'Farrell (1), has been widely used for almost 20 years in examining complex protein mixtures (2). During this time a large variety of modifications to the original technique have been employed in efforts to obtain more reproducible results with greater resolution and protein spot detection, or to visualize specific populations of polypeptides. [ABSTRACT FROM AUTHOR]

The high resolution capacity of polyacrylamide gel electrophoresis (PAGE) (seeChapter 19) has resulted in the widespread application of this group of techniques to protein separations. PAGE procedures can provide characterization of proteins in terms of their charge, size, relative hydrophobicity, and abundance. However, they provide no direct clues as to the identity or function of the separated components. A powerful approach to this problem is provided by probing the separated proteins with antibodies and other ligands specific for components of the protein mixture being analyzed. [ABSTRACT FROM AUTHOR]

Polyacrylamide gel electrophoresis (PAGE) in the presence of the anionic detergent, sodium dodecyl sulfate (SDS), is probably the most commonly used technique for the analysis of protein mixtures. SDS is a very effective solubilizing agent for a wide range of polypeptides, including membrane proteins. It has been established that the majority of proteins bind 1.4 g SDS/1 g protein (1) to form negatively charged complexes. This results in masking of the intrinsic charge of the polypeptide chains, so that net charge per unit mass becomes approximately constant. Although the majority of proteins bind SDS in the expected ratio, it is important to realize that proteins containing nonprotein groups (e.g., glycoproteins, phosphoproteins, lipoproteins) can bind varying amounts of SDS, resulting in anomalous mobility and separation artifacts during SDS-PAGE. [ABSTRACT FROM AUTHOR]

The choice of a protein assay for membranes and membrane proteins is dependent on a number of considerations: sensitivity, specificity (both with respect to variation between proteins and interference by nonprotein components), and simplicity. [ABSTRACT FROM AUTHOR]

The fatty acids (FA) of animal, plant, and microbial origin are predominantly unbranched aliphatic chains with an even number of carbon atoms and a single carboxyl group. FA can be classified as saturated, monounsaturated, or polyunsaturated (Fig. 1). Saturates have all single carbon-to-carbon bonds. Monounsaturates contain a single cis double bond, whereas polyunsaturates have two or more. A list of the commonly occurring natural FA in animal tissues is shown in Table 1. In general, FA do not exist as free carboxylic acids in living cells. In animals, they are mainly esterified into membrane phospholipids (phosphoglycerides and sphingolipids) and cytosolic neutral lipids (triglycerides and cholesteryl esters). In order to analyze the FA components of cell lipids, they must first be converted into nonpolar, volatile derivatives before they can be separated and quantified by the rapid and highly sensitive technique of gas liquid chromatography (GLC). [ABSTRACT FROM AUTHOR]

Cholesterol is an amphipathic molecule of great importance in biology. It can be measured by enzymic and chemical methods, directly or indirectly following extraction from tissues or membranes. Before determination, cholesterol esters must be hydrolyzed using either chemical methods or cholesterol ester hydrolase (EC 3.11.13) (see Note 1). Enzymic methods for cholesterol determination are more sensitive and convenient and are generally used in preference to chemical methods. [ABSTRACT FROM AUTHOR]

Phospholipids are amphipathic molecules of great importance and widespread in biological material. They play an essential role in the structure and function of biological membranes. They are classified according to the nature of the "backbone" residue (glycerol or sphingosine), the type of nitrogenous base (or hexahydric alcohol) attached to it, and the nature of the chemical bonds (ester or ester and ether) linking the hydrocarbon chains to the backbone molecule. Within the same class, an enormous number of subclasses is possible through variation in the number of carbon atoms in the hydrocarbon chains, their degree of unsaturation, and, in the case of the glycerophos-pholipids, the position (C1 or C2) of the particular fatty acid (or ether) residues on the glycerol backbone. High performance liquid chromatography (HPLC) has emerged as one of the most powerful and versatile forms of separation technique that can be applied to the efficient separation and determination of phospholipids. [ABSTRACT FROM AUTHOR]

