Eosin-5-maleimide Research Articles

An unexpectedly bizarre blood film in hemoglobin H disease

A 3-year-old boy from Saudi Arabia had been followed up with a diagnosis of hemoglobin H disease, identified in a neonatal screening programme. His parents were second cousins. Alpha globin genotyping at birth had revealed homozygosity for the non-deletional Saudi-T mutation (αTα/αTα), both parents being carriers. He had remained clinically asymptomatic. Red cell indices at the age of three years were: RBC 3.84 x 1012/l, hemoglobin concentration 80 g/l, MCV 61 fl, MCH 20.9 pg, MCHC 34 g/l, red cell distribution width 34.8 (normal range 11.8-14.3) and reticulocyte count 447 x109/l. His blood film, in addition to microcytosis, showed gross poikilocytosis with the abnormal red cells including elliptocytes and many bizarre poikilocytes, some very small (image). Although poikilocytosis can be prominent in hemoglobin H disease, we have not seen a case with this degree of abnormality. The film was reminiscent of hereditary pyropoikilocytosis. For this reason red cell membrane studies were initiated. An eosin-5 maleimide (EMA)-binding assay [1] gave a twin peak fluorescence profile: 57% of phenotypically normal red cells (MCF = 64.7 units) and 42% having reduced fluorescence (MCF = 32.4 units). Spectrin (Sp) analysis showed a Sp dimer content of 25.6 %, compared with 7.4 % for a normal control within the same assay. In limited trypsin digestion of spectrin, SpαI/50 variant was detected in the child, and absent in the mother. Neither had an increase of SpαV/41 tryptic peptide, thus excluding the presence of the polymorphic low expression allele, SpαLELY. Maternal red cells were not elliptocytic and had normal spectrin dimer content and normal EMA binding. A paternal sample was not available for testing. Although the EMA-binding assay has high specificity and sensitivity for hereditary spherocytosis, it can also be abnormal in hereditary pyropoikilocytosis, Southeast Asian ovalocytosis, cryohydrocytosis, and some cases of congenital dyserythropoietic anemia type II [2]. Hereditary elliptocytosis usually gives a single fluorescence peak within the range for normal red cells but occasionally twin peaks are observed. In this child with co-inheritance of hemoglobin H disease and hereditary elliptocytosis, the severity of the hemolytic anaemia is that expected in uncomplicated hemoglobin H disease but the bizarre blood film reflects an interaction of the hemoglobinopathy and the membrane defect. 1

Guidelines for the diagnosis and management of hereditary spherocytosis – 2011 update

Guidelines on hereditary spherocytosis (HS) published in 2004 (Bolton-Maggs et al, 2004) are here replaced to reflect changes in current opinion on the surgical management, (particularly the indications for concomitant splenectomy with cholecystectomy in children with mild HS, and concomitant cholecystectomy with splenectomy in those with asymptomatic gallstones). Further potential long term hazards of splenectomy are now recognised. Advances have been made in our understanding of the biochemistry of the red cell membrane which underpins the choice of tests. Biochemical assays of membranes proteins and genetic analysis may be indicated (rarely) to diagnose atypical cases. The diagnostic value of the eosin-5-maleimide (EMA) binding test has been validated in a number of studies with understanding of its limitations.

Analysis of microsecond relaxation dynamics of proteins and viscous media by recording relaxation shifts of phosphorescence spectra

We studied the low-temperature dynamics of the Stokes shift of instantaneous phosphorescence spectra of eosin and eosin maleimide covalently bound to hemoglobin in a 66% v/v aqueous solution of glycerol under conditions of pulsed excitation. The kinetics of the Stokes shifts are described using the Cole-Davidson distribution function. From the experimental data, we obtain the parameters τ0 and β, which describe the Cole-Davidson distribution. Values of τ0 and β agree well with data obtained by other methods, and these parameters can be used to describe electron transfer reactions in polar solutions and proteins.

