Does diabetes mellitus affect diphenylhexatriene penetration into erythrocyte membrane ghosts?

Diphenylhexatriene transverse distribution has been studied in normal and diabetic erythrocyte membrane ghosts using fluorescence polarization and fluorescence quenching methods. Acrylamide quenched the fluorescence of diphenylhexatriene according to a dynamic mechanism in agreement with Stern-Volmer equation. Nonlinear least-squares analysis based on quenching results has shown greater accessibility of fluorophore to quencher molecules in diabetic ghosts (37.2 +/- 3.2% in normal vs. 67.5 +/- 6.4% in diabetic membranes). Steady-state fluorescence anisotropy measurements evidenced the lowered membrane lipid fluidity in diabetics (anisotropy values: 0.166 +/- 0.011 in normal subjects vs. 0.193 +/- 0.018 in diabetics). A model mechanism is proposed which attributes the lowered capacity of lipid bilayer in diabetes to the increased ordering and more compact structure of membrane phospholipids. The implications of the results for the resolving of steady-state anisotropy data are discussed.     

Study of the aggregation of membrane proteins of erythrocyte ghosts using SDS-PAAG electrophoresis

Using the method of electrophoresis in SDS-PAAG the authors showed a diminution of proteins of bands I + II (spectrins) and III (major integral protein) after irradiation of erythrocyte ghosts with doses of 50 to 1000 Gy. We failed to ascertain that radiation-induced lipid peroxidation is involved into membrane protein aggregation. Among the radiolysis products, OH-radicals were shown to contribute markedly to the radiation effect observed.     

Fatty-acid spin probe interactions with erythrocyte ghosts and liposomes prepared from erythrocyte ghosts

Summary A model for the binding of 5-nitroxide stearate, I(12,3). to human erythrocyte ghosts was developed by comparing spin probe interactions with ghosts and liposomes prepared from ghosts. At low probe/lipid (P/L<1/2500), I(12,3) binds to a similar class of high-affinity, noninteracting sites in both ghosts and liposomes, indicating that lipid moieties are responsible for probe uptake. Saturation occurs in both systems with increasing P/L, and, at higher loading (e.g., P/L=1/360 for ghosts and liposomes), the probe inserts itself at initially dilute sites to form a class of low-affinity sites consisting of clusters of variable size. At still higher P/L ranges (>1/100), much increased probe uptake was observed in ghosts than in liposomes, which was attributed to another class of low-affinity sites, representing nonspecific interactions of I(12,3) with membrane proteins. The nature of the spectral components and ultrafiltration experiments with ghosts labeled at high P/L indicate that both dilute and clustered I(12,3) are due to membrane-incorporated probe.     

Infinite cis influx of cyclic AMP into human erythrocyte ghosts.

Infinite cis uptake of cyclic AMP into red blood cell ghosts has been measured. The Koiic is calculated from two different integrated rate equations that are applicable when the substrate concentration is unsufficient to cause volume changes. Values of 0.69 mM and 0.66 mM are obtained for the infinite cis Km at 30 degrees C using these procedures. These values are only slightly higher than that predicted from zero trans net flux experiments. Lowering the temperature reduces Koiic from 0.69 mM at 30 degrees C to 0.478 mM at 20 degrees C, 0.108 mM at 10 degrees C and 0.072 mM at 4 degrees C (Q10 = 2.4). The Q10 for activation of influx permeability of 10(-5) M cyclic AMP is 1.55.     

Preparation of erythrocyte ghosts by dielectric breakdown of the cell membrane.

Dielectric breakdown of membranes of red blood cells was observed in high electric fields (approx. 10-3-10-4 V/cm) using an improved Coulter Counter with hydrodynamic focussing. In making measurements of the size distributions of red blood cells as a function of increasing electric field strength it was found that a sharp discontinuity occurred in the otherwise linear relation between the pulse heights in the Coulter Counter and the electric field strength due to dielectric breakdown of the membranes. Solution of Laplace's equation for the electric field generated at breakdown in the cell membranes yeilds a mean value of about 1.6 V. for the membrane potential of red blood cells. Due to the dielectric break-down, release of hemoglobin occurred. Mechanical rupture of the red blood cells by the hydrodynamic forces in the orifice of the Coulter Counter or thermal rupture could be excluded as hemolysing mechanisms. The leaky ghost cells resealed at 37 degrees C. as shown by incorporation of 131I-labeled albumin and repeated dielctric breakdown.     

Proteolytic activity of human erythrocyte membrane during ghosts preparation

1. Proteins in human erythrocyte membranes after red blood cells hemolysis revealed relatively high rate of self-digestion. 2. This indicates hemolysis as a critical moment for membrane proteases activation. 3. The detailed pattern of band 3 protein and spectrin degradation during ghosts preparation was more complicated and reflected both the changes in proteolytic susceptibility and extraction of some proteases. 4. Further extraction of membrane proteins by alkali stripping resulted in an increase in the self-digestion rate and decrease in the degradation rate of an exogenous substrate.     

Two-dimensional separation of erythrocyte membrane proteins.

