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PubMed1995

Stabilities of the fluorescent SH-reagent eosin-5-maleimide and its adducts with sulfhydryl compounds.

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 than NEM at neutral and moderately alkaline pH values. EMA formed a succinimide-type adduct with SH-compounds, and then underwent further modification by nucleophilic attack of OH- or an amino group. The succinimide-type adducts with acetylcysteine and glutathione were converted to open-type adducts, in which the succinimide ring was cleaved, whereas the adduct with cysteine was modified to a thiazine-type adduct. Kinetic analyses showed that these open-type and thiazine-type adducts were readily formed and were stable at moderately alkaline pH values such as pH 8.0 or 9.0.

Acetylcysteine↗
PubMed2004

Role of cysteine residues of p65/NF-kappaB on the inhibition by the sesquiterpene lactone parthenolide and N-ethyl maleimide, and on its transactivating potential.

Sesquiterpene lactones (SLs) are potent anti-inflammatory substances. It was previously shown that the anti-inflammatory effect could be partly explained by the inhibition of the transcription factor NF-kappaB. Whether they inhibit the DNA binding of NF-kappaB, the activation of the IkappaB-kinase, or both is still a matter of debate. The data supporting these hypotheses were obtained using different cell systems. In this contribution we analyzed the mechanism of the sesquiterpene lactone-mediated inhibition using different cell systems, and showed that in all the cell lines analyzed, SLs inhibited both NF-kappaB binding and the IkappaB-kinase, but that the former played a more preponderant role in the inhibition. These results again confirm the importance of cysteine 38 in the inhibition and regulation of NF-kappaB's function. Moreover, we compared the selectivity of the SL parthenolide with that of N-ethyl maleimide (NEM). We showed that NEM directly alkylated p65 as well as p50 of NF-kappaB, whereas SLs possess a selectivity towards p65. Finally, we studied the transactivating properties of various p65 mutants, to analyze the effect of exchanged cysteine residues in the DNA binding domain of NF-kappaB/p65 on its function and demonstrated that the transactivating potential of the mutants did not correlate with their DNA binding strenght.

Animals↗
PubMed1997

Influence of the protein kinase C inhibitor 3-[1-[3-(amidinothio) propyl]-1H-indoyl-3-yl]-3-(1-methyl-1H-indoyl-3-yl) maleimide methane sulfonate (Ro-318220) on surfactant secretion in type II pneumocytes.

We have investigated the influence of 3-[1-[3-(amidinothio)propyl]-1H-indoyl-3-yl]-3-(1-methyl-1H- indoyl-3-yl) maleimide methane sulfonate (Ro-318220), a potent and selective inhibitor of protein kinase C, on phosphatidylcholine secretion in response to surfactant secretagogues in rat type II cells. Freshly isolated cells were cultured overnight with [3H]choline to label the phosphatidylcholine pool and were preincubated for 30 min in fresh medium with or without Ro-318220. Secretagogues were then added, and the incubation was continued for 90 min after which [3H]phosphatidylcholine secretion was measured. Ro-318220 (10 microM) almost completely abolished the stimulatory effects of 36 microM terbutaline, 10 microM ATP, and 1 microM 12-O-tetradecanoylphorbol-13-acetate (TPA) and significantly antagonized the effects of 10 microM 5'-(N-ethylcarboxyamido) adenosine (NECA), 100 microM dioctanoylglycerol, and 0.05 microM ionomycin. The effect of Ro-318220 was dependent on concentration. The IC50 values for Ro-318220 inhibition of the effects of terbutaline, NECA, TPA, and ionomycin were not significantly different. The IC50 value for Ro-318220 inhibition of the effect of TPA in the type II cell (0.05 microM) was similar to that reported for inhibition of protein kinase C in vitro (0.08 microM). We conclude that Ro-318220 antagonizes the effects of the different surfactant secretagogues by antagonizing a step common to the different signaling pathways. It remains to be established if it is protein kinase C or another step that is inhibited.

