Temperature and pH dependence of the haemolytic activity of influenza virus and of the rotational mobility of the spike glycoproteins.
AuthorsJunankar PR, Cherry RJ
JournalBiochim Biophys Acta
PubMed ID3942725
'Influenza virus (strain X-47) was labeled with the triplet probe, eosin 5-isothiocyanate. Most of the label was found to be associated with haemagglutinin, the major glycoprotein of the viral envelope. Rotational diffusion of the glycoprotein was investigated by measuring flash-induced transient dichroism of the eosin probe. The anisotropy decay curves ... More
Identification of the eosinyl-5-maleimide reaction site on the human erythrocyte anion-exchange protein: overlap with the reaction sites of other chemical probes.
AuthorsCobb CE, Beth AH
JournalBiochemistry
PubMed ID1701324
'The anion-exchange protein (band 3) reaction site in human erythrocytes for the fluorescent/phosphorescent probe eosinyl-5-maleimide (EMA) has been identified. Proteolytic dissection of band 3 in situ indicated that EMA reacts with the membrane-spanning Mr 17K peptide produced by chymotrypsin cleavage of band 3 in intact erythrocytes followed by removal of ... More
Affinity labeling of the active site of the Ca2+-ATPase of sarcoplasmic reticulum.
AuthorsMurphy AJ
JournalBiochim Biophys Acta
PubMed ID2974728
'The inactivation of sarcoplasmic reticulum ATPase by fluorescein isothiocyanate (FITC) was shown to have a hyperbolic dependence on the concentration of FITC. The results were quantitatively accounted for by a model in which the reagent first binds reversibly (Kf = 70 microM) to the ATPase and then reacts irreversibly (kmax ... More
Protein rotational motion in solution measured by polarized fluorescence depletion.
AuthorsYoshida TM, Barisas BG
JournalBiophys J
PubMed ID3730506
'A microscope-based system is described for directly measuring protein rotational motion in viscous environments such as cell membranes by polarized fluorescence depletion (PFD). Proteins labeled with fluorophores having a high quantum yield for triplet formation, such as eosin isothiocyanate (EITC), are examined anaerobically in a fluorescence microscope. An acousto-optic modulator ... More
Labeling of human erythrocyte membranes with eosin probes used for protein diffusion measurements: inhibition of anion transport and photo-oxidative inactivation of acetylcholinesterase.
AuthorsNigg E, Kessler M, Cherry RJ
JournalBiochim Biophys Acta
PubMed ID215229
'The binding of eosin-isothiocyanate (eosin-NCS) and iodoacetamido-eosin (IA-eosin) to band 3 proteins in the membrane of human erythrocytes is characterized by studying the effect of these probes on the anion transport system. Although the unbrominated fluorescein precursors do not affect anion transport, both eosin labels are strong inhibitors of sulphate ... More
Effects of villin on the polymerization and subunit exchange of actin.
AuthorsWang Y, Bonder EM, Mooseker MS, Taylor DL
JournalCell Motil
PubMed ID6883468
'We have investigated the Ca2+ -dependent interactions of villin, a protein of the intestinal microvillar core, with actin by monitoring resonance energy transfer between fluorescently labeled actin subunits. In the presence of elevated free Ca2+ (approximately 20 microM), villin affects both the nucleation and the elongation phases of actin polymerization. ... More
Lipid effects on aggregation of pulmonary surfactant protein SP-C studied by fluorescence energy transfer.
AuthorsHorowitz AD, Baatz JE, Whitsett JA
JournalBiochemistry
PubMed ID8373758
'The self-association of pulmonary surfactant protein SP-C in lipid vesicles was studied using fluorescence energy transfer. Bovine SP-C was labeled with two fluorescent probes, succinimidyl 6-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]hexanoate and eosin isothiocyanate, on the amino terminus of the protein, producing NBD-SP-C and EITC-SP-C, respectively. The N-terminus of SP-C was relatively immobile between 20 ... More
Chemical modification of matrix porin from Escherichia coli: probing the pore topology of a transmembrane protein.
AuthorsSchindler M, Rosenbusch JP
JournalJ Cell Biol
PubMed ID6177703
'Chemical modification of amino groups in matrix porin solubilized and purified from outer membranes of Escherichia coli in beta-octylglucoside was performed with eosin isothiocyanate and citraconic anhydride. At pH 7 8.5, the former reagent labeled a single amino group in the native protein, while more extensive derivatization was observed with ... More
Rotational diffusion of band 3 in the red cell membrane: measurements using triplet probes.
