Zinc concentration in erythrocyte membranes in normal volunteers and in patients with taste and smell dysfunction

Erythrocyte membrane zinc concentration was measured in 14 normal volunteers and in 36 patients with taste and smell dysfunction, and compared with zinc concentration in erythrocyte cytosol and blood plasma in the normal volunteers and with zinc concentration in erythrocyte cytosol, blood serum, urine, and parotid saliva in the patients. A A significant negative correlation was found between age and membrane zinc concentration in the normals. A significant positive correlation was found between parotid saliva zinc and erythrocyte membrane zinc concentrations in the patients. Concentration of zinc in the erythrocyte membrane is 50% greater than its concentration in cytosol, and three times greater than the zinc concentration in plasma or serum; membrane zinc concentration in humans is approximately one-half that reported in the dog or in the mouse.     

A method to quantitate Coomassie blue-stained proteins in cylindrical polyacrylamide gels

A method for the quantitation of Coomassie blue-stained proteins in cylindrical polyacrylamide gels is described. It involves an elution of the dye with an 80% methanol solution in a sealed Pyrex tube at 100 degrees C for 3 h and a measurement of its concentration at 585 nm. Using a 6.5% polyacrylamide gel and bovine serum albumin as a protein standard, the curve of absorbance of the dye solution as a function of the amount of protein was observed to be linear up to 30-40 micrograms of protein and as little as 0.8-1.0 micrograms of protein could be measured. The validity of the method was indicated by the values obtained for the relative proportions of the human erythrocyte membrane proteins. Using this method, the color yields of several proteins varying widely with respect to their size, amino acid composition, and carbohydrate content were determined in a 6.5% polyacrylamide gel. The results showed that they were generally the same except for proteins having a high carbohydrate content which were significantly lower.     

Effect of diamide on protein oxidation and physico-chemical properties of lipids in erythrocyte membranes

The effect of diamide on the physicochemical state of proteins and lipids of human erythrocyte membrane was studied. It was found that diamide at a concentration of 1 mM decreases the content of the SH-groups of membrane proteins by approximately 50%, resulting in enhanced vesiculation of erythrocytes upon metabolic exhaustion of cells. It was shown using fluorescein isothiocyanate-labeled concanavalin A and 4,4'-diisothiocyano-2,2'-stilbene disulfonate that diamide changes the structural state of the main integral protein of erythrocyte membranes, the band 3 protein. Changes in the microviscosity of the membrane lipid bilayer depending on diamide concentration were determined from the changes in the fluorescence parameters of the lipophilic probes (pyrene and 1,6-diphenyl-3,5-hexatriene). The level of lipid peroxidation products in membranes remained unchanged. It follows from these data that the SH-oxidizing agent diamide does not directly interact with the lipid bilayer of membrane and produces changes in the physicochemical state of lipids presumably by disrupting protein-lipid interactions that take place upon oxidation of the SH-groups and cross-linking of membrane proteins.     

Organic-acid transport in resealed haemoglobin-containing human erythrocyte ''ghosts''.

The transport of organic acids across the membrane of resealed haemoglobin-containing erythrocyte 'ghosts' prepared by a dialysis technique has been studied. The present work forms part of studies directed towards the use of erythrocyte cellular carriers in enzyme-replacement therapy of inherited metabolic diseases. Oxalic acid, glycollic acid and glyoxylic acid were taken as representative of aliphatic acids of low molecular mass and benzoic and cinnamic acids as representative of unsubstituted aromatic acids. These selected acids are important in the diseases with which the present work is concerned. Comparison of influx and efflux transport characteristics showed that erythrocyte 'ghosts' retain transport properties closely similar to those of normal erythrocytes. Rapid transport was observed with all organic acids studied and there was a linear relationship between initial amount of influx and external concentration of aliphatic acid. Saturation of the transport system was not observed up to 1 mM external concentration, and the presence of plasma in the external medium had no effect on transport characteristics. Transport in intact erythrocytes and prepared erythrocyte 'ghosts' from patients with hyperoxaluria was also studied.     