The membranes of animal cells contain a substantial amount of lipid, which may account for between 20 and 80% of the membrane mass. The predominant lipid components are phospholipids (PL) and cholesterol; with glycolipids, cholesterol esters, glycerides, and free fatty acids as minor constituents. PL perform a basic structural role in the cell membrane. They are arranged in a bilayer configuration, in which proteins are embedded, or associated peripherally. However, PL do not merely form an inert framework in membranes, they also play important functional roles in intracellular signaling, secretion, membrane transport, endocytosis, and cell fusion. [ABSTRACT FROM AUTHOR]

The most widely used subcellular fractionation techniques (e.g., centrifugation) are dependent on differences in physical parameters between organelles (e.g., size and density). In contrast, immunological techniques rely on biological differences, i.e., the expression of different antigens. The most popular immunological method for subcellular fractionation is immunoadsorption onto a solid phase, which offers the prospect of fast purification in high yield under conditions that do not expose organelles to unusual media or to osmotic shock. Immunoisolation techniques also have the advantage of being applicable to situations where only a small amount of cell material is available, e.g., tissue culture cells. [ABSTRACT FROM AUTHOR]

The isolation of mitochondria from plant cells that display similar biochemical and morphological characteristics to those observed in vivo requires considerable expertise. Although it is relatively easy to prepare a crude mitochondrial fraction by differential centrifugation, marked changes in functional capabilities and morphological appearance are often observed in such fractions, suggesting that some degree of structural damage has occurred during the isolation procedure. Most of the problems involved in the isolation of intact and fully functional mitochondria occur during the homogenization phase because of the high shearing forces required to rupture the plant cell wall. Such forces tend to have a deleterious effect on other subcellular organelles, such as the vacuole, resulting in the release of degradative enzymes and secondary products, such as flavonoids and phenolic compounds, which may severely impair mitochondrial integrity. Thus the effects of homogenization on mitochondrial structure and function range from undesirable to totally destructive. Obviously techniques that reduce all or any of these problems are desirable. [ABSTRACT FROM AUTHOR]

The isolation of photosynthetically active chloroplasts is the starting point for many plant metabolic studies as diverse as carbon assimilation, electron flow and phosphorylation, metabolite transport, and protein targeting. Whatever the subsequent use of the chloroplasts, it is preferable to attempt to isolate intact chloroplasts, as the organelle envelope will protect the stroma and thylakoids from the deleterious effects of degradative enzymes and phenolic compounds released from leaf tissues during the preparative procedures. [ABSTRACT FROM AUTHOR]

Perhaps the most striking feature of bacterial membranes is their multifunctional nature (1,2). Bacterial cytoplasmic membranes, for example, catalyze the reactions of respiratory and photosynthetic electron transfer and associated energy transduction, and contain numerous carriers for solute transport in and out of the cell. The cytoplasmic membrane of Escherichia coli is believed to contain more than 200 protein types, of which about 60 may be involved in transport functions. In addition, there are the many highly hydrophobic protein complexes that constitute the respiratory chains, and the multicomponent BF1 and BFo ATPase complexes. Bacterial membranes are distinctive in lacking sterols, but contain hopanes, polyterpenoids that have a role in maintaining membrane rigidity. Although variable, the major lipids are phosphatidylglycerol and phosphatidylethanolamine in gram-positive bacteria, and phosphatidylethanolamine with smaller amounts of phosphatidylglycerol in gram-negative bacteria. [ABSTRACT FROM AUTHOR]

The isolation of membranes from cultured mammalian cells poses a number of problems. First and foremost is the problem of homogenization, which is particularly difficult with suspension culture cells, as opposed to those that grow as an adherent monolayer. The choice of subcellular fractionation procedure depends on the results of the homogenization. For a review of the problems associated with the isolation of plasma membranes from tissue culture cells, see Graham (1). [ABSTRACT FROM AUTHOR]

Coated vesicles are implicated in a number of receptor-mediated transport events within eukaryotic cells. In particular, they are required for the export of newly synthesized lysosomal enzymes from the trans-Golgi network, and at the plasma membrane they are responsible for receptor-mediated endocytosis of selected ligands (1). Coated vesicles are the best characterized of all transport organelles, owing in large measure to the ease with which they can be purified. [ABSTRACT FROM AUTHOR]

