Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “ETHYLMALEIMIDE”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 73 records · Page 4Linked to original sources
PubMed1988

Purification and properties of a vanadate- and N-ethylmaleimide-sensitive ATPase from chromaffin granule membranes.

A vanadate- and N-ethylmaleimide-sensitive ATPase was purified about 500-fold from chromaffin granule membranes. The purified preparation contained a single major polypeptide with an apparent molecular mass of about 115 kDa, which was copurified with the ATPase activity. Immunological studies revealed that this polypeptide has no relation to subunit I (115 kDa) of the H+-ATPase from chromaffin granules. The ATPase activity of the enzyme is inhibited about 50% by 100 microM N-ethylmaleimide or 5 microM vanadate. The enzyme is not sensitive to dicyclohexylcarbodiimide, ouabain, SCH28080, and omeprazole, which distinguishes it from Na+/K+-ATPase and the gastric K+/H+-ATPase. ATP and 2-deoxy ATP are equally effective substrates for the enzyme. However, the enzyme exhibited only 10% activity with GTP as a substrate. UV illumination of the purified enzyme in the presence of [alpha-32P]ATP exclusively labeled the 115 kDa protein. This labeling was increased by Mg2+ and strongly inhibited by Ca2+ ions. Similarly, the ATPase activity was dependent on Mg2+ and inhibited by the presence of Ca2+ ions. The ATPase activity of the enzyme was largely insensitive to monovalent anions and cations, except for F-, which inhibited the vanadate-sensitive ATPase. Incubation of the enzyme in the presence of [14C]N-ethylmaleimide labeled the 115-kDa polypeptide, and this labeling could be prevented by the addition of ATP during the incubation. A reciprocal experiment showed that preincubation with N-ethylmaleimide inhibited the labeling of the 115-kDa polypeptide by [alpha-32P]ATP by UV illumination. This suggests a close proximity between the ATP-binding site and an essential sulfhydryl group. A possible connection between the isolated ATPase and organelle movement is discussed.

Adenosine Triphosphatases↗
PubMed1985

Sequence of the N-terminal formic acid fragment and location of the N-ethylmaleimide-binding site of the phosphate transport protein from beef heart mitochondria.

The N-terminal formic acid fragment (FA1) of the N-[3H]ethylmaleimide-labeled and carboxymethylated bovine mitochondrial phosphate transport protein (PTPN*CM) has been purified and completely sequenced: NH2-Ala-Val-Glu-Glu-Gln-Tyr-Ser-Cys-Asp-Tyr10-Gly-Ser-Gly-Arg-Phe- Phe-Ile-Leu-Cys- Gly20-Leu-Gly-Gly-Ile-Ile-Ser-Cys-Gly-Thr-Thr30-His-Thr -Ala-Leu-Val-Pro-Leu-Asp- -Leu-Val40-Lys-Cys(N-[3H]ethylmaleimide)-Arg-Met-Gln-Val-Asp- COOH. By thermolysin digestion of FA1 and high-performance liquid chromatography isolation of the radioactive subfragment Leu39-Arg43, the sole N-ethylmaleimide-binding residue has been identified as Cys42. FA1 contains a high mole percentage of cysteine (8.5%) and shows silver staining anomaly. Its sequence reveals significant homology in the triplicated gene regions (Pro27,132,229) of the mitochondrial ADP/ATP carrier from beef heart and Neurospora crassa. The hydropathic profile suggests that FA1 contains a transmembrane segment (Phe15-Val40) with only one basic (His31) and one acidic (Asp38) residue. The presence of the phosphate transport protein gene among nuclear genes is suggested from a lack of significant homology between the reverse-translated FA1 (mitochondrial codons) and the bovine mitochondrial genome. The inhibitory action of N-ethylmaleimide on the phosphate transport mechanism is discussed.

Amino Acid Sequence↗
PubMed1981

The N-ethylmaleimide-sensitive cysteine residue in the pH-dependent subunit interactions of malate dehydrogenase.

