The action of organic mercurials on the erythrocyte membrane

The solubilisation of proteins from erythrocyte membranes by treatment with organic mercurials has been studied with different species. The marked solubilisation previously reported for human membranes does not seem to be a general phenomenon. All of the other species examined showed less than 50% of the solubilisation shown by human membranes. The protein-solubilising effect seems to be dependent on hydrophobic mercury derivatives carrying a net negative charge. Uncharged compounds like phenylmercuric acetate blocked the effect, although $\text{N-}\text{ethylmaleimide}$ and iodoacetamide did not. With the aid of radioactively labelled compounds, and of atomic absorption spectrophotometry, the proteins reactive towards the mercurials were identified. The major integral protein, band 3, was the major protein capable of binding the mercurial. Reaction with the mercurial appears to disrupt interaction of band 3 with bands 2.1 and 4.2, allowing dissociation of the cytoskeleton from the membrane. In addition, band 4.9 was also found to react with the mercurials, possibly resulting in disruption of the cytoskeleton.     

Effects of dehydroabietic acid on the erythrocyte membrane

The effects of dehydroabietic acid (DHAA), a dominant resin acid in pulp and paper mill effluents, on membrane-connected events were studied in human erythrocytes. Fifty percent haemolysis was achieved by 252 microM DHAA after 1 h of incubation at +37 degrees C. At sublytic concentrations, DHAA protected erythrocytes against hypotonic haemolysis, with maximum protection occurring at 125 microM. In the lower range of sublytic concentrations, DHAA induced a slight echinocytosis; at higher sublytic concentrations erythrocytes were transformed to sphero-echinocytes and a release of acetylcholinesterase (exovesicles) occurred. Furthermore, at sublytic concentrations DHAA increased potassium efflux and passive potassium influx, while active potassium influx ((Na(+)-K+)-pump activity) and phosphate efflux were decreased. Our study indicates that DHAA acts on human erythrocytes in a way typical for amphiphilic compounds. It is proposed that DHAA by intercalating into the lipid bilayer of the membrane, affects the dynamics of the bilayer which in turn alters the permeability of the bilayer and the function of ion transporting membrane proteins.     

Effects of tannic acid on antigenicity and membrane contrast in ultrastructural immunocytochemistry.

We have modified our previous method for immunogold staining of unosmicated, plastic-embedded tissue by addition of tannic acid as a post-fixative to increase membrane contrast. Overall cell ultrastructure and organelle membranes, in particular, appeared well preserved after this treatment. We evaluated quantitatively the effect of tannic acid on the antigenicity of several membrane proteins in rat liver and intestine. For all antigens tested, significant antigenicity was retained on both intracellular and plasma membranes. However, the level of antigenicity decreased with increased concentrations of tannic acid. This effect was most apparent on the apical and basolateral membranes of hepatocytes and on the apical membrane of enterocytes, surfaces that had been in direct contact with the tannic acid fixative. The results indicate that when low concentrations of tannic acid are employed, this method yields greatly enhanced membrane contrast while preserving sufficient antigenicity to facilitate the ultrastructural localization of many membrane and other antigens.     

Ruthenium red staining and tannic acid fixation of dental basement membrane

Summary Ruthenium red staining and tannic acid fixation were used to analyse the fine structure of embryonic mouse dental basement membrane in intact first mandibular molars or in EDTA-isolated dental papillae. Preameloblasts are separated from extracellular matrix proper by a basal lamina that contains regularly arranged proteoglycan granules of about 10 nm in diameter. This distribution pattern is particularly evident in the inner and outer lamina rara of the basal lamina associated with EDTA-isolated dental papillae. The plasmalemma of preameloblasts demonstrates electron dense plaques on the inner leaflet. Ruthenium red positive granules (50 nm in diameter) coat non-striated and striated fibrils of the matrix. Hyaluronidase treatment digested the ruthenium red positive granules. Tannic acid fixation allowed the demonstration of filaments within the lamina rara interna, connecting the lamina densa with plasmalemma of preameloblasts. These observations are discussed in the context of the terminal differentiation of odontoblasts.These studies were supported by INSERM, grant n 537785 and DGRST     

Ankyrin-bound fatty acid turns over rapidly at the erythrocyte plasma membrane.