Biosynthesis of mouse erythrocyte membrane proteins by friend erythroleukemia cells

The synthesis of mouse erythrocyte membrane proteins by Friend erythroleukemia cells during dimethyl sulfoxide-induced differentiation was studied. Untreated and dimethyl sulfoxide-treated cells were incubated with l-[³H] leucine and the incorporation of radioactivity into total trichloroacetic acid-insoluble proteins and into proteins immunoprecipitated with a multivalent rabbit antibody to mouse erythrocyte membranes was determined. The immunoprecipitated membrane proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and radioactivity was detected by fluorography. The incorporation of l-[³H]leucine into total cell proteins was linear for 20 min in both untreated and treated cells. Exposure of the cells to dimethyl sulfoxide had an inhibitory effect on protein synthesis, with a significant decrease noted on the fourth day of treatment and a continued decline occurring until the seventh day when protein synthesis was 42% that of untreated cells. The synthesis of erythrocyte membrane proteins was 0.49% that of total cell proteins in untreated cells, was increased to 1.27% by the third day of treatment and remained at about 1% of total protein synthesis from the fourth to the seventh day. Untreated cells synthesized low levels of spectrin, bands 5 and 6 proteins. Treatment with dimethyl sulfoxide caused a staggered increase in synthesis of a number of erythrocyte membrane proteins. Spectrin synthesis increased 4-fold by the third day of treatment and declined thereafter. The synthesis of membrane proteins with electrophoretic mobilities similar to bands 3 and 4 was increased 2–3-fold by the fourth day, while bands 6 and 5 proteins attained maximal synthesis (4-fold) on the fifth and sixth days of treatment.     

An immunochemical approach for the analysis of membrane protein alterations in Ca2+-loaded human erythrocytes

An increase in the intracellular concentration of Ca²⁺ in human erythrocytes results in the formation of γ-glutamyl-?-lysine cross-linked membrane protein polymers. Following solubilization of the membranes with SDS, these polymers can be isolated on a Lubrol-containing sucrose gradient. Immunoelectrophoresis of the polymeric material with a polyspecific rabbit antibody against human ghosts gave rise to a single, but heterogeneous, precipitate. The polymer was amphiphilic and, on addition to Triton-solubilized erythrocyte membrane proteins, it coprecipitated with spectrin. When the antighost antibody was absorbed with the polymer prior to cross immunoelectrophoresis of normal erythrocyte membrane proteins, the precipitates of glycophorin, acetylcholinesterase, and hemoglobin were normal, whereas the anti-body liters against band 3 protein, spectrin, and ankyrin became reduced. Furthermore, a rabbit antibody raised against the isolated human polymer reacted selectively with the same three membrane proteins. No reactions occurred with lysate proteins.     

Alterations in erythrocyte membrane lipid and fatty acid composition in Chediak-Higashi syndrome

Chediak-Higashi syndrome (CHS) is an autosomal recessive disease characterized by the presence of abnormally large cytoplasmic organelles in all body granule producing cells. The molecular mechanism for this disease is still unknown. Functional disorders in membrane-related processes have been reported. Erythrocyte membranes from four CHS patients and 15 relatives including obligatory heterozygous were studied to examine potential alterations in the lipid and fatty acid profile of erythrocyte membranes associated with this syndrome. Plasma concentrations of cholesterol, triglycerides, phospholipids, and apolipoproteins AI and B100, and the lipid components of very low-, intermediate-, low- and high-density lipoproteins were also determined. CHS erythrocyte membranes were found to be enriched with lipids in relation to protein and to show: (1) an increase in cholesterol and choline-containing phospholipids (sphingomyelin and phosphatidylcholine) that predominate in the outer monolayer, which is higher than the increase in phosphatidylserine and phosphatidylethanolamine, that are chiefly limited to the inner monolayer in normal red blood cells; (2) a relative palmitic acid and saturated fatty acid increase and arachidonic acid and unsaturated fatty acid decrease, this resulting in a lower unsaturation index than controls. Changes in CHS erythrocyte membrane lipids seem to be unrelated to serum lipid disorders as plasma lipid and apolipoprotein concentrations were apparently in the normal range, with the exception of a modest hypertriglyceridemia in patients and relatives and a decreased concentration of HDL cholesterol in patients. These findings indicate that CHS erythrocyte membranes contain an abnormal lipid matrix with which membrane proteins are defectively associated. The anomalous CHS membrane composition can be explained on the postulated effects of the CHS1/Lyst gene.     