AuthorsCherry RJ, Nigg E
JournalProg Clin Biol Res
PubMed ID93746
'Rotational diffusion of band 3 in the human erythrocyte membrane has been investigated using the technique of flash photolysis. Membranes are labeled with the triplet probe eosin isothiocyanate; most of the label is found to be associated with band 3. Rotation is measured by observing the decay of dichroism of ... 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
Ca(2+)-dependent annexin self-association on membrane surfaces.
AuthorsZaks WJ, Creutz CE
JournalBiochemistry
PubMed ID1911746
'Annexin self-association was studied with 90 degrees light scattering and resonance energy transfer between fluorescein (donor) and eosin (acceptor) labeled proteins. Synexin (annexin VII), p32 (annexin IV), and p67 (annexin VI) self-associated in a Ca(2+)-dependent manner in solution. However, this activity was quite labile and, especially for p32 and p67, ... More
Protein rotation, enzyme activity and photooxidation of SH groups in sarcoplasmic reticulum Ca2+-ATPase.
AuthorsRestall CJ, Arrondo JL, Elliot DA, Jaskowska A, Weber WV, Chapman D
JournalBiochim Biophys Acta
PubMed ID6457648
'The binding of probe molecules such as fluorescein isothiocyanate, eosin isothiocyanate and erythrosin isothiocyanate to the Ca2+-ATPase of sarcoplasmic reticulum followed by illumination of the labelled protein causes substantial reductions of ATPase activity over a 1-h period. The degree of light-sensitivity induced by these probes is related to the triplet ... 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
Fluorescence energy transfer as an indicator of Ca2+-ATPase interactions in sarcoplasmic reticulum.
AuthorsPapp S, Pikula S, Martonosi A
JournalBiophys J
PubMed ID2950938
'Ca2+-ATPase molecules were labeled in intact sarcoplasmic reticulum (SR) vesicles, sequentially with a donor fluorophore, fluorescein-5''-isothiocyanate (FITC), and with an acceptor fluorophore, eosin-5''-isothiocyanate (EITC), each at a mole ratio of 0.25-0.5 mol/mol of ATPase. The resonance energy transfer was determined from the effect of acceptor on the intensity and lifetime ... More
Multiple spectral parameter imaging.
AuthorsWaggoner A, DeBiasio R, Conrad P, Bright GR, Ernst L, Ryan K, Nederlof M, Taylor D
JournalMethods Cell Biol
PubMed ID2648118
Fluorescent labeling of cell surfaces.
AuthorsEdidin M
JournalMethods Cell Biol
PubMed ID2464121
Amino acid residues complexed with eosin 5-isothiocyanate in band 3 protein of the human erythrocyte.
AuthorsChiba T, Sato Y, Suzuki Y
JournalBiochim Biophys Acta
PubMed ID2423129
'The amino-acid residues of band 3 protein taking part in the ionic interaction with an anion-transport inhibitor, eosin 5-isothiocyanate (EITC), were determined by pH titration. The plots of the absorbance of EITC-ghost system against pH reveal five equilibria, at pH 3.7, 6.4, 8.0, 11.0 and 13.1. Since the three equilibria, ... More
Characterization of eosin 5-isothiocyanate binding site in band 3 protein of the human erythrocyte.
AuthorsChiba T, Sato Y, Suzuki Y
JournalBiochim Biophys Acta
PubMed ID2432938
'The characteristics of the anion transport system in human erythrocyte, which can be modified by eosin 5-isothiocyanate (EITC), were studied using the pH titration method and by measuring the sulfate efflux. Based on the pH dependence of EITC binding to the erythrocyte ghosts, it was found that the reaction rate ... More
Effect of phospholipid:protein ratio on the state of aggregation of the (Ca2+-Mg2+)-ATPase.
AuthorsMunkonge F, Michelangeli F, Rooney EK, East JM, Lee AG
JournalBiochemistry
PubMed ID2973807
'The organization of the (Ca2+-Mg2+)-ATPase has been studied in reconstituted systems by fluorescence polarization of the ATPase labeled with fluorescein isothiocyanate (FITC) and resonance energy transfer between ATPase labeled with FITC and with eosin isothiocyanate (EITC). The fluorescence polarization of FITC-ATPase was found to decrease with increasing labeling ratio FITC:ATPase, ... More
The rotational diffusion of chloroplast phosphate translocator and of lipid molecules in bilayer membranes.
AuthorsWagner R, Apley EC, Gross A, Flügge UI
JournalEur J Biochem
PubMed ID2471643
'The rotational mobility of the phosphate translocator from the chloroplast envelope and of lipid molecules in the membrane of unilamellar azolectin liposomes has been investigated. The rotational dynamics of the liposome membrane were investigated by measuring the rotational diffusion of eosin-5-isothiocyanate(EITC)-labeled L-alpha-dipalmitoylglycerophosphoethanolamine (Pam2 GroPEtn) in the lipid phase of the ... More
Lateral mobility of band 3 in the human erythrocyte membrane studied by fluorescence photobleaching recovery: evidence for control by cytoskeletal interactions.