Detection of hereditary pyropoikilocytosis by the eosin-5-maleimide (EMA)-binding test is attributable to a marked reduction in EMA-reactive transmembrane proteins

Hereditary spherocytosis (HS) and hereditary pyropoikilocytosis (HPP, severe form of hereditary elliptocytosis) are unrelated red cell disorders caused by defects in distinct regions of the red cell cytoskeleton. The high predictive value of the eosin-5-maleimide (EMA)-binding test for the diagnosis of HS is because of its interaction with transmembrane proteins band 3, Rh protein, Rh glycoprotein and CD47, which are reduced on HS red cells. Our study was undertaken to determine why EMA-labelled HPP red cells were previously found to give much lower fluorescence readings than HS. Flow cytometry was used to determine the relative amounts of monoclonal antibodies bound to red cells from normal adults, HS and HPP groups. Confocal microscopy was used to visualise the overall staining pattern of the red cells with selected antibodies. In flow cytometry, HPP red cells gave lower antibody binding to the four EMA-reactive membrane proteins than HS red cells and bound less antibody to glycophorins A and C, and CD59. Confocal images of Rh protein and band 3 immunostaining revealed a greater number of HPP red cells having partial or no fluorescence than in HS and normal controls. Lesser amounts of EMA-reactive membrane proteins were detected in HPP than HS red cells, thus confirming their lower fluorescence readings in the EMA-binding test. The concomitant reduction in glycophorins A and C, and CD59 in HPP could have caused cellular contraction, resulting in poikilocytosis.

Flow-Assisted Differential Diagnosis of Hemolytic Anemia with Spherocytosis: A Case Report

In patients with hemolytic anemia associated with spherocytosis, differential diagnosis has to be made whether the hemolysis is immune-mediated or of non-immune origin. We report a case of hereditary spherocytosis in a 12-yr-old male child, in whom flow-assisted diagnosis was made. In this case, diagnosis was not determined because routine laboratory workups for hereditary spherocytosis yielded discrepant positive osmotic fragility test, positive direct antiglobulin test, and normal result in the red cell membrane protein sodium dodecyl succinimide polyacrylamide gel electrophoresis. However, all flow cytometry-based tests, such as osmotic fragility, direct antiglobulin, and eosin 5-maleimide binding test, yielded results compatible with hereditary spherocytosis. Additionally, in family study, the results of eosin 5-maleimide binding test suggested his disease being hereditary. In cases with diagnostic difficulties, flow cytometry may be used as an alternative tool, which can provide additional information in the differential diagnosis of hemolytic anemia with spherocytosis.

Evaluation of eosin‐5‐maleimide flow cytometric test in diagnosis of hereditary spherocytosis

A flow cytometry-based test using eosin-5-maleimide (EMA) dye was used for diagnosis of hereditary spherocytosis (HS). The mean fluorescence intensiy (MFI) of EMA tagged erythrocytes is lower in HS than that in other hemolytic and nonhemolytic anemias. We enrolled 114 subjects comprising 20 confirmed HS, 20 suspected HS/hemolytic anemia (HA), 20 normal controls, 20 other hemolytic anemias [13 autoimmune hemolytic anemia, three congenital dyserythropoietic anemia (CDA), one pyruvate kinase deficiency, two microangiopathic hemolytic anemia], 18 microcytic anemia and 16 macrocytic anemia cases. All samples were subjected to flow cytometry as per standard protocol. The mean MFI of normal control subjects was 11 861.5 (SD 883.5) and of confirmed HS was 7949.3 (SD 1304.1). Using this test, of 20 patients suspected to be HS/HA but with no confirmatory diagnosis, eight patients were diagnosed as HS. Using logistic regression analysis, the optimum cut-off MFI value between HS and normal controls was 10126. The area under the ROC curve was 0.99. The statistical significance of MFI values was obtained by t-test or Wilcoxon rank sum test as applicable. Compared with normal controls, the MFI values in HS were lower and in megaloblastic anemia were higher which was statistically highly significant (P<0.01), and the MFI values in CDA were lower which was statistically significant (P<0.05). False-positive values were obtained in three cases of AIHA and two cases of CDA. The sensitivity and specificity was 96.4% and 94.2% respectively. The EMA-based flow cytometry test is a highly sensitive and specific method for the diagnosis of HS.

