Oligomeric state of human erythrocyte band 3 measured by fluorescence resonance energy homotransfer.
AuthorsBlackman SM, Piston DW, Beth AH
JournalBiophys J
PubMed ID9675213
The oligomeric state of the erythrocyte anion exchange protein, band 3, has been assayed by resonance energy homotransfer. Homotransfer between oligomeric subunits, labeled with eosin-5-maleimide at Lys430 in the transmembrane domain, has been demonstrated by steady-state and time-resolved fluorescence spectroscopy, and is readily observed by its depolarization of the eosin ... More
Spectroscopic and kinetic characterization of eosin-5-maleimide.
AuthorsSchopfer LM, Salhany JM
JournalAnal Biochem
PubMed ID9514795
Eosin-5-maleimide (EM) is an increasingly important and widely used probe in the study of membrane protein structure and function. Yet little is known about its spectral properties in hydrophobic and hydrophilic environments. Furthermore, EM is hydrolyzed faster than the traditional N-ethylmaleimide. To offer a more solid foundation for the use ... More
Transfer of band 3, the erythrocyte anion transporter, between phospholipid vesicles and cells.
AuthorsNewton AC, Cook SL, Huestis WH
JournalBiochemistry
PubMed ID6661430
Band 3, the anion transport protein of human erythrocyte membranes, can be transferred from cells to liposomes and from liposomes back to cell membranes, retaining function and native orientation. After incubation with cells, sonicated phosphatidylcholine vesicles bind a transmembrane protein that comigrates with band 3 on sodium dodecyl sulfate-polyacrylamide gels. ... More
Structural and functional characterization of band 3 from Southeast Asian ovalocytes.
AuthorsMoriyama R, Ideguchi H, Lombardo CR, Van Dort HM, Low PS
JournalJ Biol Chem
PubMed ID1464593
'To determine why deletion of the nine amino acids joining the membrane and cytoplasmic domains of band 3 from Southeast Asian ovalocytes (SAO) renders the erythrocytes rigid, we compared the structural and functional properties of SAO and normal band 3. Calorimetric data, inhibitor binding studies, and anion transport assays all ... More
Loss of rotational mobility of band 3 proteins in human erythrocyte membranes induced by antibodies to glycophorin A.
AuthorsChe A, Cherry RJ
JournalBiophys J
PubMed ID7612830
'The effect of antibodies to glycophorin A on the rotational diffusion of band 3 in human erythrocyte membranes was investigated by transient dichrosim. Three antibodies that recognize different epitopes on the exofacial domain of glycophorin A all strongly reduce the rotational mobility of band 3. The effect is at most ... More
Effects of ATP and protons on the Na : K selectivity of the (Na+ + K+)-ATPase studied by ligand effects on intrinsic and extrinsic fluorescence.
AuthorsSkou JC, Esmann M
JournalBiochim Biophys Acta
PubMed ID6157415
'The effect of pH and of ATP on the Na : K selectivity of the (Na+ + K+)-ATPase has been tested under equilibrium conditions. The Na+ : K+-induced change in intrinsic tryptophan fluorescence and in fluorescence of eosin maleimide bound to the system has been used as a tool. 1 ... More
The orientation of eosin-5-maleimide on human erythrocyte band 3 measured by fluorescence polarization microscopy.
AuthorsBlackman SM, Cobb CE, Beth AH, Piston DW
JournalBiophys J
PubMed ID8804603
'The dominant motional mode for membrane proteins is uniaxial rotational diffusion about the membrane normal axis, and investigations of their rotational dynamics can yield insight into both the oligomeric state of the protein and its interactions with other proteins such as the cytoskeleton. However, results from the spectroscopic methods used ... More
Fluorescence energy transfer studies of purified erythrocyte Ca2+-ATPase. Ca2+-regulated activation by oligomerization.
AuthorsKosk-Kosicka D, Bzdega T, Wawrzynow A
JournalJ Biol Chem
PubMed ID2531140
'Fluorescence resonance energy transfer has been used to study oligomerization of the purified erythrocyte Ca2+-ATPase. The energy transfer efficiency has been measured at different enzyme concentrations, from fluorescein 5''-isothiocyanate attached on one enzyme molecule to eosin 5-maleimide or tetramethylrhodamine 5-isothiocyanate attached on another enzyme molecule. The energy transfer efficiency showed ... More
Interchain and intrachain disulphide bonds in human platelet glycoprotein IIb. Localization of the epitopes for several monoclonal antibodies.
'The single interchain disulphide bond in platelet glycoprotein IIb (GPIIb) is accessible to extracellular reductants, and selective cleavage does not liberate GPIIb alpha from platelet plasma membrane, confirming that non-covalent interactions contribute to maintaining attachment of this subunit to the membrane. Eosin-maleimide labelling of isolated GPIIb after selective cleavage of ... More
35Cl nuclear magnetic resonance line broadening shows that eosin-5-maleimide does not block the external anion access channel of band 3.
AuthorsLiu D, Kennedy SD, Knauf PA
JournalBiophys J
PubMed ID8527653
'It has been suggested that Lys-430 of band 3, with which eosin-5-maleimide (EM) reacts, is located in the external channel through which anions gain access to the external transport site, and that EM inhibits anion exchange by blocking this channel. To test this, we have used 35Cl nuclear magnetic resonance ... More
Chemical labelling of arginyl-residues involved in anion transport mediated by human band 3 protein and some aspects of its location in the peptide chain.
AuthorsZaki L, Böhm R, Merckel M
JournalCell Mol Biol (Noisy-le-grand)
PubMed ID8960780
'The anion exchange system of human red blood cells can be completely inhibited by a large number of arginine-specific reagents. Kinetic studies have shown that these reagents act on the substrate binding site. Complete inhibition of the transport system with 14C phenylglyoxal is accompanied by modification of 2 to 3 ... More
Inactivation of human factor VIII by activated protein C: evidence that the factor VIII light chain contains the activated protein C binding site.
AuthorsFay PJ, Walker FJ
JournalBiochim Biophys Acta
PubMed ID2521291
'Factor VIII is represented as a series of heterodimers composed of an 83(81) kDa light chain noncovalently bound to a variable size (93 to 210 kDa) heavy chain. Activated protein C inactivates factor VIII causing several cleavages of the factor VIII heavy chain(s). When factor VIII subunits were dissociated and ... More
Mechanism of hypotonic hemolysis of human erythrocytes.
AuthorsSato Y, Yamakose H, Suzuki Y
JournalBiol Pharm Bull
PubMed ID7689891
'A mechanism of hemolytic hole formation during rapid hemolysis in a hypotonic medium has been investigated using eosin-5-maleimide (EMI) as a probe. The EMI-labeled erythrocytes revealed a distinct cluster and/or ring of intense fluorescence staining in a hypotonic 5 mM Hepes buffer (pH 7.4), but not in an isotonic buffer ... More
Fluorophores at the N terminus of nascent chloramphenicol acetyltransferase peptides affect translation and movement through the ribosome.
