Ghost Preparations Research Articles

Phospholipid asymmetry in human erythrocyte ghosts.

Using phospholipase digestion and the fluorescent probe merocyanine 540 the maintenance of phospholipid asymmetry in the plasma membrane of human erythrocyte ghosts was investigated. Digestion with phospholipase A2 indicated that ghosts prepared in the presence of Mg++ as the only divalent cation retained the normal phospholipid asymmetry characteristic of intact erythrocytes. These ghosts, like normal erythrocytes, also failed to stain with merocyanine 540. However, the presence of as little as 5-10 microM Ca++ during ghost preparation resulted in ghosts in which lipid asymmetry had been abolished, as indicated by phospholipase digestion. Moreover, these ghosts stained with merocyanine 540. In contrast to ghosts, intact erythrocytes treated with ionophore required millimolar levels of Ca++ ions to disrupt membrane lipid asymmetry. To discover the reason for this difference in behavior between ghosts and intact cells, ghosts were prepared from preswollen cells using only small volumes of buffer for lysis. These experiments demonstrated that as the cellular contents of erythrocytes are diluted, the asymmetric arrangement of phospholipids becomes more sensitive to disruption by Ca++.

Human erythrocyte myosin: identification and purification.

Human erythrocytes contain an Mr 200,000 polypeptide that cross-reacts specifically with affinity-purified antibodies to the Mr 200,000 heavy chain of human platelet myosin. Immunofluorescence staining of formaldehyde-fixed erythrocytes demonstrated that the immunoreactive myosin polypeptide is present in all cells and is localized in a punctate pattern throughout the cell. Between 20-40% of the immunoreactive myosin polypeptide remained associated with the membranes after hemolysis and preparation of ghosts, suggesting that it may be bound to the membrane cytoskeleton as well as being present in the cytosol. The immunoreactive myosin polypeptide was purified from the hemolysate to approximately 85% purity by DEAE-cellulose chromatography followed by gel filtration on Sephacryl S-400. The purified protein is an authentic vertebrate myosin with two globular heads at the end of a rod-like tail approximately 150-nm long, as visualized by rotary shadowing of individual molecules, and with two light chains (Mr 25,000 and 19,500) in association with the Mr 200,000 heavy chain. Peptide maps of the Mr 200,000 heavy chains of erythrocyte and platelet myosin were seen to be nearly identical, but the proteins are distinct since the platelet myosin light chains migrate differently on SDS gels (Mr 20,000 and 17,000). The erythrocyte myosin formed bipolar filaments 0.3-0.4-micron long at physiological salt concentrations and exhibited a characteristic pattern of myosin ATPase activities with EDTA, Ca++, and Mg++-ATPase activities in 0.5 M KCl of 0.38, 0.48, and less than 0.01 mumol/min per mg. The Mg++-ATPase activity of erythrocyte myosin in 0.06 M KCl (less than 0.01 mumol/min per mg) was not stimulated by the addition of rabbit muscle F-actin. The erythrocyte myosin was present in about 6,000 copies per cell, in a ratio of 80 actin monomers for every myosin molecule, which is an amount comparable to actin/myosin ratios in other nonmuscle cells. The erythrocyte myosin could function together with tropomyosin on the erythrocyte membrane (Fowler, V.M., and V. Bennett, 1984, J. Biol. Chem., 259:5978-5989) in an actomyosin contractile apparatus responsible for ATP-dependent changes in erythrocyte shape.

Studies on exercise and an elastic protein "connectin" in hindlimb muscle of growing rat.