AuthorsGolan DE, Veatch W
JournalProc Natl Acad Sci U S A
PubMed ID6930650
'Band 3, the major intrinsic protein of the human erythrocyte membrane, was specifically labeled with the covalent fluorescent probe eosin isothiocyanate. The lateral mobility of labeled band 3 in the plane of the membrane under various conditions of ionic strength and temperature was examined by using the fluorescence photobleaching recovery ... More
Total internal reflection fluorescence study of energy transfer in surface-adsorbed and dissolved bovine serum albumin.
AuthorsBurghardt TP, Axelrod D
JournalBiochemistry
PubMed ID6838837
'A simple adaptation of a commercial spectrofluorometer allows selective excitation of fluorescent biomolecules adsorbed to a solid surface while they are in equilibrium with a bulk solution. As a demonstration of this technique, we have detected a change in the effective singlet-singlet energy transfer in fluorescence-labeled bovine serum albumin (BSA) ... More
Structural mapping of Fc receptor bound immunoglobulin E: proximity to the membrane surface of the antibody combining site and another site in the Fab segments.
AuthorsBaird B, Holowka D
JournalBiochemistry
PubMed ID4084517
'Resonance energy-transfer methods have been used to investigate the structure of immunoglobulin E (IgE) bound to its high-affinity receptor on plasma membrane vesicles derived from rat basophilic leukemia cells. The structural mapping of receptor-bound IgE was initiated in an earlier study [Holowka, D., & Baird, B. (1983) Biochemistry 22, 3475], ... More
Distribution and mobility of murine histocompatibility H-2Kk antigen in the cytoplasmic membrane.
'The topographical distributions and mobilities of the murine histocompatibility antigen H-2Kk and of concanavalin A (Con A) binding sites have been studied on a murine lymphoma cell line. The spatial distribution of H-2Kk antigens, the average distance between H-2Kk antigens and Con A binding sites, and the separation of different ... More
Coupling factor for photophosphorylation labeled with eosin isothiocyanate: activity, size, and shape in solution.
AuthorsWagner R, Junge W
JournalBiochemistry
PubMed ID6177341
Characteristics of anion transport channels in the human erythrocyte. I. Interactions between eosin 5-isothiocyanate and band 3 proteins.
AuthorsSato Y, Chiba T, Suzuki Y
JournalChem Pharm Bull (Tokyo)
PubMed ID2418991
Protein mobility in membranes.
AuthorsCherry RJ
JournalFEBS Lett
PubMed ID237788
Rotational diffusion of band 3 proteins in the human erythrocyte membrane.
AuthorsCherry RJ, Bürkli A, Busslinger M, Schneider G, Parish GR
JournalNature
PubMed ID972675
Photohemolysis of human erythrocytes labeled in band 3 with eosin-isothiocyanate.
AuthorsPooler JP, Girotti AW
JournalPhotochem Photobiol
PubMed ID2431425
Depolarization of fluorescence depletion. A microscopic method for measuring rotational diffusion of membrane proteins on the surface of a single cell.
AuthorsJohnson P, Garland PB
JournalFEBS Lett
PubMed ID6170530
Fluorescence microphotolysis. Diffusion measurements in single cells.
AuthorsPeters R
JournalNaturwissenschaften
PubMed ID6877390
Surface density determination in membranes by fluorescence energy transfer.
AuthorsFung BK, Stryer L
JournalBiochemistry
PubMed ID728398
Rotational and lateral diffusion of membrane proteins.
AuthorsCherry RJ
JournalBiochim Biophys Acta
PubMed ID391281
The reaction of fluorescein isothiocyanate with thiols: a method for assay of isothiocyanates.
AuthorsWilderspin AF, Green NM
JournalAnal Biochem
PubMed ID6414335
The reversible formation of dithiourethanes from fluorescein isothiocyanate and mercaptoethanol or mercaptoethylamine has been studied. It is shown that it is possible to use the reaction as the basis for a spectrophotometric titration of a number of isothiocyanates with mercaptoethanol. ... More
A spectroscopic technique for measuring slow rotational diffusion of macromolecules. 1: Preparation and properties of a triplet probe.
AuthorsCherry RJ, Cogoli A, Oppliger M, Schneider G, Semenza G
JournalBiochemistry
PubMed ID986163
Slow rotational diffusion may be investigated by measuring the decay of dichroism of flash-induced transient absorption changes of suitable probes. The preparation of the covalent ""triplets" probe eosin isothiocyanate is described together with investigations of spectroscopic properties of eosin-protein conjugates. Triplet state lifetimes of air-equilibrated solutions of eosin-protein conjugates are ... More
Measurement of the rotational behaviour of virus particles during adsorption to the host cell surface.