AuthorsRamachandiran V, Willms C, Kramer G, Hardesty B
JournalJ Biol Chem
PubMed ID10636875
'Structurally different fluorescent probes were covalently attached to methionyl-tRNA(f) and tested for their incorporation into nascent peptides and full-length protein using an Escherichia coli cell-free coupled transcription/translation system. Bovine rhodanese and bacterial chloramphenicol acetyltransferase (CAT) were synthesized using derivatives of cascade yellow, eosin, pyrene, or coumarin attached to [(35)S]Met-tRNA(f). All ... More
Deoxygenation affects fluorescence photobleaching recovery measurements of red cell membrane protein lateral mobility.
AuthorsCorbett JD, Cho MR, Golan DE
JournalBiophys J
PubMed ID8130343
'We have used the fluorescence photobleaching recovery technique to study the dependence on oxygen tension of the lateral mobility of fluorescently labeled band 3, the phospholipid analogue fluorescein phosphatidylethanolamine, and glycophorins in normal red blood cell membranes. Band 3 protein and sialic acid moieties on glycophorins were labeled specifically with ... More
Monopalmitoylphosphatidylcholine incorporation into human erythrocyte ghost membranes causes protein and lipid immobilization and cholesterol depletion.
AuthorsGolan DE, Furlong ST, Brown CS, Caulfield JP
JournalBiochemistry
PubMed ID3401442
'The effects of lysophosphatidylcholine (lysoPC) on human erythrocyte (RBC) ghost morphology, transmembrane protein and lipid lateral mobilities, and membrane lipid composition were studied in order to elucidate mechanisms by which lysoPC immobilizes ghost membrane components [Golan, D. E., Brown, C. S., Cianci, C. M. L., Furlong, S. T., & Caulfield, ... More
Inhibition of Na+-dependent phosphate transport by group-specific covalent reagents in rat kidney brush border membrane vesicles. Evidence for the involvement of tyrosine and sulfhydryl groups on the interior of the membrane.
AuthorsPratt RD, Pedersen PL
JournalArch Biochem Biophys
PubMed ID2912384
'The effects of tyrosine- and sulfhydryl-specific reagents on the Na+-dependent transport of phosphate in brush border membrane vesicles prepared from rat renal cortex were investigated. This study is the first to show that the tyrosine-specific reagents 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole and tetranitromethane inactivate the transporter in a concentration- and time-dependent fashion while the ... More
Conformations of IgE bound to its receptor Fc epsilon RI and in solution.
AuthorsZheng Y, Shopes B, Holowka D, Baird B
JournalBiochemistry
PubMed ID1832555
'Previous resonance energy transfer studies suggested that murine immunoglobulin E (IgE) is bent near the junction of its Fc and Fab segments when bound to its high-affinity receptor (Fc epsilon RI) on RBL cells. To examine further the conformations of IgE, both bound to this receptor and in solution, a ... More
Two-dimensional diffusion of F1F0-ATP synthase and ADP/ATP translocator. Testing a hypothesis for ATP synthesis in the mitochondrial inner membrane.
'We report here the first experimentally determined lateral diffusion coefficients of the F1F0-ATP synthase and the ADP/ATP translocator in isolated inner membranes of rat liver mitochondria. Rabbit IgG developed against the F1F0-ATP synthase isolated from rat liver mitochondria was determined to be immunospecific for the synthase subunits, notably the alpha-beta ... More
Trapping of inhibitor-induced conformational changes in the erythrocyte membrane anion exchanger AE1.
AuthorsTang XB, Casey JR
JournalBiochemistry
PubMed ID10545179
'AE1, the chloride/bicarbonate anion exchanger of the erythrocyte plasma membrane, is highly sensitive to inhibition by stilbene disulfonate compounds such as DIDS (4,4''-diisothiocyanostilbene-2, 2''-disulfonate) and DNDS (4,4''-dinitrostilbene-2,2''-disulfonate). Stilbene disulfonates recruit the anion binding site to an outward-facing conformation. We sought to identify the regions of AE1 that undergo conformational changes ... More
The capacity of basic peptides to trigger exocytosis from mast cells correlates with their capacity to immobilize band 3 proteins in erythrocyte membranes.
AuthorsDufton MJ, Cherry RJ, Coleman JW, Stanworth DR
JournalBiochem J
PubMed ID6497847
'The effect of mast-cell-triggering peptides on the rotational properties of band 3, a protein component of the human erythrocyte membrane, was measured by observing flash-induced transient dichroism of the triplet probe eosin maleimide. In the presence of melittin, polylysine and five synthetic peptides, varying degrees of retardation in the rotational ... More
Quantitation of protein 3 content of circulating erythrocytes at the single-cell level.
AuthorsJennings LK, Brown LK, Dockter ME
JournalBlood
PubMed ID2581638
'The density and size of human erythrocytes has been roughly correlated with cell age, with the denser and smaller cells being older. Observations of this type have led to a hypothesis that the membranes of circulating erythrocytes are dynamic with respect to composition and that material is lost from the ... More
Eosin-5-maleimide binding to band 3 and Rh-related proteins forms the basis of a screening test for hereditary spherocytosis.
AuthorsKing MJ, Smythe JS, Mushens R
JournalBr J Haematol
PubMed ID14675415
'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 ... More
Initiation of protein synthesis with fluorophore-Met-tRNA(f) and the involvement of IF-2.
AuthorsMcIntosh B, Ramachandiran V, Kramer G, Hardesty B
JournalBiochimie
PubMed ID10727773
'The complicity of initiation factor 2 (IF-2) in causing the observed low incorporation of N-terminal fluorophore from fluorophore-methionyl-tRNA(f) during protein synthesis in an in vitro coupled transcription/translation system was investigated. The low incorporation in comparison to formyl-methionine was not due to the lack of interaction of fluorophore-Met-tRNA(f) with IF-2. Fluorescence ... More
Interactions among red cell membrane proteins.
AuthorsPodgorski A, Alster P, Elbaum D
JournalBiochemistry
PubMed ID3126812
'Interactions between human red band 2.1 with spectrin and depleted inside-out vesicles were studied by fluorescence resonance energy transfer and batch microcalorimetry. The band 2.1-spectrin binding isotherm is consistent with a one to one mole ratio. The association constant of 1.4 X 10(8) M-1 corresponds to the association free energy ... More
Restriction by ankyrin of band 3 rotational mobility in human erythrocyte membranes and reconstituted lipid vesicles.