Adenosine↗
PubMed1995

35Cl nuclear magnetic resonance line broadening shows that eosin-5-maleimide does not block the external anion access channel of band 3.

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 (NMR) to measure Cl- binding to the external transport site in control and EM-treated human red blood cells. Intact cells were used rather than ghosts, because in this case all line broadening (LB) results from binding to external sites. In an NMR spectrometer with a 9.4-T magnetic field, red blood cells at 50% concentration (v/v) in 150 mM Cl- medium at 3 degrees C caused 19.0 +/- 1.2 Hz LB. Of this, 7.9 +/- 0.7 Hz was due to Cl- binding to the high affinity band 3 transport sites, because it was prevented by an apparently competitive inhibitor of anion exchange, 4,4'-dinitrostilbene-2,2'-disulfonate (DNDS). The LB was not due to hemoglobin released from the cells, as little LB remained in the supernatant after cells were removed by centrifugation. Saturable Cl- binding remained in EM-treated cells, although the binding was no longer DNDS-sensitive, because EM prevents binding of DNDS. The lower limit for the rate at which Cl- goes from the binding site to the external medium is 2.15 x 10(5) s-1 for control cells and 1.10 x 10(5) s-1 for EM-treated cells, far higher than the Cl- translocation rate at 3 degrees C (about 400 s-1). Thus, EM does not inhibit Cl- exchange by blocking the external access channel. EM may therefore be useful for fixing band 3 in one conformation for studies of Cl- binding to the external transport site.

Anion Exchange Protein 1, Erythrocyte↗
PubMed1996

The orientation of eosin-5-maleimide on human erythrocyte band 3 measured by fluorescence polarization microscopy.

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 to study these dynamics are dependent on the orientation of the probe relative to the axis of motion. We have employed polarized fluorescence confocal microscopy to measure the orientation of eosin-5-maleimide covalently reacted with Lys-430 of human erythrocyte band 3. Steady-state polarized fluorescence images showed distinct intensity patterns, which were fit to an orientation distribution of the eosin absorption and emission dipoles relative to the membrane normal axis. This orientation was found to be unchanged by trypsin treatment, which cleaves band 3 between the integral membrane domain and the cytoskeleton-attached domain. this result suggests that phosphorescence anisotropy changes observed after trypsin treatment are due to a rotational constraint change rather than a reorientation of eosin. By coupling time-resolved prompt fluorescence anisotropy with confocal microscopy, we calculated the expected amplitudes of the e-Dt and e-4Dt terms from the uniaxial rotational diffusion model and found that the e-4Dt term should dominate the anisotropy decay. Delayed fluorescence and phosphorescence anisotropy decays of control and trypsin-treated band 3 in ghosts, analyzed as multiple uniaxially rotating populations using the amplitudes predicted by confocal microscopy, were consistent with three motional species with uniaxial correlation times ranging from 7 microseconds to 1.4 ms.

Anion Exchange Protein 1, Erythrocyte↗
PubMed1999

Evidence from studies with N-ethyl-maleimide and 12-O-tetradecanoylphorbol-13-acetate that AP-1 and CREB are involved in the glucocorticoid activation of TRH gene expression in hypothalamic cultures.

To determine whether c-fos/c-jun (AP-1) and CREB mediate glucocorticoid stimulated TRH gene regulation, we investigated the effect of N-ethyl-maleimide (NEM), an alkylating agent, and 12-O-tetradecanoylphorbol-13-acetate (TPA) on this process. NEM decreased while TPA increased TRH levels in rat hypothalamic culture, changes similar to their effects on CREB and Fos/Jun proteins in the AtT20 cell line. This suggests that glucocorticoid stimulation of TRH gene expression may be regulated by the AP-1 complex and CREB pathway.

Animals↗
PubMed2001

Determination of biological thiols by high-performance liquid chromatography following derivatization by ThioGlo maleimide reagents.