The specific chemical modification by N-ethylmaleimide of a cysteine residue at pH 5.0 in porcine heart mitochondrial malate dehydrogenase (L-malate:NAD+ oxidoreductase, EC 1.1.1.37) has been shown to result in an enzymatically inactive, monomeric product, which does not reassociate at pH 7.5 to yield the native dimer. In this report, an investigation of proton release and uptake upon NADH binding to the native enzyme and to the N-ethylmaleimide-modified enzyme has implicated the above cysteine residue as being directly linked to the pH-dependent subunit dissociation of mitochondrial malate dehydrogenase. The results are consistent with the view that the modified cysteine residue is not located at the subunit interaction site, although it is probably near this site. A recent study from this laboratory has demonstrated that the monomeric enzyme obtained at pH 5.0 exists in a conformation which is enzymatically inactive and which has an enhanced intrinsic protein fluorescence. Interpretation of protein fluorescence data has suggested that the N-ethylmaleimide modification results in inactivation of the enzyme by preventing the pH-induced conformational change to the active dimer. However, NADH is able to induce reassociation of the N-ethylmaleimide-modified enzyme at pH 7.5 but not at pH 5.0. This reassociation at pH 7.5 is accompanied by a significant regain of enzymatic activity, indicating that NADH binding is able to partially overcome the negative effect of the cysteine modification on the pH-dependent subunit reassociation of mitochondrial malate dehydrogenase.

Animals↗
PubMed1976

Selective chemical modification of Escherichia coli elongation factor G. N-Ethylmaleimide modification of a cysteine essential for nucleotide binding.

Escherichia coli Elongation Factor G is inhibited ireversibly by the chemical modification of 1 cysteine residue with N-ethylmaleimide. At pH 5.2, this cysteine is approximately 130 times more reactive than beta-mercaptoethanol toward N-ethylmaleimide. Inhibition is not prevented by either the ribosome or GTP alone at concentrations approximately equal to that of Elongation Factor G, but in combination they reduce the inhibition by 50%. Increasing the stability of the Elongation Factor G-ribosome-GDP complex by the addition of fusidec acid, completely protects against N-ethylmaleimide inhibition. The modified protein cannot form either the Elongation Factor G-ribosome-GMP-P(CH2)P or the Elongation Factor G-ribosome-GDP-fusidic acidcomplex. However, the modification had no effect on its ability to form the Elongation Factor G-ribosome complex. These results suggest that the cysteine residue modified by N-ethylmaleimide is at or near the nucleotide binding site.

Binding Sites↗
PubMed1987

Inhibition and Labeling of the Plant Plasma Membrane H-ATPase with N-Ethylmaleimide.

H(+)-ATPase activity in plasma membranes isolated from Avena sativa root cells is inhibited by N-ethylmaleimide, a covalent modifier of protein sulfhydryl groups. The rate of inhibition is reduced by ADP, MgADP, and MgATP, but even at 40 millimolar ADP the enzyme is only partially protected against inactivation. When plasma membranes are treated wth N-[2-(3)H]ethylmaleimide and analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis, prominent radioactive bands appear at M(r)=100,000 and several other positions. However, only radioactivity in the M(r)=100,000 protein is reduced by the presence of MgADP. These results provide independent evidence that the M(r)=100,000 polypeptide which is observed in purified preparations of the enzyme is the catalytic subunit of the H(+)-ATPase. When tryptic peptides are produced from N-[2-(3)H]ethylmaleimide labeled M(r)=100,000 protein and separated by reverse phase high performance liquid chromatography, two radioactive peaks are observed for which N-[2-(3)H]ethylmaleimide incorporation is reduced in the presence of MgADP.

Journal Article↗
PubMed2004

N-ethylmaleimide-sensitive factor is required for the synaptic incorporation and removal of AMPA receptors during cerebellar long-term depression.