AuthorsChe A, Morrison IE, Pan R, Cherry RJ
JournalBiochemistry
PubMed ID9236005
'Rotational diffusion of eosin-5-maleimide-labeled band 3 was measured in erythrocyte membranes at pH 9.4-10.4. Band 3 was found to be more mobile in this pH range than at pH 7.5. Similar results were obtained with spectrin-actin-depleted membranes, where it was further shown that ankyrin is the only detectable protein released ... More
Probing the dynamic equilibrium of actin polymerization by fluorescence energy transfer.
AuthorsWang YL, Taylor DL
JournalCell
PubMed ID6101197
'Fluorescence resonance energy transfer was used to follow the dynamic equilibrium of actin polymerization. We prepared fluorescent analogs of actin by labeling actin covalently with fluorescein (as the donor) and with eosin (as the acceptor). The copolymer of donor- and acceptor-labeled actin exhibits a 60%-70% efficiency of energy transfer. We ... More
Resonance energy transfer: methods and applications.
AuthorsWu P, Brand L
JournalAnal Biochem
PubMed ID8053542
'Resonance energy transfer is widely used in studies of biomolecular structure and dynamics. It provides information about distances on the order of 10 to 100 A and is thus suitable for investigating spatial relationships of interest in biochemistry. The information available from energy transfer studies has been enhanced by the ... More
Evidence that inhibitors of anion exchange induce a transmembrane conformational change in band 3.
AuthorsMacara IG, Kuo S, Cantley LC
JournalJ Biol Chem
PubMed ID6185490
'The transport inhibitor, eosin 5-maleimide, reacts specifically at an external site on the membrane-bound domain of the anion exchange protein, Band 3, in the human erythrocyte membrane. The fluorescence of eosin-labeled resealed ghosts or intact cells was found to be resistant to quenching by CsCl, whereas the fluorescence of labeled ... More
Experience with eosin-5'-maleimide as a diagnostic tool for red cell membrane cytoskeleton disorders.
AuthorsKedar PS, Colah RB, Kulkarni S, Ghosh K, Mohanty D
JournalClin Lab Haematol
PubMed ID14641141
'The diagnosis of hereditary spherocytosis (HS) is based on red cell morphology and other conventional tests such as osmotic fragility, autohemolysis and acidified glycerol lysis. However, milder cases are at times difficult to diagnose. Confirmation by red blood cell (RBC) membrane protein analysis is not possible in most laboratories. Recently, ... More
Eosin-5-maleimide inhibits red cell Cl- exchange at a noncompetitive site that senses band 3 conformation.
AuthorsKnauf PA, Strong NM, Penikas J, Wheeler RB, Liu SQ
JournalAm J Physiol
PubMed ID7684558
'Eosin-5-maleimide (EM) has been used as a fluorescent probe for the external-facing transport site of the human erythrocyte band 3 protein. Changes in chloride concentration at both sides of the membrane have no significant effect on the inhibitory potency of EM as a reversible inhibitor of Cl- exchange at 0 ... More
Control of band 3 lateral and rotational mobility by band 4.2 in intact erythrocytes: release of band 3 oligomers from low-affinity binding sites.
AuthorsGolan DE, Corbett JD, Korsgren C, Thatte HS, Hayette S, Yawata Y, Cohen CM
JournalBiophys J
PubMed ID8785311
'Band 4.2 is a human erythrocyte membrane protein of incompletely characterized structure and function. Erythrocytes deficient in band 4.2 protein were used to examine the functional role of band 4.2 in intact erythrocyte membranes. Both the lateral and the rotational mobilities of band 3 were increased in band 4.2-deficient erythrocytes ... More
Characterization of the anion transport channel protein in human erythrocytes. Induced circular dichroism of inhibitors bound to the anion transport channel.
AuthorsSato Y, Chiba T, Suzuki Y
JournalBiochim Biophys Acta
PubMed ID2420359
'The induced circular dichroism (CD) of erythrocyte ghosts with anion-transport inhibitors has been studied. A ghost-EITC (eosin 5-isothiocyanate) system shows an induced CD spectrum at the wavelength region corresponding to the absorption bands of EITC. Also a ghost-EMI (eosin 5-maleimide) system shows induced CD, but has bands of opposite sign ... More
Rotational diffusion of biological macromolecules by time-resolved delayed luminescence (phosphorescence, fluorescence) anisotropy.
AuthorsJovin TM, Bartholdi M, Vaz WL, Austin RH
JournalAnn N Y Acad Sci
PubMed ID6942745
Inhibition and labelling of the mitochondrial 2-oxoglutarate carrier by eosin-5-maleimide.
AuthorsZara V, Palmieri F
JournalFEBS Lett
PubMed ID2457517
'Unlike hydrophobic maleimides, eosin-5-maleimide and to a lesser extent other relatively polar maleimides inhibit the 2-oxoglutarate carrier of bovine heart mitochondria. The impermeable eosin-5-maleimide labels the 2-oxoglutarate carrier in intact mitochondria but not in submitochondrial particles. 2-Oxoglutarate protects the carrier against inactivation by eosin-5-maleimide and decreases the fluorescence associated with ... More
The preparation and application of functionalised synthetic oligonucleotides: III. Use of H-phosphonate derivatives of protected amino-hexanol and mercapto-propanol or -hexanol.
AuthorsSinha ND, Cook RM
JournalNucleic Acids Res
PubMed ID3362678
'Syntheses of H-phosphonate salts (4a-e) of N/S-protected alcohols such as 6-aminohexan-1-ol, 3-mercaptopropan-1-ol and 6-mercaptohexan-1-ol are described using 2-chloro-5,6-benzo-1,3,2-phosphorin-4-one (2) as the phosphonylating agent. The H-phosphonate salts (4a-e), in the presence of pivaloyl chloride or adamantoyl chloride as an activator, were coupled to the 5''-end of synthetic oligonucleotides on solid supports ... More
Fluctuation of the first loop facing the matrix of the mitochondrial ADP/ATP carrier deduced from intermolecular cross-linking of Cys56 residues by bifunctional dimaleimides.
AuthorsHashimoto M, Majima E, Goto S, Shinohara Y, Terada H
JournalBiochemistry
PubMed ID9894001
'The effects of six thiol-specific cross-linker dimaleimides, in which the distance of the two maleimide groups ranged from 7.7 to 16. 8 A, on bovine heart mitochondria were studied at pH 7.2 and 0 degrees C. None of the dimaleimides affected mitochondrial proteins, but they caused significantly specific intermolecular cross-linking ... More
Structural and functional roles of Cys-238 and Cys-295 in Escherichia coli phosphofructokinase-2.
AuthorsBaez M, Rodríguez PH, Babul J, Guixé V
JournalBiochem J
PubMed ID12927023
'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 ... More
Diffusion properties of clathrin on the surface of isolated mouse liver nuclei by the fluorescence recovery after photobleaching technique.
