A microfluorimetric study of translational diffusion in erythrocyte membranes

A method is described which permits quantitative study of translational diffusion in the membranes of single cells. Human erythrocytes were labelled with fluorescein isothiocyanate and then hemolyzed, which yielded ghosts of normal shape and strong fluorescence. By application of sodium dodecylsulfatepolyacrylamide gel electrophoresis it was found that a very large part of fluorescein isothiocyanate was bound to the proteins of the erythrocyte membrane. In a fluorescence microscope, single ghosts were exposed to a sharply bounded intensive beam of light in such a manner that in each case only one half of the ghost was bleached. By microscopic measurements it was studied whether fluorescent material would diffuse from the unbleached part of the membrane into the bleached part and vice versa. However, within the measuring time of 20 min at room temperature a significant degree of such a diffusion could not be detected. In order to evaluate the experimental data quantitatively, the diffusion equation for a spherical surface was solved, and the obtained solution furthermore was integrated over the hemispheres. By these means a value of 3 · 10⁻¹² cm²/s was derived from the experimental data as an upper limit for the diffusion coefficient of fluorescein isothiocyanate-labelled compounds in the erythrocyte membrane at 20°–23°C.     

Increased Cation Conductance in Human Erythrocytes Artificially Aged by Glycation

Excessive glucose concentrations foster glycation and thus premature aging of erythrocytes. The present study explored whether glycation-induced erythrocyte aging is paralleled by features of suicidal erythrocyte death or eryptosis, which is characterized by cell membrane scrambling with subsequent phosphatidylserine exposure at the cell surface and cell shrinkage. Both are triggered by increases of cytosolic Ca²⁺ concentration ([Ca²⁺]_i), which may result from activation of Ca²⁺ permeable cation channels. Glycation was accomplished by exposure to high glucose concentrations (40 and 100 mM), phosphatidylserine exposure estimated from annexin binding, cell shrinkage from decrease of forward scatter, and [Ca²⁺]_i from Fluo3-fluorescence in analysis via fluorescence-activated cell sorter. Cation channel activity was determined by means of whole-cell patch clamp. Glycation of total membrane proteins, immunoprecipitated TRPC3/6/7, and immunoprecipitated L-type Ca²⁺ channel proteins was estimated by Western blot testing with polyclonal antibodies used against advanced glycation end products. A 30–48-h exposure of the cells to 40 or 100 mM glucose in Ringer solution (at 37°C) significantly increased glycation of membrane proteins, hemoglobin (HbA_1c), TRPC3/6/7, and L-type Ca²⁺ channel proteins, enhanced amiloride-sensitive, voltage-independent cation conductance, [Ca²⁺]_i, and phosphatidylserine exposure, and led to significant cell shrinkage. Ca²⁺ removal and addition of Ca²⁺ chelator EGTA prevented the glycation-induced phosphatidylserine exposure and cell shrinkage after glycation. Glycation-induced erythrocyte aging leads to eryptosis, an effect requiring Ca²⁺ entry from extracellular space.     

Location of the maltosyl isothiocyanate binding site on the human erythrocyte glucose transporter

The covalent affinity probe maltosyl isothiocyanate (MITC) has been used previously to identify the glucose transporter of human erythrocytes as a component of band 3. By use of limited proteolysis, the site on the Mr 100 000 protein to which MITC attaches has been localized to a 17 000-dalton region near the center of the polypeptide chain which is intimately associated with the membrane. The erythrocyte anion transporter, which is probably homologous to the glucose carrier, has a corresponding segment which is known to bind the covalent affinity label 4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid [Ramjeesingh, M., Gaarn, A., & Rothstein, A. (1980) Biochim. Biophys. Acta 559, 127-139]. These results suggest that, in addition to having structural features in common, the two carrier proteins may be quite similar with regard to functional organization.     