We examined the effect of physical training on the properties of an elastic protein, connectin, in the hindlimb muscles of 6 growing rats running on a treadmill, in comparison with its properties in 6 growing non-running rats. The experimental results are as follows. 1) The body weight of exercised (R-) and control (C-) rats gradually increased during housing for 31 days, from an average of 46.2 g to 275 g. The average amount of running in the 6 R-rats was 38,160 +/- 12,122 (S.D.) g X m/hr. 2) The SDS-insoluble connectin in R-rat muscle was lower in quantity than that in C-rat muscle, but the SDS-soluble connectin of R-rat muscle was greater in quantity. 3) Amino acid compositions of SDS-soluble connectin in R- and C-rats, calculated from SDS-insoluble connectin and soleus ghost, were significantly different. 4) The soleus ghost preparation consisted mainly of connectin as shown by SDS polyacrylamide gel electrophoresis and by immunohistological staining. 5) The maximum passive tension of the soleus ghost, calculated from the factors of T/phi and (dT/dx)/phi, was higher in R-rats than in C-rats. The above results suggest that physical training might alter the biosynthetic pattern of connectin to suit the developing muscle, just as physical training alters that of myosin isoenzymes.

Characterization of human red blood cell tyrosine kinase

A new tyrosine kinase in human red blood cells has been characterized and partially purified. The major substrate was a protein of molecular weight 93 K which could be phosphorylated both in whole red blood cells incubated with inorganic [ 32P] orthophosphate and in ghost preparations incubated with [γ 32P] ATP. This tyrosine kinase displayed an alkaline isoelectric pH (around 8.5), a molecular weight of 32–33 K and does not seem to be autophosphorylable. Some kinetics of the enzyme are reported. This red blood cell tyrosine kinase is unrelated to EGF and insulin or insulin-like receptor subunits. This enzyme may represent a novel class of tyrosine kinases.

A simplified procedure for estimation of lipid phosphorus in erythrocyte membranes

For the estimation of phospholipids and the other components of the erythrocyte membrane, methods are desirable that supply reproducible results, are simple and time-saving and, thus, practicable in a routine laboratory. Nowadays, nearly all procedures used for estimation of lipid phosphorus in the erythrocyte membrane require the application of extraction methods. The results of a variety of modifications of these methods are strongly affected by the composition of the solvent mixture as shown by Ways and Hanahan [l]. In addition to the extraction procedures of Folch et al [2], those of Rose and Oklander [3] have been applied in many investigations. However, the relatively considerable methodical deficiencies of all extraction procedures should not be ignored. To remove the disturbing non-covalently bound, phosphorus-containing compounds from the lipid extracts, a variety of wash procedures and specific after-treatments are necessary. Moreover, most of the methods known demand a quantitative preparation of erythrocyte ghosts before extraction. The quantitative manipulation of the ghosts during washing is difficult and high speed centrifugation (12000 X g) is necessary for the avoidance of losses. In order to overcome these difficulties, we have tested the polycationic dye Alcian blue for a quantitative preparation of erythrocyte ghosts. Some years ago this compound was used by us for haemagglutination experiments [4]. In the following, we will discuss the application of the erythrocytedye aggregates thus formed in a reproducible assay for the lipid phosphorus of the red cell membrane. This was compared with a method using an extraction procedure of a normal red cell suspension. In addition, some individual phospholipid classes were estimated by an extraction procedure of the Alcian blue ghost aggregates and also compared with our current method.

Properties of red cell ghost preparations susceptible to invasion by malaria parasites

Experimental procedures are described that lead to the formation of sealed red cell ghosts capable of efficient invasion by malaria parasites. Both human and monkey cells have been studied with respect to invasion by Plasmodium falciparum and P. knowlesi respectively. Resealed ghosts containing about half the normal concentration of haemoglobin are prepared by osmotic lysis and resealing at a haematocrit of 70%. When examined in the scanning electron microscope, populations of these ghosts contain few echinocytes, but have an abundance of stomatocytic forms. When undiluted haemolysate is substituted for the aqueous saline diluent, in which the cells are suspended for lysis and resealing, discocytes result, with a morphology very similar to that of the original cells. Invasion is somewhat, but not dramatically higher for this material. An investigation of the effect of intracellular potassium concentration revealed that this had no major effect on invasion. A small increase in invasion resulted from addition of 50 or 100 mg/ml of albumin to the medium in which the cells were resealed, but intra-erythrocytic protein content per se is evidently not a major factor in determining the efficiency of invasion. Augmentation of the cytoplasmic ATP concentration during lysis and resealing increased the level of parasitaemia significantly, and the parasites in these ghosts developed normally and gave rise to viable merozoites. It was found that albumin at certain concentrations passed freely into the lysed cells and reached equilibrium with that in the external medium. By contrast, the high molecular weight solute Blue Dextran 2000 was completely excluded from the lysed cells.