AuthorsNandi P, Wahl P
JournalBiochim Biophys Acta
PubMed ID2456787
'Clathrin labeled with eosin maleimide showed physicochemical properties similar to the native clathrin. The diffusion coefficients of clathrin protomers and cages measured at 20 degrees C by the fluorescence recovery after photobleaching technique (FRAP) were found equal to (9 +/- 1).10(-8) cm2.s-1 and (3 +/- 0.4.10(-8) cm2.s-1, respectively. After incubation ... More
Interactions between transport inhibitors at the anion binding sites of the band 3 dimer.
AuthorsMacara IG, Cantley LC
JournalBiochemistry
PubMed ID7295667
'Evidence is presented that the binding of aromatic disulfonates to the external transport sites of the red cell anion-exchange protein (band 3) can exhibit negative cooperativity. Fluorescence resonance energy transfer has been used to compare the affinities of an aromatic disulfonate 4,4'-bis-(4-nitro-2,1,3-benzoxadiazolyl)dihydrostilbene-2,2'-disulfonate[H2(NBD)2DS] for "empty" band 3 dimers (in which neither ... More
Mechanism of free radical-induced hemolysis of human erythrocytes: hemolysis by water-soluble radical initiator.
AuthorsSato Y, Kamo S, Takahashi T, Suzuki Y
JournalBiochemistry
PubMed ID7619793
'Hemolysis of human erythrocytes induced by free radicals initiated from water-soluble, 2,2''-azobis(amidinopropane) dihydrochloride (AAPH) has been investigated. The formation of the radical detected as DMPO (5,5-dimethyl-1-pyrroline N-oxide) adduct depended on temperature and AAPH concentration in a similar manner as hemolysis. The curve for the formation of DMPO--radical adduct, however, did ... More
Rotational motion and flexibility of Ca2+,Mg2+-dependent adenosine 5'-triphosphatase in sarcoplasmic reticulum membranes.
AuthorsBürkli A, Cherry RJ
JournalBiochemistry
PubMed ID6162478
'Ca2+,Mg2+-dependent adenosine 5''-triphosphatase (ATPase) in sarcoplasmic reticulum vesicles is labeled with the triplet probe, 5-iodoacetamidoesin. Rotational mobility of the ATPase is investigated by measuring flash-induced transient dichroism of the eosin probe. The absorption anisotropy measured 20 mus after the exciting flash is found to be small at 37 degrees C ... More
Regulation of band 3 mobilities in erythrocyte ghost membranes by protein association and cytoskeletal meshwork.
AuthorsTsuji A, Kawasaki K, Ohnishi S, Merkle H, Kusumi A
JournalBiochemistry
PubMed ID2462903
'Rotational diffusion of erythrocyte anion channel protein band 3 was measured in ghost membranes by observing time-resolved phosphorescence anisotropy decays of eosinyl-5-maleimide covalently attached to the protein. Experiments were carried out under conditions similar to those employed by Tsuji and Ohnishi (1986) for translational diffusion measurement of band 3 [(1986) ... More
Rotational diffusion studies of the lipoyl domain of 2-oxoacid dehydrogenase multienzyme complexes.
AuthorsHarrison JP, Morrison IE, Cherry RJ
JournalBiochemistry
PubMed ID1696836
'Rotational mobility of the lipoyl domain of a number of 2-oxoacid dehydrogenase complexes was investigated by transient dichroism after the domain had been specifically labeled with the triplet probe eosin-5-maleimide. Complexes investigated included pyruvate dehydrogenase complexes from Bacillus stearothermophilus, ox heart, and Escherichia coli (in which the E2 component had ... More
H2O2-induced uncoupling of bovine lens Na+,K+-ATPase.
AuthorsGarner WH, Garner MH, Spector A
JournalProc Natl Acad Sci U S A
PubMed ID6300890
'A 1-hr exposure of bovine lenses in organ culture to H2O2 concentrations in the range found in the aqueous fluid of patients with cataracts inhibits 86Rb+ influx. At 1 mM H2O2, complete inhibition was observed and further investigated. Membrane permeability is slightly decreased. Although lactate concentrations increase 2-fold, lens ATP ... More
Involvement of a sperm protein sensitive to sulfhydryl-depleting reagents in mouse sperm-egg fusion.
AuthorsMammoto A, Masumoto N, Tahara M, Yoneda M, Nishizaki T, Tasaka K, Miyake A
JournalJ Exp Zool
PubMed ID9181696
'Effects of various sulfhydryl (SH)-depleting reagents on sperm-egg fusion were demonstrated. When sperm were treated with three plasma membrane-permeable SH-depleting reagents, N-ethyl-maleimide, sodium tetrathionate and 5,5''-dithiobis (2-nitro-benzoic acid), the rates of cleavage of eggs were significantly lower than those with control sperm. Neither the motility, penetration of the zona pellucida, ... More
Flow cytometric analysis of band 3 protein of human erythrocytes.
AuthorsStoya G, Baumann E, Junker U, Hermann J, Linss W
JournalActa Histochem
PubMed ID9150794
'Flow cytometry was used to quantify the transmembrane anion exchanger (band 3 protein) of human erythrocytes by covalently bound eosin-5-maleimide. In vitro and in vivo vesiculated red blood cells were investigated. The fluorescence and light scatter signals of cells after heat induced vesiculation, in vivo ageing, and in patients with ... More
Interaction of fluorescein derivatives with glibenclamide binding sites in rat brain.
AuthorsHolemans S, Feron O, Octave JN, Maloteaux JM
JournalNeurosci Lett
PubMed ID7739789
'In rat brain, [3H]glibenclamide binds with high affinity to sulfonylurea receptors associated with ATP-sensitive potassium (KATP) channels. KATP channels may play a modulatory role in neurotransmitter release and are involved in acute pathological events occurring in the brain. Fluorescein derivatives, which are suitable tools for the labelling of nucleotide binding ... More
Rotational diffusion of eosin-labeled pyruvate dehydrogenase complex of Escherichia coli.
AuthorsVisser AJ, Scouten WH, Lavalette D
JournalEur J Biochem
PubMed ID6173215
'The enzymatically reduced lipoyl residues of the transacetylase component of the pyruvate dehydrogenase complex from Escherichia coli were labeled with eosin maleimide. Using eosin as triplet probe, triplet-triplet absorption dichroism measurements were performed to obtain rotational correlation times of the complex in the microsecond time domain. It was found that ... More
Characterization of cysteine residues of mitochondrial ADP/ATP carrier with the SH-reagents eosin 5-maleimide and N-ethylmaleimide.