Clinico-hematological profile of hereditary spherocytosis: experience from a tertiary care center in North India

Hereditary spherocytosis (HS) is an inherited membranopathy characterized by phenotypic and genotypic heterogeneity. This study describes the clinico-hematological profile of 70 HS patients diagnosed at a tertiary care center in North India over a period of five years. Patients commonly presented with intermittent jaundice (82·9%), pallor (80%) and dark colored urine (11·4%). The common signs were splenomegaly (92·9%), hepatomegaly (50%), cholelithiasis or choledocholithiasis (36·8%) and hemolytic facies (10%). Family history was contributory in 28·6% patients. Blood transfusion (BT) requirement was present in 35·7% patients. Unconjugated and conjugated hyperbilirubinemia was seen in 89·1 and 10·9% patients respectively. At presentation, the hemoglobin ranged from 3–14 g/dl with a mean of 9·37 g/dl (SD2·43). Spherocytes were seen in 88·6% and incubated Osmotic fragility test (OFT) was positive in 88·2% patients. The Eosin-5-maleimide (EMA) flow cytometric test was done in 28 patients. Mean fluorescence intensiy (MFI) for normal subjects was 11861·5 (SD-883·51) and for confirmed HS patients was 7949·3 (SD1304·1). Taking the MFI range of 5341·1–10 557·5 for HS, eight cases of suspected HS/undiagnosed hemolytic anemia with a negative (n=5) or equivocal (n=3) incubated OFT were diagnosed as HS. An increase in HbF level was seen in 10 cases ranging from 2·1 to 17·7% with a mean of 5·66%, three of these had associated β thalassaemia trait. Twelve patients (17%) underwent splenectomy and 91% of them did not require any BT post-splenectomy. Among the patients treated conservatively 49% had persisting pallor and 16·3% had transfusion requirement.

Evaluation of the Role of Flow Cytometry as a Diagnostic Tool for Hereditary Spherocytosis

Flow cytometric analysis of eosin-5′-maleimide-labeled red blood cells has been proposed as a method of identifying hereditary spherocytosis (HS). The flow cytometric test measures the fluorescence intensity of intact red cells labelled with the dye eosin-5-maleimide (EMA), which reacts covalently with Lys-430 on the first extra cellular loop of band 3 protein. Patients with HS have reduced fluorescence compared to other patient groups and normal controls. The aim of the present study was to assess the utility of flow cytometry in the diagnosis of hereditary spherocytosis. Fresh peripheral blood from 40 normal controls was collected in Lithium Heparin, stained with the dye EMA and analysed by flow cytometry. The normal range was established as 55–74 channel numbers. On 7 known cases of Hereditary Spherocytosis the MFI range was 35.6–44.6 channel numbers, and therefore samples falling into this range were considered to be positive for a diagnosis of HS by EMA. Equivocal results were defined when the MFI was in the range of 45–54 channel numbers. A total of 98 samples were sent for investigation or exclusion of HS. Clinical problems were grouped into the following categories: peripheral blood spherocytosis with negative Coomb's test and no family history of HS, positive family history of HS, Neonatal hyperbilirubinaemia, haemolytic anaemia of unknown origin.IndicationsCoomb's −ve, Spherocytosis, no FH (n=50) (51%)Positive family history (FH) (n=18) (17%)Neonatal hyperbilirubinemia (NNH) (n=8) (8%)Haemolytic anaemia (HA) (n=16) (17%)Results of EMAPosNegEquiPosNegEquiPosNegEquiPosNegEquiNumber1917139722420142Percentage383426503911255025087.512.5Within this cohort the group with FH of HS had the highest positive and least equivocal results. The group with suspected HS (peripheral blood spherocytosis with negative coomb's test) resulted in positive in 38% and equivocal in 26% cases. The group with NNH had positive results in 25% of cases and the group with HA had no positive results. Equivocal results occurred in 20% of cases suggesting further investigations are required to confirm or exclude HS. The EMA dye method by flow cytometry is a useful test for diagnosing red cell membrane abnormalities due to band 3 defects, but the assay produces a substantial number of equivocal results, and has poor utility in the investigation of haemolytic anaemia of unknown origin.

Using the eosin‐5‐maleimide binding test in the differential diagnosis of hereditary spherocytosis and hereditary pyropoikilocytosis

Flow cytometric analysis of eosin-5-maleimide (EMA)-labeled red blood cells (RBCs) has been used as a screening test for the diagnosis of patients with hereditary spherocytosis (HS). We assessed the fluorescence profiles for patients having HS and hereditary pyropoikilocytosis (HPP) together with their red cell indices. Flow cytometry was used to analyze EMA-labeled RBCs. Membrane protein defects and spectrin variants were identified by SDS-polyacrylamide gel electrophoresis. An overlay of single fluorescence peaks for normal individuals, and those with HS and HPP revealed a graded fluorescence intensity (normal > HS > HPP). The area under each peak defined a specific RBC subpopulation; namely, normal RBCs, spherocytes, and microspherocytes. HS RBCs having a gross reduction in band 3 or spectrin content gave fluorescence readings almost as low as those for HPP. Complex fluorescence profiles were obtained for isolated HS and HPP cases. The mean cell volume is a useful discriminator for HS and HPP. We presented evidence that a mixed RBC population could occur in some HS and HPP patients, either in a transient manner or for a long-term period. A differential diagnostic scheme for detecting HPP and HS by flow cytometry is proposed.