Calcium effects on human erythrocyte membrane proteins

The effects of Ca²⁺ on human erythrocyte membrane proteins were examined by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. Ca²⁺ had several effects on normal human erythrocyte membrane proteins. It affected the binding of cytoplasmic proteins to the membrane, produced a non-reversible aggregation of several membrane proteins and activated apparent proteolysis of membrane proteins. The Ca²⁺ effect could be obtained with isolated, washed membranes when the erythrocyte cytoplasm was added. These studies indicate that the Ca²⁺-induced membrane proteolysis and aggregation effects are not due simply to its presence at the time of hemolysis as previously suggested (Carraway, K.L., Triplett, R.B. and Anderson, D.R. (1975) Biochim. Biophys. Acta 379, 571–581), but are the result of more complex interactions between the erythrocyte membrane and cytoplasmic factors.     

Photolabeling of glucose-sensitive cytochalasin B binding proteins in erythrocyte, fibroblast and adipocyte membranes

(³H)Cytochalasin B has been photoincorporated into membrane fractions of the human erythrocyte, Rous sarcoma virus-transformed chicken embryo fibroblast and rat adipocyte. Identification of D-glucose sensitive cytochalasin B binding sites was achieved by photolyzing membranes with radioligand in the presence of 0.5–0.7M D- or L-glucose. In the erythrocyte the major labeled bands on SDS-polyacrylamide gels were at 55,000 and 46,000 daltons. In the virus-transformed fibroblasts a major labeled band was at 55,000 daltons, and in adipocyte microsomal membranes, peaks at 50,000 and 45,000 daltons were observed. Binding characteristics of these polypeptides suggest that they are the putative glucose transport proteins in these three cell types.     

A theoretical model to predict the behavior of glycosylated hemoglobin levels

The measurement of glycosylated hemoglobin as a percentage of total hemoglobin is rapidly becoming the standard method of monitoring the average blood sugar level in diabetics for research purposes and may soon become the standard for clinical care and diagnosis. Much speculation exists in the literature about the nature of the glycosylation reaction. Most experimenters expect a linear relationship between the plasma glucose level and percent glycosylated hemoglobin in whole blood; however, a curve of decreasing slope with increasing glucose concentration is found.Here, a reaction model including simple first order kinetics between glucose and hemoglobin and a finite erythrocyte life of 120 days is considered. By carrying out the integration for each erythrocyte cohort followed by an integration combining all cohorts, a curve corresponding to the experimental result is found. In addition, results on expected glycosylated hemoglobin percent as a function of erythrocyte age and plasma glucose concentration are presented as well as a plot of glucose concentration versus glycosylated hemoglobin percent for the 40-day erythrocyte life in mice. All of the results correlate with experimental values in the literature if a rate constant of k = 1·0 × 10^?5dlmg^?1 day is used.The evaluation of a radioactive iron-transferrin experiment in the literature reveals the possibility that the glycosylation reaction begins during erythropoiesis.Finally, a curve is displayed which shows the expected 120-day decay during normoglycemia, of an elevated glycosylated hemoglobin level resulting from a preceding period of constant hyperglycemia.     

Reconstitution of the glucose transport activity of rat adipocytes

Rat epididymal fat cell membrane proteins were extracted from adipocyte ghosts with octylglucoside and incorporated by detergent dialysis into unilamellar phosphatidylcholine vesicles approx. 200 nm in diameter. The rate of glucose transport into the vesicles under zero-trans conditions was substrate dependent, saturable and inhibited by phloretin and cytochalasin B. Their maximum specific transport activity was 35.6 mumol/min per mg protein, and their half saturation constant for glucose was 15 mM. Glucose transport into the reconstituted vesicles was inhibited by only those sugars which competitively inhibited glucose transport into intact adipocytes. A major protein component of the vesicles was a 100 kDa protein which we had previously found to react with the affinity label maltosyl isothiocyanate (Malchoff, D.M., Olansky, L., Pohl, S. and Langdon, R.G. (1981) Fed. Proc. 40, 1893). Removal of adipocyte ghost membrane extrinsic proteins with dimethylmaleic anhydride followed by extraction of the resulting membrane pellet with octylglucoside yielded a solution which contained two major proteins, of Mr 100 000 and 85 000, with very small quantities of lower Mr proteins. Vesicles into which these proteins were incorporated had average specific transport activities of 624 mumol/min per mg protein and half saturation constants of 22 mM. Our results strongly indicate that the native glucose transporter of the rat adipocyte, like that of the human erythrocyte (Shelton, R.L. and Langdon, R.G. (1983) Biochim. Biophys. Acta 733, 25-33), is a 100 kDa protein.     