AuthorsMajima E, Koike H, Hong YM, Shinohara Y, Terada H
JournalJ Biol Chem
PubMed ID7691823
'The effects of the membrane-impermeable fluorescent sulfhydryl reagent eosin 5-maleimide (EMA) and the membrane-permeable sulfhydryl reagent N-ethylmaleimide (NEM) on ADP transport via the ADP/ATP carrier and their labeling sites in the carrier were studied in bovine heart submitochondrial particles. Of the 4 cysteine residues in the carrier, EMA labeled Cys159 ... More
Localization of the NADH kinase in the inner membrane of yeast mitochondria.
AuthorsIwahashi Y, Nakamura T
JournalJ Biochem (Tokyo)
PubMed ID2549021
'The bulk of NADH kinase of Saccharomyces cerevisiae was recovered in the mitochondrial fraction prepared from spheroplasts. Most of the NADH kinase was localized in the inner membrane fraction, which was separated from other mitochondrial components by the combined swelling, shrinking, and sonication procedure. Treatment of mitoplasts with antiserum against ... More
The epsilon subunit of the F(1)F(0) complex of Escherichia coli. cross-linking studies show the same structure in situ as when isolated.
AuthorsSchulenberg B, Capaldi RA
JournalJ Biol Chem
PubMed ID10497194
'Four double mutants in the epsilon subunit were generated, each containing two cysteines, which, based on the NMR structure of this subunit, should form internal disulfide bonds. Two of these were designed to generate interdomain cross-links that lock the C-terminal alpha-helical domain against the beta-sandwich (epsilonM49C/A126C and epsilonF61C/V130C). The second ... More
Oxidation of specific SH protein of mitochondria by photodynamic action of hematoporphyrin. Relevance to uncoupling of oxidative phosphorylation.
AuthorsYamamoto K, Kawanishi S
JournalBiochem Pharmacol
PubMed ID1714732
'Photoexcited hematoporphyrin (Hp) induces the uncoupling of oxidative phosphorylation of mitochondria. The uncoupling was inhibited by pre-incubation of mitochondria with a fluorescent SH reagent, eosin-5-maleimide, which has been shown to react specifically with an essential SH group of the Pi/H+ symporter [Houstek and Pedersen, J Biol Chem 260: 6288-6295, 1985]. ... More
Fluorescence labeling of proteins related to ADH-induced change in frog bladder luminal membrane.
AuthorsValenti G, Guerra L, Casavola V, Svelto M
JournalBiol Cell
PubMed ID2483679
'Sulfhydryl (SH) reactive reagents, such as eosin derivatives, have been found to be useful in labeling water pathways in red cells. In the present study we used an impermeable SH-reagent, a fluorescent maleimide analogue EMA (eosin-5''-maleimide), in order to identify proteins involved in water permeability response to antidiuretic hormone (ADH). ... More
Binding of the activating ion Co(II) to myo-inositol monophosphatase monitored by fluorescence and phosphorescence spectroscopy.
AuthorsKwon OS, Churchich JE
JournalJ Protein Chem
PubMed ID9055202
'Two extrinsic probes, pyrene-maleimide and eosin-maleimide, were used to label specific SH groups of the enzyme myo-inositol monophosphatase. The fluorescence of pyrene-monophosphatase is enhanced upon addition of the activating metal ions Co(II) and Mg(II). Co(II) ions bind with a dissociation constant of 4 microM, whereas the apparent activation constant Ka ... More
Comparison of human red cell lysis by hypochlorous and hypobromous acids: insights into the mechanism of lysis.
AuthorsVissers MC, Carr AC, Chapman AL
JournalBiochem J
PubMed ID9461501
'Human red blood cells are lysed by the neutrophil-derived oxidant hypochlorous acid (HOCl), although the mechanism of lysis is unknown. Hypobromous acid (HOBr), a similarly reactive oxidant, lysed red cells approx. 10-fold faster than HOCl. Therefore we compared the effects of these oxidants on thiols, membrane lipids and proteins to ... More
Eosin, a fluorescent probe of ATP binding to the (Na+ + K+)-ATPase.
AuthorsSkou JC, Esmann M
JournalBiochim Biophys Acta
PubMed ID6170332
Rotational and lateral diffusion of membrane proteins.
AuthorsCherry RJ
JournalBiochim Biophys Acta
PubMed ID391281
Developmental changes in the lateral diffusion of Leydig cell membranes measured by the FRAP method.
AuthorsZlatanov IV, Foley M, Birmingham J, Garland PB
JournalFEBS Lett
PubMed ID3653400
A simple method for isolation and fluorescence labelling of Leydig cells (L-cells) from rat testes was developed. Lateral diffusion coefficients of both lipid and protein membrane fluorescent probes were measured by the method of fluorescence recovery after photobleaching (FRAP). Age-dependent changes in diffusibility of membrane lipids and proteins were discovered. ... More
Exposure of actin thiols by the removal of tightly held calcium ions.
AuthorsKonno K, Morales MF
JournalProc Natl Acad Sci U S A
PubMed ID3865205
The removal of bound metal ions from G-actin uncovered two thiols, Cys-10 and Cys-257. The uncovering of these thiols requires a free calcium concentration lower than 10 nM. Therefore, participation of one or both thiols in Ca2+ binding is suggested. Actin labeled with N-(5-fluoresceinyl)maleimide in the absence of calcium moves ... More
Demonstration of sulfhydryl and disulfide groups by a fluorescent maleimide procedure.
AuthorsCurtis SK, Cowden RR
JournalHistochemistry
PubMed ID7190963
Several fluorescent maleimide compounds were evaluated as possible substitutes for N-(4-aminophenyl)maleimide in the histochemical procedures developed by Sippel (1973, 1978a, b, 1980) for the demonstration of sulfhydryl and disulfide groups. The brightest and most selective fluorescence was obtained by using N-(7-dimethylamino-4-methylcoumarinyl)maleimide (DACM), although both eosin-5-maleimide and fluorescein-5-maleimide could also be ... More
Conformational change in plasma albumin due to interaction with isolated rat hepatocyte.
AuthorsHorie T, Mizuma T, Kasai S, Awazu S
JournalAm J Physiol
PubMed ID2833115
The electron spin resonance spectroscopy of 4-isothiocyanato-tempo labeled to bovine serum albumin (BSA) and the absorption spectroscopy of eosin maleimide labeled to BSA in the isolated rat hepatocyte suspension indicate conformational change occurring in the albumin molecule during interaction with the hepatocellular membrane. The conformational change in the albumin molecule ... More
Electron transfer and conformation states in bovine cytochrome c oxidase.
AuthorsWilson MT, Alleyne T, Clague M, Conroy K, el-Agez B
JournalAnn N Y Acad Sci
PubMed ID2854389
The fluorophores 1,5-I-AEDANS and eosin maleimide bind to subunit III of bovine cytochrome c oxidase. Fluorescence lifetime measurements have been made of bound AEDANS under a number of conditions. It appears that the spatial relationship between this bound probe and metal centers is unaffected by the redox changes in the ... More
Sulfhydryl groups are involved in H+ translocation via the uncoupling protein of brown adipose tissue mitochondria.