Effect of complement on the lateral mobility of erythrocyte membrane proteins. Evidence for terminal complex interaction with cytoskeletal components

The lateral mobilities of erythrocyte membrane proteins and terminal complement complexes (TCC) were measured on C-treated erythrocyte ghosts by the technique of fluorescence redistribution after photobleaching. Results showed that the lateral diffusion coefficient of the bulk membrane proteins decreased with the assembly of TCC on the membrane at low C dose and was significantly reduced with assembly of the full membrane attack complex (C5b-9), even in the absence of cell lysis. At high serum doses, the mobility of the membrane proteins increased slightly above that of the control cells. The diffusion coefficients of the TCC on the erythrocyte membrane range from 1.18 to 4.37 x 10(-11) cm2/s, values characteristic of anchored membrane proteins. Spectrin-depletion of the C-lysed erythrocytes results in 25- and 45-fold increases in the diffusion coefficients of the membrane proteins and the C5b-9 complex, respectively. Conversely, oxidative cross-linking of spectrin by diamide reduced the diffusion coefficients of both membrane and C proteins. These studies indicate that the deposition of TCC on an erythrocyte can result in a substantial change in the physical and structural properties of the target membrane, aside from the creation of functional lesions. The low mobilities of the terminal complexes on the target membrane suggest possible interactions with cytoskeletal elements or with anchored membrane proteins.     

Erythrocyte membrane proteins. Sequential accumulation in the membrane during reticulocyte maturation

Reticulocytes of increasing maturity were separated by dextran gradient centrifugation. The accumulation in the membrane of the anion transport protein and other erythrocyte membrane proteins was studied during reticulocyte maturation by separating reticulocytes after incubation with [³⁵S]methionine. The incorporation of the reticulocyte membrane proteins was shown to be sequential, the anion transport protein being inserted at a very early stage in the cells' maturation.     

Electron spin resonance, hematological, and deformability studies of erythrocytes from patients with Huntington's disease.

Electron spin resonance, hematologic, and deformability studies of erythrocytes from patients with Huntington's disease have been performed A decreased deformability of Huntington's disease erythrocytes compared to normal controls was demonstrated. No difference in erythrocyte hematologic indices, osmotic fragility, reticulocyte counts, or intracellular Na+ concentration was found. Huntington's disease serum had no demonstrable effect on electron spin resonance parameters of a protein-specific spin label attached to membrane proteins in control erythrocytes compared to the effect of control serum. This finding suggests that under the conditions employed no serum component or circulating factor is responsible for the changes in the physical state of membrane proteins in Huntington's disease erythrocytes (Butterfield, D.A., Oeswein, J.Q. and Markesbery, W.R. (1977) Nature 267, 453--455). No alteration in lipid fluidity of Huntington's disease erythrocyte membranes could be discerned suggesting that the underlying molecular defect in Huntington's disease involves a membrane protein. The results of the present studies on erythrocytes strongly support the concept that Huntington's disease is associated with a generalized membrane abnormality.     