Cerebellar long-term depression (LTD) is a persistent attenuation of synaptic transmission at the parallel fiber-Purkinje cell synapse mediated by the removal of GluR2 subunit-containing alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors. The removal of AMPA receptors requires protein kinase C phosphorylation of the GluR2 subunit within its carboxyl-terminal PSD-95/Discs Large/Zona Occludens-1 (PDZ) ligand and binding of the PDZ domain-containing protein, PICK1. The sequence of the GluR2 subunit is similar to that of the GluR3 and GluR4c subunits, which also contain PDZ ligands and protein kinase C consensus sites. Although GluR3 and GluR4c are also expressed in Purkinje cells, we have previously shown that cerebellar LTD is absent in GluR2(-/-) mice, suggesting that these subunits are unable to substitute functionally for GluR2. Here, we examine the apparent difference in the regulation of these AMPA receptor subunits by attempting to rescue LTD in GluR2(-/-) Purkinje cells with WT and mutant GluR2 and GluR3 subunits. Our results show that the selective interaction of the GluR2 subunit with the N-ethylmaleimide-sensitive factor protein is required for synaptic, but not extrasynaptic, incorporation of AMPA receptors as well as for their competence to undergo LTD. In addition, perfusion of a synthetic peptide that acutely disrupts the interaction of GluR2 with N-ethylmaleimide-sensitive factor selectively depletes GluR2-containing receptors from synapses and occludes LTD. These findings demonstrate that interaction of AMPA receptors with N-ethylmaleimide-sensitive factor plays a critical role in incorporation of AMPA receptors into synapses and for their subsequent removal during cerebellar LTD.

Amino Acid Sequence↗
PubMed1998

A highly active microsomal glutathione transferase from frog (Xenopus laevis) liver that is not activated by N-ethylmaleimide.

Microsomal glutathione transferase has hitherto only been purified from mammalian species. N-ethylmaleimide and trypsin activation (discriminating features of this enzyme) has only been observed in microsomes from mammals. In this paper we describe the first isolation and characterization of a non-mammalian microsomal glutathione transferase from frog (Xenopus laevis) liver. This protein has a molecular weight similar to that of the mammalian enzyme (approximately 17 kDa), but cannot be activated by N-ethylmaleimide or trypsin. In fact the enzyme is rapidly inactivated by this sulfhydryl reagent and protease. It thus appears that N-ethylmaleimide activation is not an obligatory property of microsomal glutathione transferase. The frog liver microsomal glutathione transferase has one of the highest specific activities towards the second substrate 1-chloro-2,4-dinitrobenzene (CDNB) (200 mumol/min mg) obtained with any glutathione transferase and accounts for the high activity found in frog liver microsomes. The kcat/K(m) for glutathione and CDNB are 0.017 and 1.1 x 10(6) M-1 s-1, respectively. The enzyme also functions as a glutathione peroxidase (dilinoleoyl phosphatidylcholine hydroperoxide is reduced (5.2 mumol/min mg)). It is now evident that a highly active microsomal glutathione transferase, with a molecular weight similar to that of the mammalian enzymes also exists in a non-mammal species.

Animals↗
PubMed1991

Apparent heterogeneity of cardiac A1 adenosine receptors as revealed by radioligand binding experiments on N-ethylmaleimide-treated membranes.