AuthorsJezek P
JournalFEBS Lett
PubMed ID3026844
Mersalyl inhibits H+ transport via the uncoupling protein (UP) in brown adipose tissue (BAT) mitochondria estimated as swelling in potassium acetate (Ki 67 microM) or as valinomycin-induced H+ extrusion in K2SO4 (Ki 55 microM) and KCl. The swelling in KCl is depressed only slightly. Some other SH-reagents (p-hydroxymercuribenzoate, 5,5'-dithiobis(2-nitrobenzoate) and ... More
Significance and redox state of SH groups in pyruvate carrier isolated from bovine heart mitochondria.
AuthorsNalecz KA, Müller M, Zambrowicz EB, Wojtczak L, Azzi A
JournalBiochim Biophys Acta
PubMed ID2317484
The role and properties of -SH groups of purified pyruvate (monocarboxylate) carrier were investigated. After isolation, this protein has all -SH groups in the oxidized state. Upon reduction, the carrier can be labelled with eosin-5-maleimide. The shift in apparent Mr after the labelling points to the presence of at least ... More
Distribution of protein sulphydryls and disulphides in fixed mammalian chromosomes, and their relationship to banding.
AuthorsSumner AT,
JournalJ Cell Sci
PubMed ID6501434
Fixed mammalian mitotic chromosomes, when stained with a variety of sensitive, sulphydryl-specific fluorochromes directly or following reduction of disulphide groups, show uniform fluorescence. Thus sulphydryl and disulphide groups are uniformly distributed along the length of the chromosomes, and do not show patterns related to chromosome bands. Performance of G- or ... More
Selective labeling of beef heart cytochrome oxidase subunit III with eosin-5-maleimide.
AuthorsMüller M, Azzi A
JournalFEBS Lett
PubMed ID2580740
Cytochrome c oxidase has been isolated from beef heart mitochondria and labeled with the fluorochrome eosin-5-maleimide (EMA) after pretreatment with mersalyl. On SDS-polyacrylamide gels, EMA fluorescence and absorption occurred at a single band corresponding to subunit III. Since only Cys 115 of the two cysteinyl residues of subunit III had ... More
Using the eosin-5-maleimide binding test in the differential diagnosis of hereditary spherocytosis and hereditary pyropoikilocytosis.
BACKGROUND: 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. METHODS: Flow cytometry was ... More
Protease-sensitive signalling by chemically engineered intramolecular fluorescent resonance energy transfer mutants of green fluorescent protein.
AuthorsSuzuki M, Ito Y, Savage HE, Husimi Y, Douglas KT,
JournalBiochim Biophys Acta
PubMed ID15358514
The native cysteine residues of green fluorescent protein (GFP) at positions 48 and 70 were replaced by non-thiolic amino acids, and new cysteine sites were introduced at specific, surface positions. Based on molecular modeling of the GFP structure, the sites chosen for mutagenesis to Cys were glutamic acid at position ... More
Caspase-3 sensitive signaling in vivo in apoptotic HeLa cells by chemically engineered intramolecular fluorescence resonance energy transfer mutants of green fluorescent protein.
AuthorsSuzuki M, Ito Y, Sakata I, Sakai T, Husimi Y, Douglas KT,
JournalBiochem Biophys Res Commun
PubMed ID15796904
Green fluorescent protein (UV5) was re-engineered to remove native cysteine residues, and a new cysteine was introduced near the C-terminus, approximately 20 A from the native fluorophore, for site-specific attachment of chemical fluorophores. The resultant efficient intramolecular FRET quenched GFP emission and gave a new emission band from the conjugated ... More
Influence of cholesterol on the rotation and self-association of band 3 in the human erythrocyte membrane.
AuthorsMühlebach T, Cherry RJ
JournalBiochemistry
PubMed ID7126539
The cholesterol/phospholipid mole ratio (C/P) in the human erythrocyte membrane was varied by incubating cells with liposomes. The rotational mobility of band 3 proteins was measured in these membranes by observing flash-induced transient dichroism of the triplet probe eosin maleimide. Measurements were performed with membranes in which associations of band ... More
Rotational dynamics of the integral membrane protein, band 3, as a probe of the membrane events associated with Plasmodium falciparum infections of human erythrocytes.
AuthorsTilley L, Foley M, Anders RF, Dluzewski AR, Gratzer WB, Jones GL, Sawyer WH
JournalBiochim Biophys Acta
PubMed ID2194565
Time-resolved phosphorescence anisotropy was used to study the molecular organisation of band 3 in the erythrocyte membrane. Three different rotational relaxation regimes of mobile band 3 were resolved. These populations may represent different aggregation states of band 3 within the membrane, or they may result from association of band 3 ... More
Stabilities of the fluorescent SH-reagent eosin-5-maleimide and its adducts with sulfhydryl compounds.
AuthorsMajima E, Goto S, Hori H, Shinohara Y, Hong YM, Terada H
JournalBiochim Biophys Acta
PubMed ID7537101
The stabilities of the SH-reagent eosin-5-maleimide (EMA) and its adducts with the SH-compounds L-cysteine, N-acetyl-L-cysteine and glutathione (reduced form) were studied under various conditions in comparison with those of the adducts of N-ethylmaleimide (NEM). Studies by reversed-phase high performance liquid chromatography and mass spectrometry showed that EMA was less stable ... More
NADP-malic enzyme from maize leaves: a fluorescence study.
AuthorsDrincovich MF, Andreo CS
JournalBiochem Mol Biol Int
PubMed ID8535301
NADP-malic enzyme from maize leaves is covalently labeled with a fluorescent-SH reactive probe eosin-5-maleimide (EMA), which reacts with groups that are totally protected by NADP against inactivation. The comparison of the emission fluorescence spectra of the native and the modified enzyme suggests the proximity of the fluorescent groups of the ... More
Rotational motions of myosin heads in myofibril studied by phosphorescence anisotropy decay measurements.
AuthorsIshiwata S, Kinosita K, Yoshimura H, Ikegami A
JournalJ Biol Chem
PubMed ID2439498
We studied the rotational Brownian motions of myosin heads, of which the sulfhydryl group was selectively labeled with the triplet probe 5-eosinylmaleimide, in myofibril by using flash-induced phosphorescence anisotropy decay measurements. The anisotropy decay curve under relaxing conditions consisted of a fast (submicrosecond) and a slow (a few microseconds) component ... More
Excitation wavelength dependent fluorescence anisotropy of eosin-myosin adducts. Evidence for anisotropic rotations.