The details of a two-dimensional separation procedure specially designed for the study of erythrocyte membranes are presented. In this highly reproducible method, the membrane proteins are dissolved in sodium dodecyl sulfate and separated first on the basis of charge by isoelectric focusing. The samples are loaded either at the cathode (CIF) or anode (AIF). The CIF samples gave better separation of the acidic proteins, while the AIF was better for the separation of the high molecular weight polypeptides of the erythrocyte. Over 90 discrete polypeptides could be detected with this method in the pH range of 5 to 8. Special attention was given to the higher molecular weight components. For example, six components could be detected within the 90,000 to 100,000 molecular weight range of protein 3, the major membrane protein. A component with the same or very nearly the same molecular weight as spectrin band 2 was detected. It is more basic than spectrin band 2, and both spectrin band 2 and the basic component are readily phosphorylated in the intact cell. However, the phosphorylation of band 2 is cAMP independent while the phosphorylation of the basic component is enhanced by cAMP. In contrast to spectrin, the basic component is not extracted from the membrane with 0.1 mm EDTA, although dilute NaOH will remove it from the membrane. The Ca²⁺-activated transferase of the erythrocyte cytoplasm will not crosslink this component. Calcium does, however, activate the conversion of this component to a lower molecular weight. This high molecular weight basic component has properties attributed to the component labeled 2.1 in Fairbanks' system of nomenclature.     

Fluidity of human erythrocyte ghosts.

The fluidity, defined by its two components, the order parameter, S, and the rotation correlation time, tau c, was studied on healthy human erythrocytes ghosts. We also measured ghost protein, cholesterol and phospholipid contents as well as acetylcholinesterase activities. No statistically significant difference was evidenced between erythrocyte ghosts from men and women. Whereas tau c values did not significantly vary among sample elements, variations of ghost order parameters about the mean were explained at 61% by changes in cholesterol contents and, to a lesser extent, in protein contents. No relationship was evidenced between ghost order parameter values and those of corresponding acetylcholinesterase activities. Liposomes prepared from ghost lipid extracts had much lower order parameter values than did corresponding ghosts. A few experiments were performed in the same way on ghosts from sickle blood. This disease appeared to decrease the bilayer lipid motionnal freedom as an increase of the order parameter values was evidenced.     

Alteration of membrane conductivity and fluidity in human erythrocyte membranes and erythrocyte ghosts following gamma-irradiation

Alterations of electrical properties of human erythrocyte membranes induced by gamma irradiation have been studied by means of conductivity measurements in the frequency range from 10 KHz to 100 MHz. The results clearly demonstrate the role played by haemoglobin in the structural modification of the membrane produced by gamma irradiation. Further support for this point of view has been derived from electron spin resonance measurements carried out on the same samples, labelled with different spin labels which probe the outer half layer of membrane at different penetration levels.     

Analysis of erythrocyte membrane proteins from dystrophic hamsters

To search for potentially mutant proteins, we have investigated erythrocyte ghost proteins from normal and dystrophic hamster by two-dimensional gel electrophoresis. No significant differences are observed between dystrophic and normal erythrocytes in their peptide patterns on SDS-polyacrylamide gel electrophoresis while on two-dimensional gels a protein spot of approximate Mr 20 000 with an approximate isoelectric point of 4.5 is found in erythrocytes from dystrophic animals and is consistently absent in normal erythrocytes. A large population of erythrocyte (60%) from dystrophic hamsters shows distorted shape as visualized by scanning electron microscopy. The nature of this protein and its relevance in hamster muscular dystrophy are at present not known.     

Removal of blood group determinants from bovine erythrocyte membranes. 1. Action of proteolytic enzymes on ghosts1

Thirty-nine blood group antigens were detected by hemolytic inhibition tests on erythrocyte ghosts prior to enzyme digestion. The ghosts, produced from erythrocytes collected from six different cattle, were digested with the proteolytic enzymes papain, protease, ficin, chymotrypsin and trypsin. Of the 39 antigens, 30 were removed from the membranes and detected in the soluble fraction resulting from the digestions. Some antigens were consistently removed by all enzymes digesting all ghosts possessing them, while the degree to which many other antigenic determinants were removed varied according to the ghosts being digested and the enzymes employed. Of the 9 remaining determinants never removed from the ghosts, some were detected in the insoluble fraction while others were not detected at all. These latter antigens were presumably destroyed by the enzyme digestion.     

Protein and protein-lipid membranes from solubilized erythrocyte ghosts

Summary Erythrocyte ghosts were solubilized by addition of acid 2-chloroethanol to an aqueous membrane suspension. The proteins were separated from the lipids by chromatography on Sephadex LH-20 (Zahler and Wallach, 1967). Both the solution of separated proteins and of the total membrane in chloroethanol-water can be spread at a benzene-water interface. By lowering a thin teflon plate with a small hole through this interface, one can form protein or protein-lipid films over the hole. After the benzene has evaporated stable thin membranes are formed which contain only protein or protein together with lipid. The morphology and thickness of these membranes were investigated by different electron microscopic techniques.     

Modulation of erythrocyte membrane proteins by membrane cholesterol and lipid fluidity.

Human erythrocyte membranes were enriched or depleted of cholesterol and effects on membrane proteins assessed with a membrane-impermeant sulfhydryl reagent, [35S]glutathione-maleimide. Reaction of the probe with intact cells quantifies exofacial sulfhydryl groups and reaction with leaky ghost membranes permits quantification of endofacial sulfhydryl groups. The mean endofacial sulfhydryl titer of cholesterol-enriched membranes exceeded that of cholesterol-depleted membrane by approximately 45 nmol/mg of protein or 64%. The corresponding exofacial titer of cholesterol-enriched cells was less than that of cholesterol-depleted cells by approximately 0.4 nmol/mg of protein, or 14%. Labeled membranes were examined by autoradiography of sodium dodecyl sulfate-polyacrylamide gel electropherograms to determine the labeling patterns of individual protein bands. Cholesterol enrichment enhanced the surface labeling of Coomassie brilliant blue stained bands 1,2,3, and 5, decreased the labeling of band 6, and did not change significantly that of band 4. The results demonstrate that changes in membrane cholesterol which influence lipid fluidity can alter the surface labeling of both intrinsic and extrinsic membrane proteins.