AuthorsBayer ME, Morrison I, Cherry R
JournalFEBS Lett
PubMed ID6479348
The rotational diffusion of bacteriophage epsilon 15 was measured before and after virus adsorption to outer membrane vesicles of the host Salmonella anatum. The virus capsid was labeled with eosin isothiocyanate, and the decay of transient dichroism following dye excitation by pulses of plane-polarized light was measured. From the data, ... 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
Synthesis, spectral properties and enzymatic hydrolysis of fluorescent derivatives of cerebroside sulfate containing long-wavelength-emission probes.
AuthorsMarchesini S, Preti A, Aleo MF, Casella A, Dagan A, Gatt S
JournalChem Phys Lipids
PubMed ID1970953
Fluorescent derivatives of cerebroside sulfate (sulfogalactosyl ceramide, sulfatide) containing long-wavelength-emission fluorophores were synthesized. For this purpose a procedure was developed for preparing a cerebroside 3-sulfate derivative with an amino group on the terminal carbon atom of its fatty acyl residue. The latter compound has been used to prepare cerebroside 3-sulfate, ... More
Surface diffusion of interacting proteins. Effect of concentration on the lateral mobility of adsorbed bovine serum albumin.
AuthorsTilton RD, Gast AP, Robertson CR
JournalBiophys J
PubMed ID2291948
Surface diffusion of bovine serum albumin absorbed from aqueous solution to poly(methylmethacrylate) surfaces is significantly hindered by protein-protein lateral interactions. The long-time self diffusion coefficient measured by fluorescence recovery after pattern photobleaching decreases by approximately one order of magnitude as the surface area fraction occupied by protein increases from 0.10 ... More
Domain structural flexibility in rhodanese examined by quenching of a phosphorescent probe.
AuthorsKoloczek H, Vanderkooi JM
JournalBiochim Biophys Acta
PubMed ID2445385
Rhodanese (thiosulfate:cyanide sulfurtransferase, EC 2.8.1.1) is an enzyme composed of two domains with the catalytic site located in the bottom of the crevice formed by the two domains. In this work, rhodanese was labeled at its catalytic site with the phosphorescence probe eosin isothiocyanide. The accessibility of molecules to the ... More
Labeling the (Ca(2+)-Mg2+)-ATPase of sarcoplasmic reticulum at Glu-439 with 5-(bromomethyl)fluorescein.
AuthorsStefanova HI, Mata AM, Gore MG, East JM, Lee AG
JournalBiochemistry
PubMed ID8099497
The (Ca(2+)-Mg2+)-ATPase of skeletal muscle sarcoplasmic reticulum was labeled with 5-(bromomethyl)fluorescein. A stoichiometry of one label per ATPase molecule was found, which was unaffected by the presence of ATP. Labeling resulted in a 60% decrease in ATPase activity. Sequencing identified the labeled residue as Glu-439. The fluorescence emission spectrum of ... More
Single-step process to reconstitute cell membranes on solid supports.
AuthorsMager MD, Melosh NA,
JournalLangmuir
PubMed ID20205459
A new technique is presented to create supported lipid bilayers from whole cell lipids without the use of detergent or solvent extraction. In a modification of the bubble collapse deposition (BCD) technique, an air bubble is created underwater and brought into contact with a population of cells. The high-energy air/water ... More
Rotational diffusion of cell surface components by time-resolved phosphorescence anisotropy.
AuthorsAustin RH, Chan SS, Jovin TM
JournalProc Natl Acad Sci U S A
PubMed ID230487
The rotational diffusion of concanavalin A receptors of viable Friend erythroleukemia cells and the band 3 anion transport system of human erythrocytes has been measured via the time-dependent phosphorescence emission intensity and anisotropy of triplet probes excited by a 5-ns laser pulse. High-quality phosphorescence decay curves with a 10-microseconds time ... More
Luteinizing hormone receptors are associated with non-receptor plasma membrane proteins on bovine luteal cell membranes.
Biophysical studies of the bovine luteinizing hormone (LH) receptor on luteal cell membranes suggest that this receptor may be part of a larger molecular weight structure. We have used 5-iodonaphthyl-1-azide (INA) to identify plasma membrane proteins near LH receptors on plasma membranes from bovine corpora lutea. Following binding of eosin ... More
Fluorescent triplet probes for measuring the rotational diffusion of membrane proteins.