AuthorsVanderMeulen DL, Nealon DG, Gratton E, Jameson DM
JournalBiophys Chem
PubMed ID1698474
Steady-state and time-resolved fluorescence anisotropy measurements of eosin in solution and eosin-5-maleimide bound to purified myosin were made to study localized motions of the "head region" of this protein. The lifetime and apparent Debye rotational relaxation times of eosin in aqueous solution are essentially invariant with changes in excitation wavelength. ... More
Two-dimensional arrangement of a functional protein by cysteine-gold interaction: enzyme activity and characterization of a protein monolayer on a gold substrate.
AuthorsSasaki YC, Yasuda K, Suzuki Y, Ishibashi T, Satoh I, Fujiki Y, Ishiwata S
JournalBiophys J
PubMed ID9083688
We have characterized the functional protein, myosin subfragment 1 (S1), attached to a gold substrate by the sulfhydryl groups of cysteine in proteins. The amino groups of the regulatory light chain (RLC) isolated from myosin were labeled with a radioisotope (125I), and the labeled RLC was incorporated into S1 from ... More
Functional and idling rotatory motion within F1-ATPase.
AuthorsSabbert D, Engelbrecht S, Junge W
JournalProc Natl Acad Sci U S A
PubMed ID9114001
ATP synthase mediates proton flow through its membrane portion, F0, which drives the synthesis of ATP in its headpiece, F1. The F1-portion contains a hexagonal array of three subunits alpha and three beta encircling a central subunit gamma, that in turn interacts with a smaller epsilon and with F0. Recently ... More
Rotational diffusion of erythrocyte membrane proteins.
AuthorsCherry RJ, Nigg EA
JournalProg Clin Biol Res
PubMed ID7022477
Rotational diffusion of band 3 proteins in human erythrocyte membranes is measured by observing flash-induced transient dichroism of the triplet probe, eosin-maleimide. At physiological temperature, both fast and slowly rotating populations of band 3 are present in the membrane. Rotational motion of band 3 is the same in membranes from ... More
Rotational diffusion of band 3 in erythrocyte membranes. 2. Binding of cytoplasmic enzymes.
AuthorsMatayoshi ED, Sawyer WH, Jovin TM
JournalBiochemistry
PubMed ID2012812
Time-resolved phosphorescence anisotropy has been used to study the rotational diffusion of eosin-labeled human erythrocyte band 3 in the presence of an enzyme bound at its cytoplasmic pole. With increasing amounts of G3PD (glyceraldehyde-3-phosphate dehydrogenase) added to ghosts, the infinite time anisotropy (r infinity) increases, and at saturating concentrations, very ... More
Binding of the fluorescein derivative eosin Y to the mitochondrial ADP/ATP carrier: characterization of the adenine nucleotide binding site.
AuthorsMajima E, Yamaguchi N, Chuman H, Shinohara Y, Ishida M, Goto S, Terada H
JournalBiochemistry
PubMed ID9425064
As the SH-reactive fluorescein derivative eosin-5-maleimide (EMA) specifically labels Cys159 in the second loop facing the matrix space (loop M2) of the ADP/ATP carrier in bovine heart submitochondrial particles [Majima, E., Koike, H., Hong, Y.-M., Shinohara, Y., and Terada, H. (1993) J. Biol. Chem. 268, 22181-22187], we studied the interaction ... More
Isolation of the mitochondrial benzodiazepine receptor: association with the voltage-dependent anion channel and the adenine nucleotide carrier.
AuthorsMcEnery MW, Snowman AM, Trifiletti RR, Snyder SH
JournalProc Natl Acad Sci U S A
PubMed ID1373486
The mitochondrial benzodiazepine receptor (mBzR) has been solubilized with retention of reversible ligand binding, and the associated subunits were characterized. mBzR comprises immunologically distinct protein subunits of 18-, 30-, and 32-kDa. The 18-kDa protein is labeled by the isoquinoline carboxamide mBzR ligand [3H]PK14105, whereas the 30- and 32-kDa subunits are ... More
Cytoskeletal restraints of band 3 rotational mobility in human erythrocyte membranes.
AuthorsClague MJ, Harrison JP, Cherry RJ
JournalBiochim Biophys Acta
PubMed ID2524215
The interaction of band 3 with cytoskeletal proteins was investigated in erythrocyte membranes by measuring the rotational mobility of band 3 using the method of transient dichroism. It was found that selective proteolysis of ankyrin, a protein known to link band 3 to the spectrin-actin network, had no significant effect ... More
Cross-linking of engineered subunit delta to (alphabeta)3 in chloroplast F-ATPase.
AuthorsLill H, Hensel F, Junge W, Engelbrecht S
JournalJ Biol Chem
PubMed ID8955107
Ser --> Cys mutations were introduced into subunit delta of spinach chloroplast F0F1-ATPase (CF0CF1) by site-directed mutagenesis. The engineered delta subunits were overexpressed in Escherichia coli, purified, and reassembled with spinach chloroplast F1-ATPase (CF1) lacking the delta subunit (CF1(-delta)). By modification with eosin-5-maleimide, it was shown that residues 10, 57, ... More
Analytical methods to investigate glutathione and related compounds in biological and pathological processes.
AuthorsCamera E, Picardo M
JournalJ Chromatogr B Analyt Technol Biomed Life Sci
PubMed ID12450659
Reduced glutathione (GSH, gamma-L-glutamyl-L-cysteinylglycine) is a fundamental low-molecular mass antioxidant that serves several biological functions. Upon enzymatic and non-enzymatic oxidation, GSH forms glutathione disulfide (GSSG) and, under particular conditions, may generate other oxidative products. The determination of GSH, its precursors, and metabolites in several bio-matrices is a useful tool in ... More
Band 3 mobility in camelid elliptocytes: implications for erythrocyte shape.
AuthorsMcPherson RA, Sawyer WH, Tilley L
JournalBiochemistry
PubMed ID8329396
Measurements of time-resolved phosphorescence anisotropy were used to monitor the rotational diffusion of eosin-labeled band 3 in membranes of the elliptocytic erythrocytes of alpacas and camels. The rotational freedom of camelid band 3 was more restricted than for human band 3. Removal of the peripheral membrane proteins from human erythrocyte ... More
Fluorescence resonance energy transfer mapping of subunit delta in spinach chloroplast F1 ATPase.
AuthorsEngelbrecht S, Giakas E, Marx O, Lill H
JournalEur J Biochem
PubMed ID9523699
Despite the considerable progress in the field of F0F1-ATPases caused by solving the 2.8-A structure of mitochondrial F1 ATPase [Abrahams, J. P., Leslie, A. G. W., Lutter, R. & Walker, J. E. (1994) Nature 370, 621-628], little is known about the position and function of the enzyme's small subunits which ... More
Specific labeling of the bovine heart mitochondrial phosphate carrier with fluorescein 5-isothiocyanate: roles of Lys185 and putative adenine nucleotide recognition site in phosphate transport.