The membrane skeletal protein ankyrin was shown to be continuously acylated and deacylated with long-chain fatty acids in mature erythrocytes. At least a fraction of the lipid bound to ankyrin turned over rapidly (half-life, approximately 50 min) compared with the polypeptide backbone, which was stable throughout the erythrocyte life. This indicates a regulatory significance of the fatty acid modification for the function of ankyrin.     

Asymmetric distribution of phosphoinositides and phosphatidic acid in the human erythrocyte membrane.

The distribution of phosphoinositides and phosphatidic acid (PA) between the outer and inner layers of the human erythrocyte membrane was investigated by using two complementary methodologies: hydrolysis by phospholipase A2 (PLA2) and immunofluorescence detection with monoclonal antibodies against polyphosphoinositides. The contents of phosphatidylinositol 4,5-bisphosphate (PIP2), phosphatidylinositol 4-phosphate (PIP) and PA were decreased by 15-20% after 60 min incubation with PLA2, while that of phosphatidylinositol (PI) was increased. Studies with 32P-labelled cells revealed that PLA2 treatment led to indirect effects on the metabolism of these phospholipids. Therefore, the asymmetric distribution of phosphoinositides and PA was inferred from the data obtained in ATP-depleted erythrocytes. In these cells with arrested phosphoinositide metabolism, the asymmetric distribution of the major phospholipids was maintained: PLA2 hydrolyzed approx. 20% of PI, PIP2 and PA (but no PIP) indicating their localization in the outer layer of the membrane. This finding was confirmed by immunofluorescence studies with antibodies specific to each phosphoinositide. External addition of anti-PIP2 but not anti-PIP gave a positive reaction both in control and in ATP-depleted erythrocytes. A pretreatment of cells with PLA2 led to a decrease in the intensity of anti-PIP2 staining. These results demonstrate that significant fractions of PIP2, PI and PA are localized on the outer surface of the erythrocyte membrane.     

The reaction of chemical probes with the erythrocyte membrane.

Trinitrobenzenesulfonate (TNBS), fluorodinitrobenzene (FDNB) and suberimidate have been reacted with intact human erythrocytes. TNBS does not penetrate the cell membrane significantly at 23 degrees C in bicarbonate-NaCl buffer, pH 8.6, as estimated by the labeling of the N-terminal valine of hemoglobin. Hence, under these conditions it can be used as a vectorial probe. However, at 37 degrees C, especially in phosphate buffer, at pH 8.6, TNBS does penetrate the cell membrane. FDNB and suberimidate both penetrate the erythrocyte membrane. The time course reaction of TNBS with intact erythrocytes over a 24-hr period at 23 degrees C is complex and shows transition zones for both membrane phosphatidylethanolamine (PE) and membrane proteins. No significant cell lysis occurs up to 10 hr. The fraction of total PE or phosphatidylserine (PS) which reacts with TNBS by this time period can be considered to be located on the outer surface of the cell membrane. Under these conditions it can be located on the outer surface of the cell membrane. Under these conditions it can be shown that 10 to 20% of the total PE and no PS is located on the outer surface of the membrane and hence these amino phospholipids are asymmetrically arranged. The pH gradient between the inside and outside of the cell in our system is 0.4 pH units. Nigericin has no effect on the extent of labeling of PE or PS by TNBS. Isotonic sucrose gives a slight enhancement of the labeling of PE by TNBS. Hence, the inability of PE and PS to react with the TNBS is considered not due to the inside of the cell having a lower pH. The extent of reaction of TNBS with PE is not influenced by changing the osmolarity of the medium or by treatment of cells with pronase, trypsin, phospholipase A or phospholipase D. However, bovine serum albumin (BSA) does protect some of the PE molecules from reacting with TNBS. Cels treated with suberimidate were suspended in either isotonic NaCl or in distilled water. In both cases the suberimidate-treated cells became refractory to hypotonic lysis. Pretreatment of cells with TNBS did not prevent them from interacting with suberimidate and becoming refractory to lysis. However, pretreatment of cells with the penetrating probe FDNB abolished the suberimidate effect. Electron-microscopic analysis of the cells showed a continuous membrane in the case of cells suspended in isotonic saline. The cells suspended in water did not lyse but their membranes had many large holes, sufficient to let the hemoglobin leak out. Since the hemoglobin did not leak out we know that the hemoglobin is cross-linked into a large supramolecular aggregate.