[125I]diiodofluorescein isothiocyanate: Its synthesis and use as a reagent for labeling proteins and cells to high specific radioactivity

We have synthesized a radioactive derivative of fluorescein isothiocyanate (PITC) by lactoperoxidase-catalyzed iodination of fluorescein amine using ¹²⁵I. The iodinated amine was purified by thin-layer chromatography and converted to the isothiocyanate by reaction with thiophosgene. The product was inferred to be the diiodo derivative of FITC by comparing its absorbance and fluorescence emission spectra with those of known standards. This reagent, [¹²⁵I]diI-FITC, shares many of the useful features of its congener, FITC. Specifically, it may be used to label under mild conditions of temperature and pH, and it is chemically stable. When erythrocytes were labeled with [¹²⁵I]diI-FITC, radioactivity was found principally in a major exposed protein of the cell surface, and very little hemoglobin was labeled. [¹²⁵I]diI-FITC may prove generally useful as a means of labeling proteins and cell surfaces to high specific radioactivity.     

Different binding sites for glucose and sorbose at the erythrocyte membrane,studied by gel filtration and infrared spectroscopy

Summary Human red blood cell membranes were solubilized with sodium dodecylsulfate and incubated with various concentrations of¹⁴C-glucose and¹⁴C-sorbose. After gel filtration on Sephadex G-100, which separated lipoproteins of differing lipid content, it was observed that the radioactivity of the bound glucose coincided with the protein peak. Radioactivity of bound sorbose was found mainly before and after the protein peak. This distribution of bound sugars was confirmed by double labeling experiments in which³H-glucose and¹⁴C-sorbose were applied simultaneously. Infrared spectroscopy revealed differences between the membranes loaded with sorbose and glucose. Particularly, the band in the C–O–C and P=O region at 1,225 cm^–1 was intensified in the sorbose-loaded membranes. Compared to serum albumin, the erythrocyte membranes were found to bind 4 times as much¹⁴C-glucose per mg of protein. It is concluded from the results obtained by gel filtration that glucose and sorbose preferentially bind at different sites of the erythrocyte membrane. The results obtained by infrared spectroscopy correspond with this conclusion.     

The effect of erythrocyte associated light scattering on membrane fluorescence polarization

Summary The apparent membrane fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene has been reported to be lower in intact erythrocytes than in isolated erythrocyte membranes. Although this difference was once suggested to be caused by the fluidizing effect associated with the loss of erythrocyte proteins during membrane isolation, it is currently thought to be an artifact resulting from intense light scattering properties of intact erythrocytes which overwhelm extrapolation methods of correcting for light scattering. This study confirmed that, at erythrocyte concentrations greater than 10⁷ cells/ml, this difference was caused by intense light scattering; however, at erythrocyte concentrations less than 4.0 × 10⁶ cells/ml, the anisotropy values for erythrocytes and isolated membranes are identical, demonstrating that intense light scattering can be overcome with dilute suspensions of cells.     

Stopped-Flow Circular Dichroism Study on Conformational Change of Alpha-Chymotrypsin by Sodium Dodecyl Sulfate

Protein synthesis in dispersed cells from fetal liver was studied by fluorography of SDS-polyacrylamide gel electrophoresis of a [³⁵S]methionine labeled cell lysate. Synthesis of several proteins with molecular weights ranging from 45,000 to 220,000 was observed during erythropoiesis in fetal liver. Some of these proteins were demonstrated to be erythrocyte membrane proteins because they were immunoprecipitated with antiserum against rat red blood cells and the immunoprecipitation was competitive with non-radioactive proteins solubilized from erythrocyte ghosts. The same antiserum caused agglutination of dispersed cells from fetal liver. This supported the possibility that these proteins are translocated onto plasma membranes of the dispersed cells.     