An improved method of harvesting haemoglobin-free human erythrocyte ghosts.

The ghosts (stromas) of human erythrocytes bearing Hb of different phenotypes were separately isolated by incubating the erythrocytes in hypotonic (20 imOsm) phosphate buffer saline (PBS) for 1 h at pH 7.4. All of these washed ghosts were found to react with benzidine indicating the presence of Hb. In the preparation of Hb free ghosts, the pH 8.2 of the phosphate buffer, incubation time (4 h) and extra oxygen supply to the medium were noticed to be very effective. The results shows that certain ghosts bound to SS, SF, and S-Thal type of Hb were more resistant to the clearing procedure than the ghosts bound to AA, AF, ASF and AC types of Hb. The combined effect of above variables for the optimum isolation of clear ghosts with minimum or no loss of membrane components has been described.

25] Proteins involved in membrane—cytoskeleton association in human erythrocytes: Spectrin, ankyrin, and band 3

This chapter focuses on the proteins involved in association of spectrin with the membrane. The chapter describes the methods used for preparation of erythrocyte ghosts and inside-out vesicles, and for purification of spectrin, ankyrin, a spectrin-binding fragment of ankyrin, and a proteolytic fragment containing the cytoplasmic domain of band 3. The chapter also discusses the conditions for measurement of binding of spectrin and ankyrin to inside-out vesicles. Spectrin is associated with the cytoplasmic surface of the membrane by attachment to ankyrin. Ankyrin, which is a peripheral membrane protein, is linked to the membrane by association with the cytoplasmic domain of band 3, a major membrane-spanning integral protein. Spectrin is extracted by incubation of ghosts at low ionic strength and can then be purified by gel filtration or by sedimentation on preparative sucrose gradients. Ankyrin can be purified by DEAE-chromatography and sedimentation on preparative sucrose gradients.

Enzyme replacement therapy in porphyrias—III: Potential use of erythrocyte ghosts as carriers of δ-aminolaevulinate dehydratase

1. 1. Resealed erythrocytes ghosts are potential in vivo carriers of exogenous therapeutic enzymes. The present work is part of studies directed toward the use of enzymes encapsulated within artificial or natural cellular carriers, in enzyme replacement therapy in porphyrias. 2. 2. Lead intoxication was chosen as a model system for these experiments. 3. 3. The optimum conditions for both the preparation of erythrocyte ghosts and encapsulation of δ-aminolaevulinate dehydratase (ALA-D) were studied in detail and a very good yield procedure for entrapment of exogenous highly purified human blood ALA-D, into both normal and defective erythrocytes was developed. 4. 4. The capacity of ALA-D defective cells of lead intoxicated patients and animals was brought within the normal range, in vitro, either by introducing ALA-D into them or by mixing with ALA-D loaded erythrocyte ghosts. 5. 5. Red blood cell ghosts entrapment is therefore a promising approach to the delivery of ALA-D when applying enzyme replacement therapy in lead poisoning.

Thermodynamic behaviour of membrane enzymes in Duchenne muscular dystrophy

Erythrocyte ghost preparations have been prepared from blood of Duchenne patients (DMD), female carriers of the disease and controls. Arrhenius plots of Na +,K +-ATPase activity of these membrane preparations show a biphasic response for controls. For 75% of DMD and carriers the response is monophasic. This is not an inherent property of the membrane since it can vary over time in the one individual and it can be induced in normal membranes by preincubation with DMD plasma. Arrhenius plots of AChE activity showed no such difference between the three sources of blood.