While G protein-coupled receptors are often studied by analyzing antagonist radioligand: "cold" agonist inhibition curves using an independent site model, it is now clear that KL and KH values determined in these analyses are not reliable estimates of the affinities of the agonists for "free" and G protein-coupled forms of the receptor. Thus, such experiments cannot be used to contrast the characteristics of a given type of receptor in different tissues, i.e., to probe for the existence of receptor subtypes. Since treatment with N-ethylmaleimide treatment blocks receptor: Gi/Go protein interactions, such analyses on N-ethylmaleimide-pretreated membranes should allow direct assessment of the affinities of competing ligands for the free receptor or for multiple receptor subtypes. As A1 adenosine receptors couple to Gi, and perhaps to Go, we have performed A1 adenosine receptor radioligand "competition" studies first on control, then on N-ethylmaleimide-pretreated bovine cardiac and cerebral cortical membranes. Results of experiments with the antagonist radioligand [3H]xanthine amine congener appeared to be confounded by ligand binding to A2 adenosine receptors present in the cardiac membrane preparations. Further experiments utilized the A1-specific radioligand [3H]1,3-dipropyl-8-cyclopentylxanthine. These experiments confirmed once more that the KL values determined by computer analysis of "competition" curves performed on control membranes are not reliable estimates of the affinities of the competing ligand for free receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine↗
PubMed2006

Mechanism of N-ethylmaleimide-induced contraction of the frog sartorius muscle.

We studied parameters of the frog sartorius muscle contraction initiated by ryanodine receptor agonists in the presence of ROS donors. We hypothesized that sodium nitroprusside and hydrogen peroxide inhibit initiation of contractions by N-ethylmaleimide and that this effect of ROS donors on parameters of N-ethylmaleimide-induced contractions is due to a direct effects of sodium nitroprusside and hydrogen peroxide on N-ethylmaleimide, but not to inactivation of ryanodine receptors in the sarcoplasmatic reticulum of frog skeletal muscle.

Animals↗
PubMed1981

Transport of methotrexate in L1210 cells. Mechanism for inhibition by p-chloromercuriphenylsulfonate and N-ethylmaleimide.

Methotrexate transport in L1210 cells is highly sensitive to inhibition by p-chloromercuriphenylsulfonate (CMPS) and, to a lesser extent, by N-ethylmaleimide. A 50% reduction in the methotrexate influx rate occurred upon exposure of cells to 3 microM CMPS or 175 microM N-ethylmaleimide, while complete inhibition was achieved at higher levels of these agents. Dithiothreitol reversed the inhibition by CMPS, suggesting that a sulfhydryl residue is involved. This residue is apparently not located at the substrate binding site of the transport protein, since methotrexate failed to protect the system from inactivation by either CMPS or N-ethylmaleimide, and the transport protein retained the ability to bind substrate (at 4 degrees C) after exposure to these inhibitors (at 37 degrees C). Methotrexate efflux was also inhibition by CMPS (50% at 4 microM), indicating that both the uptake and efflux of methotrexate in L1210 cells occur via the same transport system. High concentrations of CMPS (greater than 20 microM) increased the efflux rate, apparently by damaging the cell membrane and allowing the passive diffusion of methotrexate out of the cell.

4-Chloromercuribenzenesulfonate↗
PubMed1975

Characterization of N-ethylmaleimide-reactive proteins from human tonsillar ribosomes by two-dimensional polyacrylamide gel electrophoresis.

Human tonsillar 80-S ribosomes were 17% and 43% inactivated by 1 mM N-ethylmaleimide after 12 min at 30 or 37 degrees C, respectively. The ribosomes were unaffected by the reagent during the same period of time at 0 or 20 degrees C. 4, 12, 27 and 59 sulfhydryl groups per 80-S ribosomes were found labeled by 1 mM N-ethyl[14C] maleimide after 12 min at 0, 20, 30 or 37 degrees C, respectively. The analysis of radioactively labeled proteins by two-dimensional gel electrophoresis revealed the following: after 3 min at 37 degrees C only two 40-S proteins, S3 and S7, displayed a significant amount of label. After 12 min at 37 degrees C, there was a several-fold increase in the extent of radioactivity found in each of these proteins and, additionally, S1, S2, S4, S5, S15, S22 and S31 were also found among labeled 40-S proteins. S3 appeared to be the most N-ethylmaleimide-reactive 40S protein. After 3 min at 37 degrees C, L10, L17, L20 (and/or S20), L26, L32 and L33, and after 12 min at 37 degrees C, additionally L1, L2, L7, L9, L11, L15, L16, L18, and L25 were labeled among 60-S proteins. l17 and 32 were the most N-ethylmaleimide-reactive proteins under these conditions. After 12 min at 37 degrees C, approx. 26% and 39% of the radioactivity incorporated into the 80 S or 60 S ribosomal protein, respectively, was found in these two proteins. After 12 min at 0 degrees C, S3, L17, L32 and L33 were the only labeled proteins.