Identification of differences between the surface proteins and glycoproteins of normal mouse (Balb/c) and human erythrocytes

Summary The topography of the external surface of the Balb/c mouse erythrocyte has been investigated and compared to the human erythrocyte by using a series of protein radiolabeling probes. After sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the pattern of Coomassie Blue stained proteins was very similar for mouse and human erythrocyte ghosts, as was the distribution of radioactivity in protein bands after lactoperoxidase catalyzed radioiodination. The mouse erythrocyte glycoproteins identified by periodic-acid-Schiff and Stains-All reagents, sialic acid analysis of gel slices, binding of¹²⁵I-wheat germ agglutinin and¹²⁵I-concanavalin A to the gels, and glycoprotein radiolabeling techniques, differed markedly from the sets of proteins labeled by radioiodination, and also differed from the human erythrocyte glycoproteins. Instead of the PAS I to PAS IV series of sialoglycoproteins characteristic of human erythrocytes, the mouse erythrocyte possesses a broad band of sialoglycoproteins with several peaks ranging in mol wt from 65,000 to 32,000. The same group of sialoglycoproteins were labeled by the periodate/B³H₄ ^– technique specific for terminal sialic acid, and the galactose oxidase/B³H₄ ^– method (plus neuraminidase) specific for galactosyl/N-acetylgalactosaminyl residues penultimate to sialic acid. These results emphasize the necessity to employ a variety of protein radiolabeling probes based on different labeling specificities, to study the membrane topography of cells which are poorly understood compared to the human erythrocyte membrane.     

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.     

Preparation and characterization of chick erythrocyte nuclei from heterokaryons

A method for the isolation of reactivated chick erythrocyte nuclei from heterokaryons was developed. The heterokaryons were produced by fusing chick erythrocytes with HeLa or L cells in the presence of inactivated Sendai virus. At various time intervals after fusion nuclei were isolated directly from the monolayer by treatment with an acidic detergent solution. Chick erythrocyte nuclei were then separated from other nuclei (HeLa or L cell) by centrifugation on sucrose gradients. The purified preparation of reactivated chick erythrocyte nuclei was shown to be free from other nuclei and cytoplasmic contamination. By using L cells which had been labelled with ³H-leucine before fusion or heterokaryons labelled after fusion it was demonstrated that labelled mouse proteins migrate from the cytoplasm of the heterokaryons into the reactivating chick erythrocyte nuclei. ³H-uridine labelling of heterokaryons made by fusing UV-irradiated chick erythrocytes with L cells failed to reveal any significant migration of mouse RNA into the chick erythrocyte nuclei.     

Induction of increased calcium uptake in liposomes having membrane proteins of chicken erythrocytes by S-adenosylmethionine

Liposomes having membrane proteins of chicken erythrocytes were prepared and the effect of S-adenosylmethionine on ⁴⁵Ca²⁺ uptake into the liposomes was investigated. S-Adenosylmethionine, a donor of methyl groups in enzymatic methylation, induced an increase of ⁴⁵Ca²⁺ uptake into the proteoliposomes with membrane proteins but not into the liposomes without membrane proteins. Increased release of ⁴⁵Ca²⁺ from the inside of the proteoliposomes was also induced by S-adenosylmethionine. These increases of uptake and release of ⁴⁵Ca²⁺ were inhibited by S-adenosylhomocystein, an inhibitor of enzymatic methylation. Furthermore, membrane proteins from chicken erythrocytes showed protein and phospholipid methyltransferase activities. The uptake of other materials, 3-0-[methyl-³H]glucose, α-[1-¹⁴C]aminoisobutyric acid, ⁴²K⁺ and ⁵⁴Mn²⁺, into the proteoliposomes was not increased by S-adenosylmethionine. These results suggest that enzymatic methylation of membrane components may have an important role in the regulation of calcium transport in the chicken erythrocyte membrane and this regulation is rather specific for calcium.     

