Ghost protein, erythrocyte

The amount of decrease of d.c. resistance is not or only insignificantly dependent upon polyelectrolyte concentration, as long as this is over 0.05%. The most drastic decrease in d.c. resistance may be obtained by combining polyphosphate and bovine erythrocyte ghost protein. Resistances as low as 5 X 103 ohms per sq. cm. have been obtained at pH 6.8. 

From the experiments it is clear that poly electrolyte is adsorbed on the surface of the black lipid film. This applies both to the experiments with gelatin and bovine serum albumin, which gave no decrease of film resistance, and to the experiments with bovine erythrocyte ghost protein and polyphosphate. The adsorption of protein on the phospholipid-water interface may be controlled independently by investigating the electrophoretic behavior of emulsion droplets, stabilized by phospholipid, in a protein solution, as a function of pH. In this way Haydon (3) established protein adsorption on the phospholipid-water interface. If the high resistance (107 ohms per sq. cm.) of black lipid films is to be ascribed to the continuous layer of hydrocarbon chains in the interior of the film, as is generally done, an increase in film conductivity is not expected from adsorption without penetration. 

Polyphosphate was chosen as a polyelectrolyte in addition to the erythrocyte ghost protein, because van Steveninck demonstrated (17) that polyphosphate plays an important role in membrane transport in yeast cells. The results obtained with polyphosphate, especially on protein-covered membranes, indicate that the possibility of ionic transport is strongly enhanced. 

If preparative or instrumental artifact is ruled out, the universal occurrence of red-shifted Cotton effects with a-helical character in all the membranes studied points to a common property of the proteins in biological membranes. The ORD results from lipid-free mitochondrial structural protein and erythrocyte ghost protein are consistent with assigning the red shift in these membranes to aggregated protein. It is, therefore, reasonable that similar protein-protein association may occur in all membranes. Ionic requirements for membrane stability could then reflect in part the requirements for protein-protein association. To some extent the molecular associations which stabilize membranes, therefore, may be protein-protein as well as lipid-lipid in nature. 

Strambini and Galley have used tryptophan anisotropy to measure the rotation of proteins in glassy solvents as a function of temperature. They found that the anisotropy of tryptophan phosphorescence reflected the size of globular proteins in glycerol buffer in the temperature range -90 to -70°C.(84 85) Tryptophan phosphorescence of erythrocyte ghosts depolarized discontinuously as a function of temperature. These authors interpreted the complex temperature dependence to indicate protein-protein interactions in the membrane. 

The influence of adsorption of polyelectrolytes on bimolecular phospholipid leaflets was studied. All polyelectrolytes studied were adsorbed on the surface of the film, as demonstrated by greatly increased drainage times. Only some of the polyelectrolytes investigated are able to decrease the d.c. resistance, notably a protein derived from ox erythrocyte ghosts and a Na-K polyphosphate. The combination of these latter substances proved particularly effective. It is concluded that the decrease of d.c. resistance is caused by adsorption and penetration of the polyelectrolytes into the membrane, resulting in the formation of pores or water channels, and not by the possibility of transport of charged macromolecules through the membrane. 

The observation by Maddy and Malcolm (53) that the amide I band of bovine erythrocyte ghosts in D20 is not shifted is remarkable because it implies that all of the membrane protein is either deeply buried in an environment of hydrophobic lipids or exists in a tightly folded a-helical conformation. We have examined extensively the infrared spectra of bovine erythrocyte ghosts, both as dry films and as intact ghosts in D20 and H20 (73). The results for dry films essentially agree with those of other workers and show no evidence of f3 structure. Little change occurs in water. In D20, however, we consistently obtained a shift in the amide I band and a considerable decrease in absorption of the amide II band. 

Although the majority of the lipids in M. laidlawii membranes appear to be in a liquid-crystalline state, the system possesses the same physical properties that many other membranes possess. The ORD is that of a red-shifted a-helix high resolution NMR does not show obvious absorption by hydrocarbon protons, and infrared spectroscopy shows no ft structure. Like erythrocyte ghosts, treatment with pronase leaves an enzyme-resistant core containing about 20% of the protein of the intact membrane (56). This residual core retains the membrane lipid and appears membranous in the electron microscope (56). Like many others, M. laidlawii membranes are solubilized by detergents and can be reconstituted by removal of detergent. Apparently all of these properties can be consistent with a structure in which the lipids are predominantly in the bilayer conformation. The spectroscopic data are therefore insufficient to reject the concept of a phospholipid bilayer structure or to... 

First, a mixture of synthetic or natural phospholipids, polymerizable lipids, and proteins can be converted to liposomes and then be polymerized. Second, polymerizable lipids are introduced into e.g. erythrocyte ghost cells by controlled hemolysis and subsequent polymerization as described by Zimmermann et al.61). This hemolysis technique is based on a reversible dielectric breakdown of the cell membrane. Dielectric breakdown provides a third possible path to the production of bi omembrane models. Zimmermann et al. could show that under certain conditions cells can be fused with other cells or liposomes61). Thus, lipids from artificial liposomes could be incorporated into a cell membrane. A fourth approach has been published by Chapman et al.20). Bacterial cells incorporate polymerizable diacetylene fatty acids into their membrane lipids. The diacetylene units can be photopolymerized in vivo. The investigations discussed in more detail below are based on approaches 1. and 3. 

Figure 2. Loss of membrane proteins during reticulocyte maturation. Reticulocyte-poor (A) and reticulocyte-rich (B) erythrocyte membrane ghost proteins were subjected to 2DE and stained with Coomassie blue (0.5%). Selected spots identical between the protein samples are identified with black boxes. Several of the many spots that are unique to reticulocyte-rich erythrocytes are shown with the black circles. (Reproduced with permission from Prenni and Olver Proteomics A review and an illustration. Veterinary Clinical Pathology, in press.)...

Rabilloud, X, Blisnick, T., Heller, M., Luche, S., Aebersold, R., Limardi, J. and Braun-Breton, C. (1999) Analysis of membrane proteins by two-dimensional electrophoresis comparison of the proteins extracted from normal or Plasmodium falciparam-infected erythrocyte ghosts. Electrophoresis 20, 3603-3610. 

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