The importance of thiols has stimulated the development of a number of methods for determining glutathione and other biologically significant thiols. Methods that are currently available, however have some limitations, such as being time consuming and complex. In the present study, a new high-performance liquid chromatography (HPLC) method for determining biological thiols was developed by using 9-Acetoxy-2-(4-(2,5-dihydro-2,5-dioxo-1H-pyrrol-1-yl)phenyl)-3-oxo-3H-naphtho[2,1-b]pyran (ThioGlo3) as a derivatizing agent. ThioGlo reacts selectively and rapidly with the thiols to yield fluorescent adducts which can be detected fluorimetrically (lambda(ex) = 365 nm, lambda(em) = 445 nm). The within-run coefficient of variation for glutathione (GSH) by this method ranges from 1.08 to 2.94% whereas the between-run coefficient of variation for GSH is 4.31-8.61%. For GSH, the detection limit is around 50 fmol and the GSH derivatives remain stable for 1 month, if kept at 4 degrees C. Results for GSSG and cysteine are also included. The ThioGlo method is compared to our previous method in which N-(1-pyrenyl)maleimide (NPM) is used to derivatize thiol-containing compounds. The present method offers various advantages over the currently accepted techniques, including speed and sensitivity.

Animals↗
PubMed1995

Evidence that eosin-5-maleimide binds close to the anion transport site of human erythrocyte band 3: a fluorescence quenching study.

The interaction between eosin-5-maleimide (EMA), an inhibitor of the anion transport protein, band 3, and I-, a transportable substrate, was investigated by fluorescence quenching. The Stern-Volmer plot for the quenching reaction between EMA-labeled band 3 and I- exhibits downward curvature both in human erythrocyte ghosts and in purified band 3. The quenching reaction is insensitive to the viscosity of the bulk phase. The shape of the Stern-Volmer plot becomes more linear with increasing temperature. Following the approach of Blatt et al. [(1986) Biophys. J. 50, 349-356], we have developed a binding-diffusion model which is in good agreement with the quenching data. The model supposes that EMA is located in a compartment or "pocket" in band 3 which is separate from the bulk phase and contains a binding site or sites for the quencher. Quenching of band 3-bound EMA fluorescence by I- is inhibited by DIDS and by the transportable anions Cl-, HCO3-, and Br-. Analysis of these experiments yields dissociation constants for the anions which are in reasonable agreement with those determined from transport kinetics and by NMR. We thus deduce that the quencher binding site is the anion binding/transport site on band 3. We propose that EMA is located in the wall of the anion access channel such that it does not inhibit anion binding. The methods described in this report should facilitate detailed studies of anion binding to the transport site on band 3 under a variety of experimental conditions.

Anion Exchange Protein 1, Erythrocyte↗
PubMed1994

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.

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 the carrier, the labeling of Cys159 by EMA progressed predominantly and rapidly, and those of Cys56 and Cys256 moderately, but Cys128 was not labeled, as we reported previously [Majima, E., et al. (1993) J. Biol. Chem. 268, 22181-22187]. ADP inhibited the labelings of Cys56, Cys159, and Cys256 by EMA. BKA markedly inhibited the labeling of Cys159 by EMA, and also the labeling of Cys256, but did not affect the labeling of Cys56, suggesting that it binds from the matrix side to a region close to Cys159 in the second loop facing the matrix space. CATR completely inhibited the labeling by EMA when added on the cytosolic side, but had no effect when added on the matrix side. From these results, the conformational changes of the carrier induced by CATR, BKA, and ADP are discussed. Furthermore, a mechanism of adenine nucleotide transport through the ADP/ATP carrier in association with change in its conformation is proposed.

Adenine Nucleotides↗
PubMed1990

Identification of the eosinyl-5-maleimide reaction site on the human erythrocyte anion-exchange protein: overlap with the reaction sites of other chemical probes.