A new method for the preparation of band 3, the main integral protein of the human erythrocyte membrane

Band-3 protein from human erythrocyte membranes was isolated, without using detergents, by a two-step procedure: (1) The peripheral proteins were removed from the membrane by treatment with 10% acetic acid. (2) The remaining lipoprotein complex was solubilized in approximately 92% (v/v) acetic acid and then separated into its components by preparative zonal electrophoresis in a gradient made up of acetic acid, water and sucrose. Band 3 was recovered from the gradient at a yield of 60 - 70% and purity of about 95%. Approximately 25 mg of band 3 could be prepared in one run. The protein is soluble in aqueous solutions, even in the absence of organic solvents or detergents. In addition to band 3, the proteins stained by periodic acid/Schiff's reagent (the sialoglycoproteins) are also separated from the other proteins.     

Quantitation of lysine-bound glucose of normal and diabetic erythrocyte membranes by HPLC analysis of furosine [ epsilon-N(L-furoylmethyl)-L-lysine]

Non-enzymatic glycosylation of erythrocyte membranes was studied using a non-radioactive and sensitive procedure for specific quantitation of lysine-bound glucose in proteins. About 2 nmol lysine-bound glucose/mg protein were found in ghosts from normal erythrocytes, and this value was about doubled in diabetic patients. In vitro incubation of normal ghosts with glucose gave rise to levels of lysine-bound glucose similar to those found in diabetics. There was a linear correlation between the amount of lysine-bound glucose of total hemoglobin and of membrane proteins. Membrane glycosylation also depended on the age of erythrocytes displaying significantly higher values in old cell populations.     

Speciation of Al in human serum by convective-interaction media fast-monolithic chromatography with inductively coupled plasma mass spectrometric detection

A new analytical procedure using anion-exchange separation support based on convective-interaction media (CIM) was developed for the speciation of Al in human serum. The separation of proteins was performed on a weak anion-exchange CIM diethylamine (DEAE) fast-monolithic disk. To prevent co-elution of low molecular mass (LMM) Al species with high molecular mass (HMM) Al compounds on CIM disk serum proteins were first separated from LMM-Al species by the use of size exclusion chromatography (SEC). For this purpose 1 mL of serum was injected onto SEC (Superdex 75 HR 10/30) column. Isocratic elution using 0.05 M TRIS-HCl+0.03 M NaHCO(3) was applied and separation of proteins was followed by UV detection at 278 nm. It was experimentally proven that proteins were eluted in 5.5 mL peak that was collected into a polyethylene cup. A 0.1 mL of the sample aliquot was then injected onto the CIM DEAE disk. The separation of serum proteins was obtained in 10 min by applying linear gradient elution from 100% buffer A (0.05 M TRIS-HCl+0.03 M NaHCO(3)) to 100% buffer B (A+1M NH(4)Cl) and followed by UV detection at 278 nm. Separated Al species were detected on-line by inductively coupled plasma mass spectrometry (ICP-MS). Well-resolved protein peaks were obtained. It was experimentally proven that 90+/-3% of Al in spiked serum of renal patient was eluted under the transferrin peak. The proposed speciation procedure removes LMM-Al species and enables reliable determination of the concentration and composition of Al bound to proteins by CIM DEAE-ICP-MS when the concentration of Al in serum is higher than 5 ng mL(-1). In comparison to chromatographic columns CIM disks enable faster separation and simpler manipulation during cleaning procedure and coupling to ICP-MS.     

Increase of the negative charge of erythrocyte membrane proteins in hereditary neuromuscular diseases

The parameters of erythrocyte ghost protein's fluorophores by nitrate's anions were studied in patients with various hereditary myodystrophy. In all the groups under examination the share of fluorophores accessible to a quencher was close to 1. In erythrocyte membranes of healthy donors the relevant constant quenchering was about 17.3 +/- 1.9 M-1 while those of patients were decreased by 3.1 (Duchenne's myodystrophy) and by about 2.0 (other forms of primary and secondary progressive muscular dystrophies). The most probable reason for the decreasing constant of quenchering is the increase of negative charges on the erythrocyte membrane proteins.     