AuthorsMajima E, Ishida M, Miki S, Shinohara Y, Terada H
JournalJ Biol Chem
PubMed ID11133984
The amine/SH-modifying fluorescein 5-isothiocyanate (FITC) specifically labeled Lys(185) in the putative membrane-spanning region of the phosphate carrier from both the cytosolic and matrix sides of bovine heart mitochondria at 0 degrees C and pH 7.2, and the labeling inhibited the phosphate transport. Nonmodifying fluorescein derivatives having similar structural features to ... More
Selective labeling and rotational diffusion of the ADP/ATP translocator in the inner mitochondrial membrane.
AuthorsMüller M, Krebs JJ, Cherry RJ, Kawato S
JournalJ Biol Chem
PubMed ID6173378
Submitochondrial particles were labeled with the triplet probe eosin-5-maleimide (EMA) after pretreatment with N-ethylmaleimide. On sodium dodecyl sulfate-polyacrylamide gels, eosin fluorescence occurred in a single band of Mr approximately 30,000. The labeled band was identified as the ADP/ATP translocator, since EMA binding was completely inhibited by carboxyatractylate. Furthermore, the EMA-labeled ... More
Importance of loops of mitochondrial ADP/ATP carrier for its transport activity deduced from reactivities of its cysteine residues with the sulfhydryl reagent eosin-5-maleimide.
AuthorsMajima E, Shinohara Y, Yamaguchi N, Hong YM, Terada H
JournalBiochemistry
PubMed ID7520750
The effects of various compounds such as the transport substrate ADP and the transport inhibitors carboxyatractyloside (CATR) and bongkrekic acid (BKA) on the labeling of cysteine residues in the ADP/ATP carrier of bovine heart submitochondrial particles by the SH reagent eosin-5-maleimide (EMA) were studied. Of the four cysteine residues in ... More
Anchorage of a band 3 population at the erythrocyte cytoplasmic membrane surface: protein rotational diffusion measurements.
AuthorsNigg EA, Cherry RJ
JournalProc Natl Acad Sci U S A
PubMed ID6933517
Direct physical evidence for the linkage of a band 3 population to the cytoskeleton in the erythrocyte ghost membrane is presented. The rotational diffusion of band 3 proteins was mesured by observing flash-induced transient dichroism of a covalently bound eosin probe. After proteolytic release of a 40,000-dalton cytoplasmic segment of ... More
Detection of Cl- binding to band 3 by double-quantum-filtered 35Cl nuclear magnetic resonance.
AuthorsLiu D, Knauf PA, Kennedy SD
JournalBiophys J
PubMed ID8789088
We have applied double-quantum-filtered (DQF) NMR of 35Cl to study binding of Cl- to external sites on intact red blood cells, including the outward-facing anion transport sites of band 3, an integral membrane protein. A DQF 35Cl NMR signal was observed in cell suspensions containing 150 mM KCl, but the ... More
Rotational diffusion of band 3 in erythrocyte membranes. 1. Comparison of ghosts and intact cells.
AuthorsMatayoshi ED, Jovin TM
JournalBiochemistry
PubMed ID1707311
The rotational diffusion of eosin-labeled 3 in human erythrocyte cells and hemoglobin-free ghosts at 37 degrees C has been studied in detail by polarized delayed luminescence. The time-resolved anisotropy with both cells and freshly prepared ghosts is similar, decaying with well-resolved rotational correlation times of 0.03, 0.2, and greater than ... More
Proteolytic digestion of band 3 at an external site alters the erythrocyte membrane organisation and may facilitate malarial invasion.
AuthorsMcPherson RA, Donald DR, Sawyer WH, Tilley L
JournalMol Biochem Parasitol
PubMed ID8139616
Invasion of human erythrocytes by Plasmodium falciparum is inhibited by chymostatin. This suggests that digestion of erythrocyte surface proteins by a protease with chymotrypsin-like activity may be involved in the invasion process. We find that treatment of intact erythrocytes with chymotrypsin cleaves the integral membrane protein, band 3, generating a ... More
Influence of temperature and cholesterol on the rotational diffusion of band 3 in the human erythrocyte membrane.
AuthorsNigg EA, Cherry RJ
JournalBiochemistry
PubMed ID224909
Band 3 rotation in the human erythrocyte membrane is measured by observing flash-induced dichroism of eosin probes. The decay of the absorption anisotropy is found to be strongly dependent on temperature. The results are analyzed on the assumption that rotation of band 3 only occurs about the membrane normal. It ... More
Altered structure and anion transport properties of band 3 (AE1, SLC4A1) in human red cells lacking glycophorin A.
AuthorsBruce LJ, Pan RJ, Cope DL, Uchikawa M, Gunn RB, Cherry RJ, Tanner MJ
JournalJ Biol Chem
PubMed ID14604989
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 ... More
Rotational diffusion of band 3 proteins in membranes from En(a-) and neuraminidase-treated normal human erythrocytes.
AuthorsNigg EA, Gahmberg CG, Cherry RJ
JournalBiochim Biophys Acta
PubMed ID7407136
Recent experiments have demonstrated an association between band 3 and glycophorin A in the human eythrocyte membrane (Nigg, E.A., Bron, C., Girardet, M. and Cherry, R.J. (1980) Biochemistry 19, 1887-1893). Here, the influence of sialoglycoproteins on the rotational diffusion of band 3 in the human erythrocyte membrane was investigated by ... More
Microsecond rotational dynamics of phosphorescent-labeled muscle cross-bridges.
AuthorsLudescher RD, Thomas DD
JournalBiochemistry
PubMed ID2455541
We have measured the microsecond rotational motions of myosin heads in muscle cross-bridges under physiological ionic conditions at 4 degrees C, by detecting the time-resolved phosphorescence of eosin-maleimide covalently attached to heads in skeletal muscle myofibrils. The anisotropy decay of heads in rigor (no ATP) is constant over the time ... More
Activation and inhibition of ATP-sensitive K+ channels by fluorescein derivatives.
Authorsde Weille JR, Müller M, Lazdunski M
JournalJ Biol Chem
PubMed ID1311312
Fluorescein derivatives are known to bind to nucleotide-binding sites on transport ATPases. In this study, they have been used as ligands to nucleotide-binding sites on ATP-sensitive K+ channels in insulinoma cells. Their effect on channel activity has been studied using 86Rb+ efflux and patch-clamp techniques. Fluorescein derivatives have two opposite ... More
Comparison of reagents for shape analysis of fixed cells by automated fluorescence microscopy.
AuthorsElliott JT, Tona A, Plant AL
JournalCytometry A
PubMed ID12655652
BACKGROUND: Cell size and shape have been implicated as potentiators of intracellular signaling events and as indicators of abnormal cell behavior. Automated microscopy and image analysis can provide quantitative information about the size and shape of cultured cells, but it requires that the edge of a cell be clearly identified. ... More