AuthorsJohnson P, Garland PB
JournalBiochem J
PubMed ID7103944
We have previously described a method for measuring the rotational diffusion of membrane proteins by using fluorescent triplet probes [Johnson & Garland (1981) FEBS Lett. 135, 252-256]. We now describe the criteria by which the suitability of such probes may be judged. In general, the greatest sensitivity is achievable with ... More
Avidin-EITC: an alternative to avidin-FITC in confocal scanning laser microscopy.
Detection of fluorescein-5-isothiocyanate (FITC)-labeled conjugates is suboptimal in two-color confocal scanning laser microscopy (CLSM). This limits the detection of small, dimly fluorescent targets. We explored the possible advantages of applying eosin-5-isothiocyanate (EITC) conjugated to avidin (Av-EITC) as an alternative for Av-FITC in CSLM. Despite the lower quantum efficiency of EITC, ... More
Rotational motion and evidence for oligomeric structures of sarcoplasmic reticulum Ca2+-activated ATPase.
AuthorsHoffmann W, Sarzala MG, Chapman D
JournalProc Natl Acad Sci U S A
PubMed ID158763
The rotational motion of the sarcoplasmic reticulum Ca2+-activated ATPase (ATP phosphohydrolase, EC 3.6.1.3) has been investigated by measuring the decay of laser flash-induced dichroism with the covalently attached triplet probe eosin isothiocyanate. The Arrhenius plot for rotational mobility indicates two discontinuities at approximately 15 degrees C and approximately 35 degrees ... More
Major histocompatibility complex independent T cell receptor-antigen interaction: functional analysis using fluorescein derivatives.
We have isolated T cell receptor (TCR) cDNAs from fluorescein (FL)-specific human T cell clones (alpha FL beta FL), and transferred them to TCR beta- Jurkat cells in order to study direct FL-binding to the TCR. Using either FL-conjugated polymers (FL-polymer) or FL-substituted Sepharose beads, we are able to demonstrate ... More
A spectroscopic technique for measuring slow rotational diffusion of macromolecules. 2: Determination of rotational correlation times of proteins in solution.
AuthorsCherry RJ, Schneider G
JournalBiochemistry
PubMed ID986164
Experiments have been performed to test the validity of measurements of slow rotational diffusion using eosin isothiocyanate as a probe. When eosin-protein conjugates are covalently bound to Sepharose 4B, the dichroism of the flash-induced absorption transient does not decay with time. The magnitude of the dichroism is smaller than for ... More
Xanthene-dye-labelled phosphatidylethanolamines as probes of interfacial pH. Studies in phospholipid vesicles.
AuthorsKnight CG, Stephens T
JournalBiochem J
PubMed ID2471509
We have been developing the use of plasma-membrane-bound fluorescent probes to measure the pH values at the surfaces of living chondrocytes. For this purpose, three lipophilic pH indicators were made by covalently binding the xanthene dyes fluorescein, eosin or dichlorofluorescein to the amino group of phosphatidylethanolamine. The probes were incorporated ... More
Fluorescence photooxidation with eosin: a method for high resolution immunolocalization and in situ hybridization detection for light and electron microscopy.
A simple method is described for high-resolution light and electron microscopic immunolocalization of proteins in cells and tissues by immunofluorescence and subsequent photooxidation of diaminobenzidine tetrahydrochloride into an insoluble osmiophilic polymer. By using eosin as the fluorescent marker, a substantial improvement in sensitivity is achieved in the photooxidation process over ... 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
Studies on the mechanism of polyethylene glycol-mediated cell fusion using fluorescent membrane and cytoplasmic probes.
AuthorsWojcieszyn JW, Schlegel RA, Lumley-Sapanski K, Jacobson KA
JournalJ Cell Biol
PubMed ID6826645
The mechanism by which polyethylene glycol (PEG) mediates cell fusion has been studied by examining the movements of membrane lipids and proteins, as well as cytoplasmic markers, from erythrocytes to monolayers of cultured cells to which they have been fused. Fluorescence and freeze-fracture electron microscopy and fluorescence recovery after photobleaching ... More
Triplet-state detection of labeled proteins using fluorescence recovery spectroscopy.
AuthorsCorin AF, Blatt E, Jovin TM
JournalBiochemistry
PubMed ID2441744
The experimental procedures for detecting the triplet states of chromophores in solutions (cuvettes) by fluorescence recovery spectroscopy (FRS) are described in detail, together with applications in studies of protein structure and protein-cell interactions in the microsecond to millisecond time domain. The experimental configuration has been characterized by measuring the emission ... More
Inhibition of the red blood cell calcium pump by eosin and other fluorescein analogues.
