Surface labeling erythrocyte membranes

Besides the common fluorescent probes used for studying protein structure (ANS, TNS, etc.), other labels suitable espa ially for the modification of cell surfaces and the membranes of intracellular structures have been introduced in research. ANDS, for instance, is such a label (3-azido[l, 7]napthalene disulphonate) It is used for nonspecific covalent modification of hydrophilic cell surfaces. Another is FDNB (l-fluoro-2,4-dinitrobenzen) > which is used for identifying membrane proteins associated with glucose transport in human erythrocytes. More detailed information can be found elsewhere... 

Several other techniques have been exploited to answer queries about asymmetrical compositions of plasma membranes. To make an observation of the transverse asymmetry in the distribution of certain membrane molecules requires that one side of the membrane be excluded from a reaction medium while a constituent is chemically altered, labeled or identified by its properties. Many such experiments have been conducted with intact erythrocytes which were reacted and then lysed and reacted again. The reactant saw initially only the outer surface of the membrane and eventually both sides. Qualitative or quantitative differences observed under these conditions allowed conclusions about the... 

Following the observation that proteins located on the external surface of human erythrocyte membranes contain carbohydrate, lactoperoxidase-catalysed iodination has been used to label the external surface of human tumour KB cells. Only a relatively small number of protein subunits of the plasma membrane were accessible to enzymic iodination, and most, if not all, of these molecules appear to be glycosylated. A cell-surface glycoprotein containing a D-galactosyl residue at the non-reducing terminus has been isolated from culture fluids of I29/J mouse ascites teratoma cells, and it may be responsible for cell-to-cell adhesion. ... 

The amoeba Acanthamoeba castellani has been shown to bind to equine erythrocytes by interaction with carbohydrates on the surface of the red cells. 4-Nitrophenyl tri-A-methyltyrosinate [ I]iodide has been used to label the outer surface of mature chicken erythrocytes. Two component glycoproteins, with subunits of molecular weight 5 x 10 and 1 x 10 , were identified. Neuraminidase-treated rat and rabbit erythrocytes were shown to be rapidly sequestered by the liver. Chemical studies of glycoproteins obtained from erythrocyte membranes from a number of sources have been reported. ... 

A notable feature of the lipid regions of biological membranes is that the different phospholipid types may be asymmetrically distributed across the bilayer. For the erythrocyte membrane for example, it has been demonstrated by surface labelling and phospholipase digestion that the sphingomyelin and phosphatidylcholine are located in the outer half of the bilayer, whereas the phosphatidylethanolamine and phosphatidylserine are localized to the inner half (Zwaal et al., 1973). 

A method for selective, radioactive labelling of sialic acids, especially in cell membranes, by mild oxidation with periodate followed by reduction with borotritide, has heen described by Gahmberg and Andersson.200 This procedure can be used either for isolation and characterization of the labelled, cell-surface glycoconjugates (see, for example, Ref. 201) or for autoradiography of tissues and cells (for example, erythrocytes).142... 

The method of biosynthetic incorporation of spin label, rather than mechanical addition to isolated material, is a convenient way of ensuring that the results obtained are biologically meaningful and has also been used with such systems as the mould Neurospora crassa [158], Mycoplasma laidlawii [159], human leucocytes, and mouse L cells [160]. The spectra from these two mammalian cells showed distinct similarities for a variety of spin labels, but different spectra were obtained when the labels were incorporated in human erythrocytes. Fractionation of the cell components showed the stearic acid (C, n = 3) spin label in all the major fractions, but by far the largest concentration was in the nuclear membrane. The ESR spectrum underwent a time and temperature dependent decay and spin labels on the surface membrane were reactivated with K3Fe(CN)6. 

N- (4-Azido-2-nitro-phenyl)taurine Erythrocytes (internal surface) After allowing transport of reagent inside at 37°, labeling of cytoplasmic membrane proteins no... 

The first natural membrane to be examined for asymmetry was that from erythrocytes. Such cells are very convenient for membrane studies since erythrocytes can be easily purified in large quantities and contain only a single membrane. In order to study the two leaflets of such membranes, intact cells were probed (or labelled) in order to establish those components which were in the outer leaflet (i.e. that were accessible). In a second experiment, the erythrocytes were broken by a suitable method (e.g. osmotic shock, sonication) to allow access of the probe to the inner surface as well (Figure 6.12). Control experiments are also necessary to evaluate the total membrane proteins and lipids, since not all of these may be accessible to the probes used. 

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