Age-dependence of polyadenylate stimulation of nuclear-envelope nucleoside triphosphatase

Nuclear envelopes of mammalian cells contain a nucleoside triphosphatase which is probably involved in mRNA transport through the nuclear membrane. The activity of the enzyme, studied in RNA-depleted nuclear ghosts, can be stimulated by poly(A) or by poly(A) (+)mRNA. Using nuclear ghost preparations from mature (8–10 months' old) and old (40–42 months' old) Wistar rats, it was shown that in “old” preparations the basal activity of the enzyme is significantly reduced (by 15%). In addition, the enzyme from old animals responds only very little to poly(A) or poly(A) (+)mRNA, compared to preparations from mature animals. Using a concentration of 6.8 × 10 11 poly(A) (+)mRNA molecules per μg of enzyme preparation, the nucleoside triphosphatase from mature animals is stimulated by 77% and the enzyme from old animals by only 26%. Binding studies of poly(A) to pore laminae revealed that the number of binding sites in unphosphorylated preparations from old animals is significantly reduced (by 24%) compared to “mature” preparations. As a consequence of in vitro phosphorylation, no difference is observable in the number of binding sites between the two age groups. The values for half-maximal saturation binding constants for poly(A) are identical in unphosphorylated and phosphorylated pore-laminae preparations, irrespective of the age group studied. The results presented indicate that in old animals the pathway from the phosphorylated to the dephosphorylated nuclear-envelope protein which is controlled by poly(A) is impaired in the proposed cycle for mRNA efflux from nuclei.

Evidence for restricted oligosaccharide mobility at the erythrocyte membrane surface: A fluorescence study

The glycophorins of whole, human erythrocytes were labeled at their sialic acid residues with one of three fluorescent probes. After preparation of the erythrocyte ghosts, the mobility of each fluorescent probe on the intact membrane was compared with its mobility on the isolated, labeled glycopeptides dissolved in aqueous buffer. A four- to ninefold decrease in the rotational relaxation time, as defined by the Perrin equation, accompanied the proteolytic removal of the labeled glycopeptides from the membrane. This suggests that the fluorescent probes, and by extrapolation, the sugars to which they are immediately attached, are restricted in their mobility at the membrane surface. A crude model of the carbohydrate layer of the erythrocyte surface was constructed by incorporating the labeled, tryptic glycopeptides into agarose gels of different agarose content. A decrease in the probe's mobility was observed as agarose content was raised. This indicates that the high oligosaccharide density at the erythrocyte membrane surface may contribute to the observed immobilization of the fluorescent probes in situ.

A Technique to Detect Reduced Mechanical Stability of Red Cell Membranes: Relevance to Elliptocytic Disorders

A method has been developed for determining the relative tendency of red cell membranes to fragment under shear stress. For these measurements, resealed ghosts were subjected to constant, high fluid shear stress in an ektacy-tometer. The deformability signal, which this instrument provides, progressively declined as the deformable cells were transformed into less deformable, more spherical cell fragments. The deformability signal for normal ghost preparations decayed over a narrowly defined time period; this decay was accompanied by the production of cell fragments, as shown by microscopic examination and analysis of particle volume distribution. The method was used to study cells from patients with various hemolytic disorders. Membrane preparations from patients with various types of nonhemolytic congenital elliptocytoses showed a reduction in the time required for fragmentation to about half the normal value. Membrane preparations from patients with hemolytic elliptocytosis associated with a severe deficiency of the membrane skeletal protein band 4.1 showed an even greater reduction in fragmentation time, distinct from the values for nonhemolytic samples. In elliptocytoses secondary to myelofibrosis, the fragmentation times were within the normal range. In hereditary spherocytosis (HS), heterogeneity in fragmentation times was observed; marginally decreased or normal fragmentation times were seen for cells from 8 patients, but a pronounced shortening in fragmentation time was found for one other HS patient. A defect was previously found in spectrin-band 4.1 binding for this same patient. These observations suggest that this membrane fragmentation technique may be useful for the identification of certain specific disorders or their clinical variants in which the integrity of membrane skeletal interactions has been perturbed in such a way that the mechanical stability of the membrane is reduced.