Titration of cardiolipin by either 10-N-nonyl acridine orange or acridine orange sensitizes the adenine nucleotide carrier to permeability transition

Under the action of carboxyatractyloside or fatty acids, adenine nucleotide translocase switches its function from nucleotide carrier to modulator of the opening of a non-specific pore. In addition to the effect of these agents, this modification in activity is, in some way, dependent on the influence of the lipid milieu of the membrane. Cardiolipin is, among other membrane phospholipids, the one that interacts the most with the translocase. This work shows that 10-N-nonyl acridine orange and acridine orange, probes for this phospholipid, modify the sensitivity of the translocase to carboxyatractyloside, oleate, and palmitate to induce permeability transition. The results also show that these probes stimulate the release of mitochondrial cytochrome c, and increase labeling of the carrier by eosin 5-maleimide. Based on the aforementioned it is proposed that the increase in sensitivity is due to a conformational change in the translocase, induced by the binding of the probe to cardiolipin.

Functionally important conserved length of C-terminal regions of yeast and bovine ADP/ATP carriers, identified by deletion mutants studies, and water accessibility of the amino acids at the C-terminal region of the yeast carrier

Comparison of the amino acid sequence of yeast type 2 ADP/ATP carrier (yAAC2) with that of bovine type 1 AAC (bAAC1) revealed that the N- and C-terminus of yAAC2 are 15- and 6-amino acids longer, respectively, than those of bAAC1. In the present study, we focused on the difference in the C-terminal region between yAAC2 and bAAC1. Deletion of first six residues of C-terminus of yAAC did not markedly affect the function of yAAC2; however, further deletion of 1 amino acid (7th amino acid from the C-terminus) destroyed its function. On the contrary, deletion of the first amino acid residue of the C-terminus of bAAC1 caused failure of its functional expression in yeast mitochondria. Based on these results, we concluded that the 6-amino acid residue extension of the C-terminus of yAAC2 was not necessary for the function of this carrier and that the remainder of the C-terminal region of yAAC2, having a length conserved with that of bAAC1, is important for the transport function of AACs. We next prepared various single-Cys mutants in which each of 32 residues in the C-terminus of yAAC2 was replaced by a Cys residue. Since all mutants were successfully expressed in yeast mitochondria, we examined the reactivity of these cysteine residues with the membrane-impermeable sulfhydryl reagent eosin 5-maleimide (EMA). As a result, all cysteine residues that replaced the 9 continuous amino acids in Met310-Lys318 showed high reactivity with EMA regardless of the presence of carboxyatractyloside or bongkrekic acid; and so this region was concluded to be exposed to the water-accessible environment. Furthermore, based on the reactivities of cysteine residues that replaced amino acids in the sixth transmembrane segment, the probable structural features of the C-terminal region of this carrier in the presence of bongkrekic acid were discussed.

1,1‐bis(3′‐indolyl)‐1‐(p‐substituted phenyl)methanes decrease mitochondrial membrane potential and induce apoptosis in endometrial and other cancer cell lines