AuthorsGatto C, Milanick MA
JournalAm J Physiol
PubMed ID7687411
This paper addresses the mechanism of inhibition of the plasma membrane Ca pump by fluorescein analogues and their isothiocyanate derivatives. Eosin (i.e., tetrabromofluorescein) was found to be one of the most potent reversible inhibitors of the erythrocyte Ca pump [half-maximal inhibitory concentration (IC50) < 0.2 microM]; fluorescein itself was about ... More
Rotational molecular dynamics of the membrane-bound acetylcholine receptor revealed by phosphorescence spectroscopy.
AuthorsBartholdi M, Barrantes FJ, Jovin TM
JournalEur J Biochem
PubMed ID6172277
The rotational mobility of the membrane-bound acetylcholine receptor from Torpedo marmorata has been studied by phosphorescence anisotropy techniques using eosin-5'-isothiocyanate and eosin-5'-iodoacetamide derivatives of the alpha-neurotoxin of Bungarus multicinctus (alpha-bungarotoxin). Normal membranes, those depleted of nonreceptor peripheral peptides by alkaline treatment, as well as membranes subjected to various chemical modifications ... More
Characterization of redox state and reductase activity of protein disulfide isomerase under different redox environments using a sensitive fluorescent assay.
AuthorsRaturi A, Mutus B
JournalFree Radic Biol Med
PubMed ID17561094
In this study, dieosin glutathione disulfide (Di-E-GSSG) was synthesized by the reaction of eosin isothiocyanate with GSSG. Di-E-GSSG had low fluorescence which increased approximately 70-fold on reduction of its disulfide bond. The substrate was used to monitor the disulfide reductase activity of PDI. Di-E-GSSG is the most sensitive pseudo substrate ... More
In vivo analysis of the stability and transport of nuclear poly(A)+ RNA.
We have studied the distribution of poly(A)+ RNA in the mammalian cell nucleus and its transport through nuclear pores by fluorescence and electron microscopic in situ hybridization. Poly(A)+ RNA was detected in the nucleus as a speckled pattern which includes interchromatin granule clusters and perichromatin fibrils. When cells are fractionated ... 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
Requirement of ryanodine receptor subtypes 1 and 2 for Ca(2+)-induced Ca(2+) release in vascular myocytes.
AuthorsCoussin F, Macrez N, Morel JL, Mironneau J
JournalJ Biol Chem
PubMed ID10734110
While the roles of subtypes 1 and 2 of the ryanodine receptors (RYRs) have been studied in cellular systems expressing specifically one or the other of these subtypes (i.e. skeletal and cardiac muscle), the function of these receptors has not been evaluated in smooth muscles. We have previously reported RYR-mediated ... More
Use of resonance energy transfer to determine the proximity of the guanine nucleotide binding site of transducin relative to a conformationally-sensitive site on the gamma subunit of the cyclic GMP phosphodiesterase.
AuthorsErickson JW, Mittal R, Cerione RA
JournalBiochemistry
PubMed ID7542027
In this work, we have used resonance energy transfer to determine the relative positions of a reactive cysteine residue on the gamma subunit of the retinal cyclic GMP phosphodiesterase (gamma PDE) and a reactive lysine residue on the alpha subunit of transducin (alpha T). The single cysteine residue on gamma ... More
Resonance energy transfer as a direct monitor of GTP-binding protein-effector interactions: activated alpha-transducin binding to the cGMP phosphodiesterase in the bovine phototransduction cascade.
AuthorsErickson JW, Cerione RA
JournalBiochemistry
PubMed ID1713060
Resonance energy-transfer approaches have been used to directly monitor the interactions of the GTP gamma S-bound alpha subunit of transducin (alpha T GTP gamma S) with the retinal cyclic GMP phosphodiesterase (PDE). The PDE was labeled with 5-(iodoacetamido) fluorescein (IAF-PDE) and served as the fluorescence donor in these experiments while ... More
Fluorescence resonance energy transfer study of the associative state of membrane-bound complexes of complement proteins C5b-8.
AuthorsCheng KH, Wiedmer T, Sims PJ
JournalJ Immunol
PubMed ID3923109
Human complement protein C8 was labeled with the fluorescent chromophores fluorescein-5-isothiocyanate (FITC), 3-(4-isothiocyanatophenyl)-7-diethylamine-4-methyl coumarin (IPM), eosin-5-isothiocyanate (EOS), or Texas Red (sulforhodamine-101-sulfonyl chloride; TR) with only minor reduction in the specific hemolytic activity of the protein. The distribution of C5b-8 complexes bound to sheep erythrocyte membranes was investigated by monitoring fluorescence ... More
Aluminum fluoride activation of bovine transducin induces two distinct conformational changes in the alpha subunit.
AuthorsMittal R, Cerione RA, Erickson JW
JournalBiochemistry
PubMed ID7520280
We have used resonance energy transfer to read out the interactions of the alpha subunit of transducin (alpha T) with the transducin beta gamma subunit complex (beta gamma T) and to compare the rate of aluminum fluoride-induced alpha T activation, as reflected by the enhancement of the alpha T tryptophan ... More
Structural significance of the plasma membrane calcium pump oligomerization.