Membrane raft actin deficiency and altered Ca2+-induced vesiculation in stomatin-deficient overhydrated hereditary stomatocytosis

In overhydrated hereditary stomatocytosis (OHSt), the membrane raft-associated stomatin is deficient from the erythrocyte membrane. We have investigated two aspects of raft structure and function in OHSt erythrocytes. First, we have studied the distribution of other membrane and cytoskeletal proteins in rafts by analysis of detergent-resistant membranes (DRMs). In normal erythrocytes, 29% of the actin was DRM-associated, whereas in two unrelated OHSt patients the DRM-associated actin was reduced to <10%. In addition, there was a reduction in the amount of the actin-associated protein tropomodulin in DRMs from these OHSt cells. When stomatin was expressed in Madin-Darby canine kidney cells, actin association with the membrane was increased. Second, we have studied Ca2+-dependent exovesiculation from the erythrocyte membrane. Using atomic force microscopy and proteomics analysis, exovesicles derived from OHSt cells were found to be increased in number and abnormal in size, and contained greatly increased amounts of the raft proteins flotillin-1 and -2 and the calcium binding proteins annexin VII, sorcin and copine 1, while the concentrations of stomatin and annexin V were diminished. Together these observations imply that the stomatin-actin association is important in maintaining the structure and in modulating the function of stomatin-containing membrane rafts in red cells.     

Erythrocyte 2,3-DPG,ATP and oxygen affinity in hemodialysis patients

Patients on a chronic hemodialysis regimen were studied with respect to their erythrocyte adaptation to anemia. Erythrocyte 2,3-diphosphoglycerate (DPG) concentration was suboptimal compared with that of anemic patients who were not uremic. In uremic patients erythrocyte 2,3-DPG correlated poorly with hemoglobin level but more strongly with plasma pH. Differences between observed levels of erythrocyte 2,3-DPG and the values predicted using data from other anemic patients also correlated with pH. Gradual correction of plasma pH with oral sodium bicarbonate resulted in a substantial increase in erythrocyte 2,3-DPG and a decrease in oxygen affinity. Therefore, maintenance of normal pH in uremic subjects may improve tissue oxygenation. On the other hand, the rapid correction of acidosis during dialysis resulted in increased oxygen affinity. This response was due to the direct effect of pH on oxygen affinity in the absence of a significant change in erythrocyte 2,3-DPG or adenosine triphosphate (ATP) during hemodialysis. Erythrocyte ATP but not 2,3-DPG correlated with serum inorganic phosphate in uremic subjects. A 21% reduction of serum phosphate produced by ingestion of aluminum hydroxide gel had no significant effect on these variables.     

Effects of simvastatin and pravastatin on peroxidation of erythrocyte plasma membrane lipids in patients with type 2 hypercholesterolemia

Since hypercholesterolemia directly modifies the composition of erythrocytes plasma membrane, the influence of statins on erythrocytes has been researched. The beneficial effects of statins on clinical events may involve mechanisms that modify endothelial dysfunction, plaque stability, thrombus formation and inflammatory responses. The aim of the study was to evaluate the hypolipemic efficacy and effects of pravastatin and simvastatin on erythrocyte membrane fluidity and damage of erythrocytes in patients with type 2 hypercholesterolemia in comparison with a control group of healthy subjects. The study involved 53 patients affected by type 2 hypercholesterolemia (mean age, 53.3 +/- 10.3) with initial total serum cholesterol (TC) levels > 250 mg/dL, LDL-cholesterol (LDL-C) levels > 170 mg/dL, and triglycerides (TG) levels < 400 mg/dL. The control group consisted of 30 healthy individuals (mean age 56.9 +/- 6.3). Statins were given for 12 weeks. The dosages for oral administration of simvastatin and pravastatin were 20 mg/day. Laboratory tests were carried out before and after 4 and 12 weeks of the pharmacological treatment. The damage to plasma membrane of erythrocytes was measured on the basis of lipid peroxidation. The fluidity of plasma membrane of erythrocytes was determined by electron paramagnetic resonance (EPR) spectroscopy, using two spin labels: 5-DSA and 16-DSA. The cholesterol level in the membrane of red blood cells was estimated. Simvastatin and pravastatin reduced the total cholesterol concentration and LDL-cholesterol in plasma, as well as the cholesterol concentration in erythrocytes membranes. Hypercholesterolemia induced changes in the basic properties of human erythrocyte plasma membrane, including its fluidity and the intensity of lipid peroxidation. These results indicate that the simvastatin and pravastatin therapy reverses the alteration in the erythrocyte plasma membrane properties.     