1,1-Bis(3'-indolyl)-1-(p-substituted phenyl)methanes, containing p-t-butyl (DIM-C-pPhtBu) and phenyl (DIM-C-pPhC(6)H(5)) substituents, are peroxisome proliferator-activated receptor gamma (PPARgamma) agonists; however, DIM-C-pPhtBu-induced growth inhibition and cell death in human HEC1A endometrial cancer cells is PPARgamma-independent. DIM-C-pPhtBu decreased mitochondrial membrane potential (MMP) and promoted the release of cytochrome c and caspase activation and nuclear uptake of endonuclease G leading to apoptosis of HEC1A cells. DIM-C-pPhtBu specifically targeted the mitochondrial permeability transition pore complex (PTPC) because the DIM-C-pPhtBu-induced pro-apoptotic responses were inhibited by atractyloside (Atra), a compound that specifically interacts with the inner mitochondrial membrane adenine nucleotide transport (ANT) proteins. At the dose of Atra used in this study (300 microM), this compound alone did not alter the PTPC but inhibited the mitochondriotoxic effects of DIM-C-pPhtBu. DIM-C-pPhtBu/DIM-C-pPhC(6)H(5) and Atra also differentially affected the ability of eosin-5-maleimide (EMA) to alkylate Cys160 in the ANT protein and Atra, but not DIM-C-pPhtBu, inhibited the exchange of ATP/ADP in isolated mitochondria suggesting that these pharmacophores act on different sites on the ANT protein. Results of this study show that the receptor-independent proapoptotic activity of DIM-C-pPhtBu and DIM-C-pPhC(6)H(5) were related to novel mitochondriotoxic activities involving inner mitochondrial ANT proteins.

Augmented erythrocyte band-3 phosphorylation in septic mice

Infection-induced RBC dysfunction has been shown to play a role in the modulation of host response to injury and infection. The underlying biochemical mechanisms are not known. This study investigated alterations in RBC band-3 phosphorylation status and its relationship to anion exchange activity in vitro as well as under in vivo septic conditions induced by cecal ligation and puncture (CLP) in mice. Pervanadate treatment in vitro increased band-3 tyrosine phosphorylation that was accompanied by decreased RBC deformability and anion exchange activity. Following sepsis, band-3 tyrosine phosphorylation in whole RBC ghosts as well as in cytoskeleton-bound or soluble RBC protein fractions were elevated as compared to controls. Although anion exchange activity was similar in RBCs from septic and control animals, band-3 interaction with eosin-5-maleimide (EMA), which binds to band-3 lysine moieties, was increased in cells from septic animals as compared to controls, indicating that sepsis altered band 3 organization within the RBC membrane. Since glucose-6-phosphate dehydrogenase is a major antioxidant enzyme in RBC, in order to assess the potential role of oxidative stress in band-3 tyrosine phosphorylation, sepsis-induced RBC responses were also compared between WT and (G6PD) mutant animals (20% of normal G6PD activity). Band-3 membrane content and EMA staining were elevated in G6PD mutant mice compared to WT under control non-septic conditions. Following sepsis, G6PD mutant animals showed lessened responses in band-3 tyrosine phosphorylation and EMA staining compared to WT. RBC anion exchange activity was similar between mutant and WT animals under all tested conditions. In summary, these studies indicate that sepsis results in elevated band-3 tyrosine phosphorylation and alters band-3 membrane organization without grossly affecting RBC anion exchange activity. The observations also suggest that factors other than oxidative stress are responsible for the sepsis-induced increase in RBC band-3 tyrosine phosphorylation.

The β4-β8 Groove Is an ATP-interactive Site in the α Crystallin Core Domain of the Small Heat Shock Protein, Human αB Crystallin

The site for ATP interactions in human αB crystallin, the archetype of small heat-shock proteins, was identified and characterized to resolve the controversial role of ATP in the function of small heat-shock proteins. Comparative sequence alignments identified the αB crystallin sequence, 82 KHFSPEELKVK VLGD 96 as a Walker-B ATP-binding motif that is found in several ATP-binding proteins, including five molecular chaperones. Fluorescence resonance energy transfer and mass spectrometry using a novel fluorescent ATP analog, 8-azido-ATP-[γ]-1-naphthalenesulfonic acid-5(2-aminoethylamide) (azido-ATP-EDANS) and a cysteine mutant of human αB crystallin (S135C) conjugated with a fluorescent acceptor, eosin-5-maleimide (EMA) identified the β4-β8 groove as the ATP interactive site in αB crystallin. A 44% decrease in the emitted fluorescence of azido-ATP-EDANS at the absorption maximum of S135C-EMA and a corresponding 50% increase in the fluorescence emission of S135C-EMA indicated a close spatial relationship between azido-ATP-EDANS and the center of the β8 strand ( 131LTITSSLS 138). Liquid chromatography, electrospray ionization mass spectrometry identified two peptide fragments of the αB crystallin Walker-B motif photo-affinity-labeled with azido-ATP-EDANS confirming the β4-β8 groove as an ATP interactive site. The results presented here clearly establish the β4-β8 groove as the ATP interactive region in αB crystallin, and are in contrast to the existing paradigm that classifies small heat-shock proteins as ATP-independent chaperones.