Erythrocyte-ghost Ca2+-stimulated Mg2+-dependent adenosine triphosphatase in Duchenne muscular dystrophy

The Ca2+-stimulated Mg2-dependent ATPase activities (Ca2+-ATPase) of erythrocyte-ghost membranes from patients with Duchenne muscular dystrophy (DMD) and carriers of DMD were compared with activities of normal controls. The Ca2+-ATPase activity of DMD-patient ghost preparations was found to follow the same pattern of activation by Ca2+ as the control membranes. However, the Ca2+-ATPase activity in DMD and some DMD-carrier preparations was substantially elevated compared with controls. To characterize further the elevated Ca2+-ATPase activity found in DMD-patient ghost membrane preparations, we estimated kinetic parameters using both fine adjustment and weighting methods to analyse our experimental data. It was established that in both DMD and DMD-carrier preparations the increase in Ca2+-ATPase activity was reflected by a significant increase in Vmax. rather than by any change in Km. The response of the membrane Ca2+-ATPase activity to changes in temperature was also investigated. In all preparations a break in the Arrhenius plot occurred at 20 degrees C, and in DMD and DMD-carrier preparations an elevated Ca2+-ATPase activity was detected at all temperatures. Above 20 degrees C the activation energy for all types of preparation was the same, whereas below this temperature there appeared to be an elevated activation in DMD and DMD-carrier preparations compared with normal controls. The concept that a generalized alteration in the physicochemical nature of the membrane lipid domain may be responsible for the many abnormal membrane properties reported in DMD is discussed.

Binding of dihydrotestosterone to a nuclear-envelope fraction from the male rat liver.

Intact nuclear 'ghosts' containing small amounts of DNA were obtained from rat liver. Incubation of radiolabelled dihydrotestosterone with isolated nuclear-envelope fraction from male rat liver resulted in specific binding of the dihydrotestosterone to the membranes. Optimal binding occurred at 20 degrees C after 20h incubation. Storage for 2 weeks at -80 degrees C resulted in little loss of specific binding. Scatchard analysis revealed a class of binding sites with a KD of 23.2 nM. Pronase and heat treatment destroyed the binding site. Androgens and glucocorticoids competed for labelled dihydrotestosterone binding to the ghosts, whereas oestrogens did not compete. Castration 24h before preparation of ghosts did not alter the binding site, and a similar class of binding sites was identified on female rat liver nuclear envelopes.

A basement membrane‐associated glycoprotein from skeletal muscle

We have isolated a major glycoprotein that appears to be associated with rat skeletal muscle basement membrane. We determined that the glycoprotein was part of the muscle cell surface complex when we found it to be enriched in preparations of muscle ghosts. We isolate the glycoprotein from homogenized muscle preextracted with 4 M and 8 M urea. It elutes as a major component in the presence of 8 M urea/50 mM 2-mercaptoethanol. Its apparent molecular weight on sodium dodecyl sulfate gels is 130,000. Amino acid analysis indicates that it is not a collagen but that it does contain small amounts of hydroxyproline and hydroxylysine. There may be collagenous domains in the glycoprotein molecule, for it is cleaved into three fragments by purified bacterial collagenase. Immunoperoxidase staining confirms that the 130,000-dalton protein is localized at the surface of adult skeletal muscle cells. It is probably a general basement membrane-associated glycoprotein because we found material immunologically cross-reactive with the muscle glycoprotein in basement membrane regions of kidney, liver, brain, and small intestine. We have shown the glycoprotein to be distinct from fibronectin, laminin, and types I, III, IV, and V collagens in enzyme-linked immunosorbent assays.

