Ghost cells preparation

Figure 2. Distributions of cell volumes of typical red cell and ghost cell preparations, obtained by Coulter counter. Key to mean cell volumes red cells, 87 ixm3 and ghost cells, 76 ixm3.

In addition to SEM, size analysis of the ghost cells was performed with a Coulter counter, and as shown in Figure 2, the size distribution is similar to that of the washed red cell preparation. 

Hemoglobin-free white membrne ghosts were prepared from normal human erythrocytes according to the methods in [ll,12]. The red blood cells were washed three times with 150 mM NaCl saline solution buffered with 5 mM sodium phosphate at pH 8 ( 1 volume of cell + 10 volume of buffer solution). 

Here, we discuss a solid-state 19F-NMR approach that has been developed for structural studies of MAPs in lipid bilayers, and how this can be translated to measurements in native biomembranes. We review the essentials of the methodology and discuss key objectives in the practice of 19F-labelling of peptides. Furthermore, the preparation of macroscopically oriented biomembranes on solid supports is discussed in the context of other membrane models. Two native biomembrane systems are presented as examples human erythrocyte ghosts as representatives of eukaryotic cell membranes, and protoplasts from Micrococcus luteus as membranes... 

Schwoch G, Passow H (1973) Preparation and properties of human erythrocyte-ghosts. Mol Cell Biochem 2 197-218... 

Hanahan DJ, Ekholm JE (1974) The preparation of red cell ghosts (membranes). Methods Enzymol 31 168-172... 

Figure 8-14 SDS-polyacrylamide gel electrophoresis of human erythrocyte ghosts. (A) From untreated cells. (B) From cells digested externally with chymotrypsin. (C) Inside-out vesicles prepared from cells pretreated with chymotrypsin. (D) The same inside-out vesicles after further treatment with chymotrypsin. (E) Polypeptides released hy the chymotryptic treatment of the inside-out vesides. The peptides are numbered according to the system of Steck232 Hb, hemoglobin. From Luna et al233...

In contrast to the ion exchange theory, much evidence indicates that cells have an active ion pump that removes Na+ from cells and introduces K+. For example, the cytoplasm of the giant axons of nerves of squid can be squeezed out and replaced by ionic solutions. Erythrocyte ghosts can be allowed to reseal with various materials inside. Ion transport into or out of cells has been demonstrated with such preparations and also with intact cells of many types. Such transport is blocked by such inhibitors as cyanide ion, which prevents nearly all oxidative metabolism. However, the cyanide block can be relieved by introduction into the cells of ATP and other phosphate compounds of high group-transfer potential. 

Pyran Copolymer. Pyran copolymer used In calorimetric studies of red cell ghosts was NSC-46015, a gift of Dr. David S. Breslow of Hercules, Inc. For studies of pyran-llpld mixtures, the polymer was prepared by radical copolymerization of maleic anhydride and dlvlnyl ether according to the technique of Breslow (1), using 9/1 acetone/tetrahydrofuran mixture as solvent, and azoblslsobutyronltrlle as Initiator. The Inherent viscosity of the sample was 0.189 dl/g (0.5 g/100 ml In 0.05H NaCl, 30 C). 

Erythrocytes (red blood cells) can be easily lysed to release their contents and yield membrane ghosts. These are large vesicles that represent a nearly pure preparation of the plasma membrane of the cells. The lipid composition and distribution between the inner and outer leaflets of the bilayer membrane are shown in Table 10.3 and Figure 10.15, respectively. 

Arricta et at. (2003) produced a preparation from bovine red blood cell ghosts as a standard for AChE a.ssays. The activity of this standard was stable at —70°C for up to 3 years. 

The erythrocyte and erythrocyte ghost suspensions are very similar systems. They differ in their inner solution (in the case of erythrocytes it is an ionic hemoglobin solution in the case of ghosts it is almost like the surrounding solution they were in while they were sealed). The cell sizes in a prepared suspension depend both on the ion concentration in the supernatant and in the cell interior (70). Thus, the dielectric spectra of erythrocytes and erythrocyte ghost suspensions have the same shape, which means that there are no additional (except Maxwell-Wagner) relaxation processes in the erythrocyte cytoplasm thus, the singleshell model (Eq. 89) can be applied. 

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