The structure of the second cytosolic loop of the yeast mitochondrial ADP/ATP carrier AAC2 is dependent on the conformational state

To detect structural changes in the second cytosolic loop of the mitochondrial ADP/ATP carrier of Saccharomyces cerevisiae AAC2, we prepared 20 single cysteine mutants by replacing each amino acid in the S213 to L232 region. All single cysteine mutants were fully functional, because they could restore growth on glycerol of a yeast strain lacking functional ADP/ATP carriers. First, these single-Cys mutants were treated with carboxyatractyloside to lock the carrier in the cytosolic state or with bongkrekic acid to generate the matrix state, and then with the membrane-impermeable SH reagent eosin-5-maleimide (EMA) to probe accessibility. The amino acid residues S213C, L214C, F231C and L232C were not labeled, indicating that these 4 residues must have been buried in the membrane, whereas the region between residues K215 and S230 is accessible to labeling and must, therefore, have protruded into the aqueous phase. Residue L218C showed strong resistance against EMA labeling regardless of the state of the carrier, but the reason for such behavior is unclear. On the contrary, the labeling of the residues between F227C and S230C was strongly dependent on the state of the carrier. Thus, the C-terminal region of the second cytosolic loop in AAC2 changes its environment when the carrier cycles between the matrix and cytosolic state.

Cysteine Labeling Studies Detect Conformational Changes in Region 106−132 of the Mitochondrial ADP/ATP Carrier of Saccharomyces cerevisiae

To know the structural and functional features of the cytosolic-facing first loop (LC1) including its surrounding region of the mitochondrial ADP/ATP carrier (AAC), we prepared 27 mutants, in which each amino acid residue between residues 106 and 132 of the yeast type 2 AAC (yAAC2) was replaced by a cysteine residue. For mutant preparation, we used a Cys-less AAC mutant, in which all four intrinsic cysteine residues were substituted with alanine residues, as a template [Hatanaka, T., Kihira, Y., Shinohara, Y., Majima, E., and Terada, H. (2001) Biochem. Biophys. Res. Commun. 286, 936-942]. From the labeling intensities of the membrane-impermeable SH-reagent eosin-5-maleimide (EMA), sequence Lys(108)-Phe(127) was suggested to constitute the LC1. The N-terminal half of this region (Lys(108)-Phe(115)) was suggested to change its location from the cytosol to a region close to the membrane on conversion from the c-state to the m-state in association with disruption or unwinding of its alpha-helical structure, whereas the C-terminal half region (Gly(116)-Phe(127)) was considered to extrude essentially into the cytosol, while keeping its alpha-helical structure. Hence, the conformation of m-state LC1 is greatly different from that of c-state LC1. Possibly the LC1 changes its location between the membranous region and the cytosol during ADP/ATP transport. Lys(108) in the LC1 of the yAAC2 was found to be associated with binding of the transport substrates, and its -NH(3)(+) moiety, to be of importance for the transport function. On the basis of these results, possible roles of the conformational changes of the LC1 in the transport activity are discussed.

Twisting of the Second Transmembrane α-Helix of the Mitochondrial ADP/ATP Carrier during the Transition between Two Carrier Conformational States

To investigate the structural and functional features of the second alpha-helical transmembrane segment (TM2) of the mitochondrial ADP/ATP carrier (AAC), we adopted cysteine scanning mutagenesis analysis. Single-cysteine mutations of yeast AAC were systematically introduced at residues 98-106 in TM2, and the mutants were treated with the fluorescent SH reagent eosin-5-maleimide (EMA). EMA modified different amino acid residues of alpha-helical TM2 between the two distinct carrier conformations, called the m-state and the c-state, in which the substrate recognition site faces the matrix and cytosol, respectively. When amino acids in the helix were projected on a wheel plot, these EMA-modified amino acids were observed at distinct sides of the wheel. Since the SH reagent specifically modified cysteine in the water-accessible environment, these results indicate that distinct helical surfaces of TM2 faced the water-accessible space between the two conformations, possibly as a result of twisting of this helix. In the recently reported crystal structure of bovine AAC, several amino acids faced cocrystallized carboxyatractyloside (CATR), a specific inhibitor of the carrier. These residues correspond to those modified with EMA in the yeast carrier in the c-state. Since the binding site of CATR is known to overlap that of the transport substrate, the water-accessible space was thought to be a substrate transport pathway, and hence, the observed twisting of TM2 between the m-state and the c-state may be involved in the process of substrate translocation. On the basis of the results, the roles of TM2 in the transport function of AAC were discussed.