Growth of human malaria parasites in biotinylated erythrocytes

The adaptation of the biotin-avidin system for the analysis of membrane pathobiology in Plasmodium falciparum malaria is described. Biotin was linked covalently via the succinimide ester derivative (biotinyl- N-hydroxysuccinimide ester, BNHS) to intact human erythrocytes, prior to inoculation and in vitro cultivation of falciparum parasites. Growth experiments indicated that incubation concentrations of ⪕ 1.0 mg BNHS/1.0 ml erythrocyte packed cell volume could yield biotinylated erythrocytes capable of sustaining parasite growth at levels comparable to control cultures. Using a synthesized [ 14C]BNHS compound at optimal incubation concentration, it was determined that 1.32 × 10 −4 mmol [ 14C]BNHS were bound per 1.0 mg of erythrocyte stromal protein. In addition, analysis of [ 14C]biotinylated red blood cell ghost preparations by polyacrylamide gel electrophoresis demonstrated that band 3 (a heterogeneous glycoprotein) was the principal site of membrane labeling. Approximately 77% of total membrane-associated [ 14C]BNHS was localized to this polypeptide. The unique properties of the specific, ligand-protein interaction of the biotin-avidin complex suggest that the biotinylation procedure described in this report will provide a useful analytical tool in host cell-plasmodial parasite, membrane studies.

Reduced erythrocyte membrane protein methylation in sickle cell anemia.

The methylation of erythrocyte membrane components in sickle cell anemia has been studied and found to differ considerably from that of normal erythrocytes. When sickle erythrocytes were incubated under physiological conditions (pH = 7.4, 37 degrees C) in the presence of L-[methyl-3H]methionine or S-adenosyl-L-[methyl-3H] methionine, a 50% decrease in the protein-carboxyl methylation was observed compared to the normal erythrocyte. This reduction in degree of methylation was reflected in all of the major methylated protein bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Since this methylation is catalyzed by the cytosolic protein methylase II (S-adenosylmethionine:protein-carboxyl O-methyltransferase, EC 2.1.1.24), the in vitro substrate capability of sickle erythrocyte ghosts and pH 11 treated ghosts were tested by incubation with purified protein methylase II and S-adenosyl-L-[methyl-14C]methionine. Both types of sickle cell ghost preparations showed the same 50% decrease in methylation as was seen in the intact cells. Since it was also shown that the protein methylase II and methyl acceptor membrane protein levels in the sickle erythrocytes are the same as the normal control, the data suggest that the observed reduced methylation may be due to an altered membrane conformation. The methylation of phospholipid was also studied and found to be decreased in sickle cell erythrocytes. However, this methylation was extremely minor in comparison to protein-carboxyl methylation which represents the bulk of the membrane methylation.

Lack of effect of phenytoin on functions related to Na+,K+-ATPase activity of the intact erythrocytes of epileptic patients.