The oligomerization of the plasma membrane calcium pump (PMCA) in phospholipid/detergent micelles was evaluated using a combined spectroscopic and kinetic approach and related to the enzyme stability. Energy transfer between fluorescein-5'-isothiocyanate and eosin-5'-isothiocyanate attached to different PMCA molecules was used to determine the dissociation constant of dimeric PMCA (140 +/- ... More
Analysis of fluorescence energy transfer in duplex and branched DNA molecules.
AuthorsCooper JP, Hagerman PJ
JournalBiochemistry
PubMed ID2271593
Nonradiative fluorescence energy transfer (FET) is thought to be a highly sensitive measure of distance, occurring through a dipole coupling (Forster) mechanism in which the efficiency of FET depends on the inverse sixth power of the distance between fluorophores. The current work assesses the utility of FET for measuring distances ... More
Inter-active-site distance and solution dynamics of a bivalent-bispecific single-chain antibody molecule.
AuthorsMallender WD, Ferreira ST, Voss EW, Coelho-Sampaio T
JournalBiochemistry
PubMed ID8060979
The solution dynamics of a bivalent bispecific single-chain antibody (BiSCA) specific against fluorescein (Fl) and single-stranded DNA (ssDNA) were investigated. Fluorescence resonance energy transfer (FRET) studies were performed in order to estimate the average distances, R, between the anti-Fl and the anti-ssDNA active sites. In separate experiments, either 2-(dimethylamino)naphthalene-5-sulfonyl chloride ... More
Fluorescence polarization and energy-transfer studies on the pyruvate dehydrogenase complex of Escherichia coli.
AuthorsScouten WH, Visser AJ, Grande HJ, De Kok A, De Graaf-Hess AC, Veeger C
JournalEur J Biochem
PubMed ID6161006
We have attached eosin maleimide specifically to the lipoyl group of the pyruvate dehydrogenase complex isolated from Escherichia coli. Using this as the fluorescence acceptor and the intrinsic FAD of the lipoamide dehydrogenase subunit as the fluorescence donor, we confirmed previous measurements with other probes, in which it was suggested ... More
Erythrocyte Lii-Nao countertransport system. Inhibition by N-ethylmaleimide probes for a conformational change of the transport system.
AuthorsLevy R, Livne A
JournalBiochim Biophys Acta
PubMed ID6487623
Human erythrocytes were treated by a series of SH-reagents, including maleimides, iodo compounds, mercurials and oxidizing agents. Rates of Li efflux into Na-rich medium, Li leak and Lii-Nao countertransport were then determined. Of the 13 different reagents studied, only N-ethylmaleimide, iodoacetamide and iodoacetate inhibited selectively the countertransport activity. The effect ... More
Potentiation of hyperthermia-induced haemolysis of human erythrocytes by photodynamic treatment. Evidence for the involvement of the anion transporter in this synergistic interaction.
AuthorsPrinsze C, Tijssen K, Dubbelman TM, Van Steveninck J
JournalBiochem J
PubMed ID1713033
Heat treatment of human erythrocytes led to increased passive cation permeability, followed by haemolysis. K+ leakage was linear up to a loss of about 80% in the temperature range 46-54 degrees C. Kinetic analysis of the results revealed an activation energy of 246 kJ/mol, implicating a transition in the membrane ... More
Induced circular dichroism of eosin-5-maleimide bound to band 3 of human erythrocytes.
AuthorsChiba T, Sato Y, Suzuki Y
JournalBiochim Biophys Acta
PubMed ID1694686
The inhibition of anion exchange in human erythrocyte membrane by eosin-5-maleimide (EMI) was examined at various pH values. At the pH region between pH 6.0 and 8.0, EMI inhibited the sulfate efflux by about 90%. Further, the interaction of EMI molecules with erythrocyte ghosts was studied by induced circular dichroism ... More
Real time conformational changes in the retinal phosphodiesterase gamma subunit monitored by resonance energy transfer.
AuthorsBerger AL, Cerione RA, Erickson JW
JournalJ Biol Chem
PubMed ID9006909
The gamma subunit of the retinal cGMP phosphodiesterase (gammaPDE) acts as an inhibitor of phosphodiesterase (PDE) catalytic activity and mediates enzyme regulation by the alpha subunit of the GTP-binding protein transducin (alphaT). In order to characterize conformational changes in the 87-amino acid gammaPDE subunit that may accompany the activation of ... More
Proximity between fluorescent probes attached to four essential lysyl residues in phosphoenolpyruvate carboxylase. A resonance energy transfer study.