Altered Structure and Anion Transport Properties of Band 3 (AE1, SLC4A1) in Human Red Cells Lacking Glycophorin A

We have studied the properties of band 3 in different glycophorin A (GPA)-deficient red cells. These red cells lack either both GPA and glycophorin B (GPB) (M(k)M(k) cells) or GPA (En(a-) cells) or contain a hybrid of GPA and GPB (MiV cells). Sulfate transport was reduced in all three red cell types to approximately 60% of that in normal control red cells as a result of an increased apparent K(m) for sulfate. Transport of the monovalent anions iodide and chloride was also reduced. The reduced iodide transport resulted from a reduction in the V(max) for iodide transport. The anion transport site was investigated by measuring iodide fluorescence quenching of eosin-5-maleimide (EMA)-labeled band 3. The GPA-deficient cells had a normal K(d) for iodide binding, in agreement with the unchanged K(m) found in transport studies. However, the apparent diffusion quenching constant (K(q)) was increased, and the fluorescence polarization of band 3-bound EMA decreased in the variant cells, suggesting increased flexibility of the protein in the region of the EMA-binding site. This increased flexibility is probably associated with the decrease in V(max) observed for iodide transport. Our results suggest that band 3 in the red cell can take up two different structures: one with high anion transport activity when GPA is present and one with lower anion transport activity when GPA is absent.

Eosin-5-maleimide binding to band 3 and Rh-related proteins forms the basis of a screening test for hereditary spherocytosis.

Flow cytometric analysis of eosin-5-maleimide (EMA) binding to red cells is a screening test for the diagnosis of hereditary spherocytosis (HS). The present study used chemical modifications to determine the integral membrane proteins that react with EMA. The predominant interaction of EMA, contributing c. 80% of fluorescence, was with the epsilon-NH2 group of lysine in band 3 protein, as previously reported. The remainder of the EMA fluorescence was attributable to labelling of accessible sulfhydryl groups on intact red cells. This reaction was heat labile. Three molecules containing sulfhydryl groups were shown to be associated with the Rh blood group protein complex by sodium dodecyl sulphate polyacrylamide gel electrophoresis. These were CD47 and the Rh-associated glycoprotein, both in the Mr 40-60 kD region, and the Rh blood group proteins in the 30-32 kD region. Immunoprecipitation, using specific monoclonal antibodies and antibody binding studies by flow cytometry, showed that the relative content of these three membrane proteins was lower in HS than normal red cells. Thus, the high predictive value of the EMA binding test for HS reflects changes in the relative amounts of the Rh-related integral membrane proteins as well as band 3 in HS red cells.

Structural and functional roles of Cys-238 and Cys-295 in Escherichia coli phosphofructokinase-2.

Modification of Escherichia coli phosphofructokinase-2 (Pfk-2) with pyrene maleimide (PM) results in a rapid inactivation of the enzyme. The loss of enzyme activity correlates with the incorporation of 2 mol of PM/mol of subunit and the concomitant dissociation of the dimeric enzyme. The two modified residues were identified as Cys-238 and Cys-295. In the presence of the negative allosteric effector, MgATP, Cys-238 was the only modified cysteine residue. Kinetic characterization of the Cys-238-labelled Pfk-2 indicates that the enzyme is fully active, with the kinetic constants ( K(m), kcat) being almost identical to the ones obtained for the native enzyme. The modified enzyme is a monomer in the absence of ligands and, like the native enzyme, behaves as a tetramer in the presence of the nucleotide. However, in the presence of fructose-6-phosphate (fru-6-P) and ATP(-4), the enzyme behaves as a dimer, suggesting that the monomers undergo re-association in the presence of the substrates and that the active species is a dimer. Modification of Pfk-2 with eosin-5-maleimide (EM) results in the labelling of Cys-295. This modified enzyme is inactive and is not able to bind to the allosteric effector, remaining as a dimer in its presence. Nonetheless, Cys-295-labelled Pfk-2 is able to bind to the substrate fru-6-P in an hyperbolic fashion with a K(d) value that is 6-fold higher than the one determined for the native enzyme. These are the first residues to be implicated in the activity and/or structure of the Pfk-2.