British Journal of Clinical PharmacologyVolume 12, Issue 1 p. 79-81 Free Access Lack of effect of phenytoin on functions related to Na+,K+-ATPase activity of the intact erythrocytes of epileptic patients. JK Aronson, JK AronsonSearch for more papers by this authorDP Geaney, DP GeaneySearch for more papers by this authorDG Grahame-Smith, DG Grahame-SmithSearch for more papers by this authorJC Carver, JC CarverSearch for more papers by this authorKF Hallis, KF HallisSearch for more papers by this author JK Aronson, JK AronsonSearch for more papers by this authorDP Geaney, DP GeaneySearch for more papers by this authorDG Grahame-Smith, DG Grahame-SmithSearch for more papers by this authorJC Carver, JC CarverSearch for more papers by this authorKF Hallis, KF HallisSearch for more papers by this author First published: July 1981 https://doi.org/10.1111/j.1365-2125.1981.tb01858.xCitations: 1AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat References ARONSON, J.K., GRAHAMESMITH, D.G., HALLIS, K.F., HIBBLE, A. & WIGLEY, F. (1977). Monitoring digoxin therapy: I. Plasma concentrations and an in vitro assay of tissue response. Br. J. clin. Pharmac., 4, 213– 221. CUMBERBATCH, M., ZAREIAN, K., DAVIDSON, C., MORGAN, D.B. & SWAMINATHAN, R. (1981). The early and late effects of digoxin treatment on the sodium transport, sodium content and Na+, K+-ATPase of erythrocytes. Br. J. clin. Pharmac. (In press). DEUPREE, J.D. (1977). The role or non-role of ATPase activation by phenytoin in the stabilization of excitable membranes. Epilepsia, 18, 309– 315. FORD, A.R., ARONSON, J.K., GRAHAMESMITH, D.G., ROSE, J.A. (1979a). The characteristics of the binding of 12-α-[3H]-digoxin to the membranes of intact human erythrocytes: relevance to digoxin therapy. Br. J. clin. Pharmac., 8, 115– 124. FORD, A.R., ARONSON, J.K., GRAHAMESMITH, D.G. & CARVER, J.G. (1979b). Changes in cardiac glycoside receptor sites 86rubidium uptake and intracellular sodium concentrations in the erythrocytes of patients receiving digoxin during the early phases of treatment of cardiac failure in regular rhythm and of atrial fibrillation. Br. J. clin. Pharmac., 8, 125– 134. KURATA, D. & WILKINSON, G.R. (1974). Erythrocyte uptake and plasma binding of diphenylhydantoin. Clin. Pharmac. Ther., 16, 355– 362. SCHWOCH, G. & PASSOW, H. (1973). Preparation and properties of human erythrocyte ghosts. Mol. Cell. Biochem., 2, 197– 218. SCOTT, A.K., GILBERT, J.C. & WEBSTER, J. (1980). Anticonvulsants and human erythrocyte ATPase activities. Br. J. clin. Pharmac., 10, 175– 177. Citing Literature Volume12, Issue1July 1981Pages 79-81 ReferencesRelatedInformation

Radioimmunochemical measurement of the transferrin receptor in human trophoblast and reticulocyte membranes with a specific anti-receptor antibody.

A radioimmunoassay was developed to directly assay the presence of transferrin receptors in human tissues. Antisera developed in a goat against purified human placental transferrin binding protein was purified by fractional sodium sulfate precipitation and adsorption against Sepharose-bound transferrin to remove trace anti-transferrin activity. The antisera immunoprecipitates a Mr 94,000 peptide on 125I-iodinated syncytial trophoblast membranes from placentae. This polypeptide has been identified previously as the transferrin binding protein of the placenta [Wada, H. G., Hass, P. E. & Sussman, H. H. (1979) J. Biol. Chem. 254, 12629-12635]. A standard curve using purified 125I-iodinated placental transferrin receptor and various amounts of the purified noniodinated receptor is sensitive from 5 to 900 ng. A reticulocyte-enriched membrane ghost preparation (5% reticulocyte) gives a value of 9.5 micrograms of receptor per mg of protein. Normal erythrocyte membrane ghosts show binding (0.57 micrograms of receptor per mg of protein) proportional to the amount of reticulocytes normally present in blood (0.5-1.0%). In other tissues in which the transferrin receptor binding has been reported, purified syncytial trophoblastic membranes are found to have 34.5 micrograms of receptor per mg of protein, and BeWo cells, a choriocarcinoma cell line, are found to have 15.7 micrograms of receptor per mg of protein. In contrast, normal breast tissue, which has no demonstrated transferrin binding, contains only 0.18 micrograms of receptor per mg of protein by this method.