Phosphoenolpyruvate carboxylase, purified from maize leaves, is rapidly inactivated by the fluorescence probe dansyl chloride. The loss of activity can be ascribed to the covalent modification of an R-NH2 group, presumably the epsilon-NH2 group of lysine. Analysis of the data by the statistical method of Tsou [Sci. Sin. 11, 1535-1558 ... More
Microsecond rotational motions of eosin-labeled myosin measured by time-resolved anisotropy of absorption and phosphorescence.
AuthorsEads TM, Thomas DD, Austin RH
JournalJ Mol Biol
PubMed ID6209402
We have studied submicrosecond and microsecond rotational motions within the contractile protein myosin by observing the time-resolved anisotropy of both absorption and emission from the long-lived triplet state of eosin-5-iodoacetamide covalently bound to a specific site on the myosin head. These results, reporting anisotropy data up to 50 microseconds after ... More
Correlation of rotational mobility and flexibility of Sendai virus spike glycoproteins with fusion activity.
AuthorsLee PM, Cherry RJ, Bächi T
JournalVirology
PubMed ID6192588
The rotational mobility of Sendai virus glycoprotein spikes was measured by flash-induced transient dichroism of eosin triplet probes. The possible importance of this molecular motion for function was investigated by parallel assays of hemagglutination and fusion with erythrocytes. For mobility measurements, the glycoproteins were labeled on amino groups with eosin-5-isothiocyanate ... More
Three-dimensional structure in solution of the calcium channel blocker omega-conotoxin MVIIA.
AuthorsKohno T, Kim JI, Kobayashi K, Kodera Y, Maeda T, Sato K
JournalBiochemistry
PubMed ID7640281
The three-dimensional solution structure of omega-conotoxin MVIIA, a 25-mer peptide antagonist of N-type calcium channels, was determined by two-dimensional 1H NMR spectroscopy with simulated annealing calculations. A total of 13 converged structures of omega-conotoxin MVIIA were obtained on the basis of 273 experimental constraints, including 232 distance constraints obtained from ... More
Rotational diffusion of Escherichia coli RNA polymerase free and bound to deoxyribonucleic acid in nonspecific complexes.
AuthorsAustin RH, Karohl J, Jovin TM
JournalBiochemistry
PubMed ID6349679
We have studied the rotational diffusion of Escherichia coli RNA polymerase free in solution and bound nonspecifically to DNA fragments. The rotational motion was measured by the decay in anisotropy of the triplet-triplet absorption by using as probes either the liganded enzyme inhibitor Rose Bengal or eosin 5'-isothiocyanate conjugated to ... More
Class I major histocompatibility complex antigens are not associated with the LH/CG receptor on ovine luteal cells.
AuthorsKenny N, Rahman NA, Barisas BG, Roess DA
JournalBiochim Biophys Acta
PubMed ID8268250
We have examined the rotational dynamics of the luteinizing hormone (LH) receptor on day 10 intact ovine small luteal cells and isolated plasma membranes using polarized fluorescence depletion (PFD). This technique measures rotational correlation times which are proportional to the in-membrane volume of a protein and are useful for examining ... More
Measurement of protein rotational motion using frequency domain polarized fluorescence depletion.
AuthorsYoshida TM, Zarrin F, Barisas BG
JournalBiophys J
PubMed ID3207826
Polarized fluorescence depletion (PFD) methods (Yoshida, T. M. and B. G. Barisas. Biophys. J. 1986. 50:41-53) are approximately 10(3)-10(4) fold more sensitive than other techniques for measuring protein rotational motions in cell membranes and other viscous environments. Proteins labeled with fluorophores having a high quantum yield for triplet formation are ... More
Determining the extent of labeling for tetramethylrhodamine protein conjugates.
AuthorsMeadows DL, Shafer JS, Schultz JS
JournalJ Immunol Methods
PubMed ID1719100
A new, relatively simple, spectrophotometric technique has been developed which is useful for accurately determining the extent of chromophore labeling of proteins. Often the absorbance spectra and extinction coefficients of dye/protein conjugates are strongly affected by changes in the chromophore microenvironment that may occur at high dye/protein ratios. In the ... More
Rotational dynamics of type I Fc epsilon receptors on individually-selected rat mast cells studied by polarized fluorescence depletion.
AuthorsRahman NA, Pecht I, Roess DA, Barisas BG
JournalBiophys J
PubMed ID1547323
We report the first application of polarized fluorescence depletion (PFD), a technique which combines the sensitivity of fluorescence detection with the long lifetimes of triplet probes, to the measurement of membrane protein rotational diffusion on individually selected, intact mammalian cells. We have examined the rotation of type I Fc epsilon ... More