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 the cytoplasmic domain by mild trypsin digestion of ghost membranes. Sequencing of the major eosin-labeled peptide obtained from HPLC purification of an extensive chymotrypsin digest of purified Mr 17K peptide allowed assignment of the covalent reaction site for EMA to lysine-430 of the human erythrocyte protein [Tanner et al. (1988) Biochem. J. 256, 703-712]. Hydropathy plots based upon the primary structure of the protein [Lux et al. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 9089-9093] suggest that this residue is in an extracellularly accessible loop connecting membrane-spanning segments 1 and 2 of native band 3 in the erythrocyte membrane. Inhibition of sequential labeling of intact erythrocytes by pairs of chemical probes including EMA, the anion transport inhibitor 4,4'-diisothiocyanodihydrostilbene-2,2'-disulfonate (H2-DIDS), and the reactively bifunctional spin-label bis(sulfo-N-succinimidyl) doxyl-2-spiro-5'-azelate (BSSDA) has also been investigated. Each of these reagents affinity labels band 3 when added separately to a suspension of intact human erythrocytes by formation of one or more stable covalent bonds. Prelabeling of intact erythrocytes with EMA reduced subsequent labeling of band 3 by H2-DIDS by approximately 95% and by BSSDA by 90%. Similarly, prelabeling with H2-DIDS reduced subsequent labeling of band 3 by EMA by over 90%, and BSSDA prelabeling reduced EMA labeling by approximately 95%. Therefore, though having widely divergent chemical structures and protein modification reactivities, each of these negatively charged reagents may be competing for reaction with spatially overlapping sites on band 3 which are accessible from the extracellular space.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1998

The eosin-5-maleimide binding site on human erythrocyte band 3: investigation of membrane sidedness and location of charged residues by triplet state quenching.

The triplet probe eosin-5-maleimide (EMA) is a specific inhibitor of anion transport mediated by the erythrocyte membrane protein, band 3. It was previously shown that the eosin moiety is located close to the anion binding site when EMA is covalently bound to band 3 [Pan, R.-j., and Cherry, R. J. (1995) Biochemistry 34, 4880-4888]. In the present study the electrostatic properties and membrane sidedness of the EMA binding site of band 3 were further investigated by triplet state quenching. A series of stable nitroxyl free radicals, which are characterized by different charges, and I- were used as the quenchers. Time-resolved laser spectroscopy was employed to measure the triplet lifetime of EMA. It was found that the quenching reaction between the quenchers and band 3-bound EMA follows a linear Stern-Volmer plot. The quenching rate constants (Kq) of the quenchers are in the order of NH3+-TEMPO (Kq = 6.34 x 10(6) M-1 s-1) > TEMPO-Choline+ (Kq = 2.18 x 10(6) M-1 s-1) > TEMPO (Kq = 1.13 x 10(6) M-1 s-1) > I- (Kq = 2.46 x 10(5) M-1 s-1) > pyrroline-COO- (Kq = 2.18 x 10(4) M-1 s-1). Experiments with resealed ghosts and inside-out vesicles revealed that negatively charged quenchers can only access the EMA binding site from the extracellular side of the membrane while the positively charged quenchers acted from the cytoplasmic side. The ionic strength dependence of the quenching rate constants and the effects of pH on the quenching reaction were also studied. For both TEMPO-Choline+ and I-, the Kq values decreased as the ionic strength increased, but quenching by TEMPO was independent of the ionic strength variation over the same range. It was also found that at lower pH, the I- quenching rate constant increases but the TEMPO-choline+ quenching rate constant decreases. In both cases, the dependence of quenching on pH exhibited an apparent pKa of about 6.5, which suggests the involvement of one or more histidine residues. This notion gained further support from the finding that modification of His residues of band 3 by DEPC reduced I- quenching at pH 6. On the basis of these results, it is proposed that eosin is located in the anion transport channel such that it is accessible from both sides of the membrane. Histidine residues, which have previously been proposed to lie in the anion channel, probably are located on either side of the eosin probe where they contribute to electrostatic interactions which determine the Kq values for the charged quenchers.

Ammonia↗
PubMed2006

Rhodium-catalyzed asymmetric construction of quaternary carbon stereocenters: ligand-dependent regiocontrol in the 1,4-addition to substituted maleimides.