Characterization of carbonic anhydrase and anion exchange in the erythrocytes of bowfin (Amia calva), a primitive air-breathing fish

The purpose of this study was to investigate the characteristics of carbonic anhydrase (CA) and the Cl⁻/HCO₃⁻ exchanger (Band 3; AE1) in the erythrocytes of bowfin (Amia calva), a primitive air-breathing fish, in order to further understand the strategies of blood CO₂ transport in lower vertebrates and gain insights into the evolution of the vertebrate erythrocyte proteins, CA and Band 3. A significant amount of CA activity was measured in the erythrocytes of bowfin (70 mmol CO₂ min⁻¹ ml⁻¹), although it appeared to be lower than that in the erythrocytes of teleost fish. The turnover number (K_cat) of bowfin erythrocyte CA was intermediate between that of the slow type I CA isozyme in agnathans and elasmobranchs and the fast type II CA in the erythrocytes of the more recent teleost fishes, but the inhibition properties of bowfin erythrocyte CA were similar to the fast mammalian CA isozyme, CA II. In contrast to previous findings, a plasma CA inhibitor was found to be present in the blood of bowfin. Bowfin erythrocytes were also found to possess a high rate of Cl⁻/HCO₃⁻ exchange (6 nmol HCO₃⁻ s⁻¹ cm⁻²) that was sensitive to DIDS. Visualization of erythrocyte membrane proteins by SDS-PAGE revealed a major band in the 100 kDa range for the trout, which would be consistent with the anion exchanger. In contrast, the closest major band for the membranes of bowfin erythrocytes was around the 140 kDa range. Taken together, these results suggest that the strategy for blood CO₂ transport in bowfin is probably similar to that in most other vertebrates despite several unique characteristics of erythrocyte CA and Band 3 in these primitive fish.     

Inhibition of glucose transport into brain by phlorizin, phloretin and glucose analogues

An indicator dilution technique with ²²Na⁺ as the intravascular marker was used to measure unidirectional transport of d-[6-³H]glucose from blood into the isolated, perfused dog brain. 18 compounds which are structurally related to glucose were tested for their ability to inhibit glucose transport. The data suggest that no single hydroxyl group is absolutely required for glucose transport, but rather that glucose binding to the carrier probably occurs through hydrogen bonding at several sites (hydroxyls on carbons 1, 3, 4 and 6). In addition, α-d-glucose has higher affinity for the carrier than does β-d-glucose.A separate series of experiments demonstrated that phlorizin and phloretin are competitive inhibitors of glucose transport into brain; however, phloretin is partially competitive and inhibits at lower concentrations than does phlorizin. Inhibition by phlorizin and phloretin is mutually competitive, indicating that these compounds compete for binding to the glucose carrier. Comparison with the results reported in the literature for similar studies using the human erythrocyte demonstrates a fundamental similarity between glucose transport systems in the blood-brain barrier and erythrocyte.     

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.     

Cyclic AMP increase the Na+ permeability of the avian erythrocyte membrane by a process which does not involve protein phosphorylation

Summary Preparations of avian erythrocyte plasma membranes have been made which are in the form of sealed vesicles. Using these preparations the permeability of the membranes to Na⁺ K⁺, Mg⁺ and Ca⁺ was measured. Monobutyryl cyclic AMP and cyclic AMP increased the permeability to Na⁺ and Ca⁺ under conditions where no protein phosphorylation could occur. The only effect of phosphorylation of membrane proteins was to reduce Ca⁺ permeability. It is thus concluded that cyclic AMP increases Na⁺ permeability in the avian erythrocyte by a direct effect which does not involve protein phosphorylation.