Molecular organization of glutamate-sensitive, chemoexcitatory membranes of nerve cells. Physico-chemical characteristics of the glutamate-binding synaptic membrane proteins of the rat cerebral cortex

Some physico-chemical properties of glutamate-binding proteins solubilized from rat cerebral cortex synaptic membranes and purified by affinity chromatography were studied. Purified proteins were shown to be homogenous during SDS polyacrylamide gel electrophoresis (Mr 14000). The Scatchard plots for L-[3H]glutamate binding to the purified membrane proteins revealed the presence of one type of binding sites with Kd 800-1000 nM and Bmax 180-200 pmol/mg of protein. Ultracentrifugation of the glutamate-binding membrane protein in sucrose linear gradient demonstrated that the position of the protein peak depends on protein concentration, i.e. after dilution of the sample the protein peak is shifted from 28 000-30 000 to 12 000-15 000. The values of sedimentation coefficients decrease correspondingly to 2.1S. Presumably, these processes are due to dissociation of receptor macromolecules. The glutamate receptor is a glycoprotein-lipid complex made up of several low molecular weight subunits.     

Membrane raft actin deficiency and altered Ca-induced vesiculation in stomatin-deficient overhydrated hereditary stomatocytosis

In overhydrated hereditary stomatocytosis (OHSt), the membrane raft-associated stomatin is deficient from the erythrocyte membrane. We have investigated two aspects of raft structure and function in OHSt erythrocytes. First, we have studied the distribution of other membrane and cytoskeletal proteins in rafts by analysis of detergent-resistant membranes (DRMs). In normal erythrocytes, 29% of the actin was DRM-associated, whereas in two unrelated OHSt patients the DRM-associated actin was reduced to < 10%. In addition, there was a reduction in the amount of the actin-associated protein tropomodulin in DRMs from these OHSt cells. When stomatin was expressed in Madin-Darby canine kidney cells, actin association with the membrane was increased. Second, we have studied Ca²⁺-dependent exovesiculation from the erythrocyte membrane. Using atomic force microscopy and proteomics analysis, exovesicles derived from OHSt cells were found to be increased in number and abnormal in size, and contained greatly increased amounts of the raft proteins flotillin-1 and -2 and the calcium binding proteins annexin VII, sorcin and copine 1, while the concentrations of stomatin and annexin V were diminished. Together these observations imply that the stomatin-actin association is important in maintaining the structure and in modulating the function of stomatin-containing membrane rafts in red cells.     

The effect of plasma viscosity on the resistance of erythrocyte movement along capillaries

By means of three-dimensional mathematical model describing movement of an asymmetric erythrocyte along the capillary of a small section the effect of the viscosity of the blood plasma on the resistance value to the erythrocyte movement in microvessels was studied. The model takes into account the elastic properties of the erythrocyte, the volume and area of its surface and the mobility of the membrane performing the rotation. It is shown that for characteristic conditions of hemodynamics experimentally observed in the microcirculation system the pressure gradient on the erythrocyte ends is 3-5-fold above the difference of pressures at the ends of plasma column of similar length. The dependence of the pressure gradient at the erythrocyte ends, which accounts for its movement along the capillary, on plasma viscosity is determined at different values of erythrocyte velocity.