A rhodium-catalyzed asymmetric 1,4-addition of arylboronic acids to substituted maleimides has been described. The regioselectivity in this reaction is controlled by the choice of ligand (dienes or bisphosphines), and 1,4-adducts with a quaternary stereocenter can be obtained with high regio- and enantioselectivity by the use of (R)-H8-binap.

Journal Article↗
PubMed2006

Ruthenium-catalyzed [2 + 2 + 1] cocyclization of isocyanates, alkynes, and CO enables the rapid synthesis of polysubstituted maleimides.

Intermolecular [2 + 2 + 1] cocyclization of isocyanates, alkynes, and CO (1 atm) proceeded smoothly in the presence of a catalytic amount of Ru3(CO)12 (3.3 mol %) in mesitylene at 130 degrees C for 3 approximately 42 h to give a variety of polysubstituted maleimides in excellent yields with high selectivity. The reaction may involve an azaruthenacyclopentenone intermediate derived from oxidative cyclization of an isocyanate and an alkyne on an active ruthenium species.

Journal Article↗
PubMed2005

An experimental and theoretical study of stereoselectivity of furan-maleic anhydride and furan-maleimide diels-alder reactions.

The stereoselectivity of the reaction of furan (1) with maleic anhydride (2) and maleimide (3) was studied experimentally and theoretically. Although the two reactions are highly similar with regard to their preference for endo and exo steroisomers, notable differences were experimentally observed and explained on the basis of calculated reaction-free energies and transition-state barriers. The experimental values of rate constants (k(1+2endo) = (1.75 +/- 0.48) x 10(-5); mol(-1) l s(-1); k(1+2exo) = (3.10 +/- 0.55) x 10(-5); mol(-1) l s(-1); k(1+3endo) = (1.93 +/- 0.082) x 10(-5); mol(-1) l s(-1), k(1+3exo) = (1.38 +/- 0.055) x 10(-5); mol(-1) l s(-1) all at 300 K) and the observed reaction course clearly confirm that neither of these reactions are prototypical examples of Diels-Alder [4 + 2] cycloadditions, whose dominant preference is for endo isomers. However, only by comparing their energetics-calculated at the CCSD(T) level of theory-with the analogous reactions involving cyclopentadiene (8) as a diene can these observations be understood. The low thermodynamic stability of furan [4 + 2] adducts opens retro-Diels-Alder reaction channels and overrules the very small kinetic preference (calculated and measured here) of initial formation for endo stereoisomers. On a macroscopic scale "an irregular"-thermodynamically more stable-exo stereoisomer was consequently observed as a dominant species.

Journal Article↗
PubMed2003

Diels-Alder reactions of fused pyran-2-ones with maleimides: efficient syntheses of benz[e]isoindoles and related systems.

[reaction: see text] The Diels-Alder reaction of some substituted 5,6,7,8-tetrahydro-2H-1-benzopyran-2-ones (1a-f) with N-substituted maleimides (2a-c) leading to fused isoindole derivatives (5a-n, 7) or, in a few cases, to bridged double cycloadducts (fused bicyclo[2.2.2]octene derivatives) (6a-f) is presented. When X = CO, the first efficient, substituent-driven aromatization of an intermediary-formed cycloadduct was observed, resulting in substituted benz[e]isoindoles (5a-k). The same type of aromatization can also be achieved in an unprecedented catalysis with Rh/C.

Journal Article↗
PubMed2006

Turrapubesins A and B, first examples of halogenated and maleimide-bearing limonoids in nature from Turraea pubescens.

[structure: see text] Two novel tetranortriterpenoids, turrapubesins A (1) and B (2), representing the first examples of halogenated and maleimide-bearing limonoids, were isolated from the twigs and leaves of Turraea pubescens. The structures of 1 and 2 were elucidated by extensive spectroscopic analysis. Their absolute configurations were determined by X-ray crystallography of 1 and by CD analysis of a dihydrogenated derivative of 2. Turrapubesin A (1) exhibited weak cytotoxicity against the P-388 tumor cell line.

Animals↗