Receptor-mediated endocytosis is an important mechanism by which the cell is able to take in macromolecules from the external environment and carry them through the necessary metabolic processes. Studies of the sequential steps of these events require methods of following the fate of the molecule from its internalization to its degradation in lysosomes or its transfer to other intracellular compartments. [ABSTRACT FROM AUTHOR]

The plasma membrane (PM) of polarized epithelial cells is composed of two physically continuous, but compositionally and functionally distinct domains: basolateral and apical. In hepatocytes, the basolateral domain includes the sinusoidal front, which faces the space of Disse and is involved in exchange of metabolites with the circulation, and the lateral surface, which is contiguous to neighboring hepatocytes. The apical or bile canalicular domain is specialized for the transport of bile acids and is separated from the basolateral domain by tight junctions, which prevent lateral diffusion of membrane proteins and outer leaflet lipids. Biochemical analysis of hepatocyte PM constituents requires the isolation of substantial amounts of this organelle. [ABSTRACT FROM AUTHOR]

The membrane organelles of an organized tissue, such as rat liver, that will always form closed vesicles, irrespective of the mode of homogenization, are the tubular membranes of the smooth and rough endoplasmic reticulum. Almost without exception, these vesicles retain their normal orientation, i.e., the cytoplasmic face is outermost. This is particularly clear in the case of the rough endoplasmic reticulum, for the ribosomes remain attached on the outside face. The microsomal (vesicular) fraction of a homogenate will also contain vesicles derived from any domain of the plasma membrane that, in vivo, is not stabilized by structures, such as tight junctions, desmosomes, or an extensive cytoskeleton. Thus, the blood sinusoidal membrane of hepatocytes and the basolateral membranes of enterocytes both tend to form vesicles, whereas hepatocyte contiguous membranes and enterocyte microvillar membranes will remain intact, as long as the homogenization is relatively mild (seeChapter 6). [ABSTRACT FROM AUTHOR]

Continuous-flow electrophoresis (CFE), which separates particles on the basis of surface charge density, should be regarded as an adjunct to centrifugation rather than as an alternative for membrane fractionation. The equipment is expensive and has been used for relatively specialized tasks, not for the routine isolations from a total homogenate. [ABSTRACT FROM AUTHOR]

This fraction is defined broadly as the material that will sediment at about 3500g for 10 min from a postnuclear supernatant. It contains the nuclei that failed to sediment at 1000g for 5-10 min, the largest mitochondria, and very few other organelles, such as lysosomes and peroxisomes. [ABSTRACT FROM AUTHOR]

Of all the organdies in rat hepatocytes, the nucleus is the largest and the most dense. It is therefore relatively easy to isolate in high purity and high yield. The majority of methods (1,2) involve the homogenization of the liver in isoosmotic sucrose followed by differential centrifugation and isopycnic purification through a sucrose density barrier. Because of their high density (>1.30 g/cm3), the sucrose density barrier concentration is about 2.25M or 60% (w/w) and, consequently, very hyperosmotic. This is one of the disadvantages of the method in that the nuclei collapse significantly during their passage through the sucrose barrier. They are subsequently resuspended in an isoosmotic medium in which they swell. The hydration of the DNA within the nucleus therefore changes during the purification process. Moreover, the high viscosity of the barrier causes a considerable reduction in the rate of sedimentation of the nuclei, which is the reason for the use of high relative centrifugal forces (rcf) (normally 100,000g). Furthermore, high viscosity sucrose solutions are difficult to prepare and measure out accurately. [ABSTRACT FROM AUTHOR]

The chapters in the two volumes of Biomembrane Protocols are broadly devoted to the methodologies involved in the structural and functional characterization of cell membranes. In many cases, a study of a particular membrane presupposes that it can be isolated by disruption of the cells and fractionation of the resulting homogenate, and then identified unambiguously. The early chapters of this volume, Biomembrane Protocols: I. Isolation and Analysis, are concerned with this fractionation process. Later chapters are concerned with the determination of composition, whereas in the second volume, Biomembrane Protocols: II. Architecture and Function, the methods associated with architecture and function predominate. [ABSTRACT FROM AUTHOR]