Electrophoresis, Polyacrylamide Gel↗
PubMed1981

Template-binding site of AMV reverse transcriptase and inactivation of the enzyme by N-ethylmaleimide.

N-Ethylmaleimide, a sulfhydryl-specific reagent, strongly inhibits AMV reverse transcriptase by specifically interfering with the template-binding site of the enzyme. However, the kinetics of inhibition differ widely with the composition and structure of the templates employed. The copying of templates with multiple 3'-hydroxyl termini appeared to be more susceptible to N-ethylmaleimide treatment, suggesting that the reagent may interfere with initiation of DNA synthesis. The ability of a template bound to enzyme prior to N-ethylmaleimide treatment to protect against inactivation of copying of other templates also, implies a common binding site for the different templates. Template exchange experiments demonstrated competition between activated calf thymus DNA and rAn . dT12--18 for binding to the enzyme. Thus, templates varying widely in composition and conformation appear to bind at a common site on reverse transcriptase. The experimental data also show suggestive evidence for small but finite differences in the requirements for optimal binding for templates of different structures.

Animals↗
PubMed1983

Inactivation of regulatory volume decrease in human peripheral blood lymphocytes by N-ethylmaleimide.

The sulfhydryl group reagent N-ethylmaleimide was found to inhibit in a dose dependent manner regulatory volume decrease of human peripheral lymphocytes swollen in buffered hyposmotic NaCl media. In hyposmotic KCl media NEM treated lymphocytes prevented an additional secondary swelling seen in control lymphocytes. The data suggest that N-ethylmaleimide acts on ion transport mechanisms involved in volume regulatory changes. This effect contrasts with the stimulation by N-ethylmaleimide of apparently volume sensitive K/Cl fluxes in certain mammalian red cells.

Adult↗
PubMed1982

Dual effect of N-ethylmaleimide on agonist-mediated conformational changes of beta-adrenergic receptors.

Beta-Adrenergic agonists cause adenylate cyclase activation via a conformational change of their receptor. This was earlier revealed by the use of N-ethylmaleimide as a structural probe: agonist-bound receptors are rapidly inactivated by 0.1 mM N-ethylmaleimide while free and antagonist-bound receptors remain unaffected. Furthermore, the beta-adrenergic receptors only change conformation when coupled to the guanine nucleotide regulatory component of the adenylate cyclase system. It is shown in this report that treatment of turkey erythrocyte membranes with the sulphydryl-specific agent 2,2'-dinitro-5,5'-dithiodibenzoic acid and elevated concentrations of N-ethylmaleimide (1-10 mM) do not affect the total receptor number, but prevent the ability of beta-adrenergic agonists to mediate conformational changes. This effect is mimicked by GTP. These three compounds cause also a two- to four-fold decrease in agonist affinity. Both phenomena may be explained by the ability of the reagents to prevent, and of GTP to reverse, the functional coupling between the receptors and the regulatory component of the adenylate cyclase system. Removal of Mg2+ from the incubation medium (i.e. presence of 1 mM EDTA) produces a similar decrease in agonist affinity, but does not impair the ability of agonist/N-ethylmaleimide to inactivate the receptors. This suggests that Mg2+ increases the agonist affinity for the receptor-regulatory component complex, but is not required for its formation.

Adrenergic beta-Agonists↗
PubMed1990

N-ethylmaleimide irreversibly inhibits the binding of [3H]threo-(+-)-methylphenidate to the stimulant recognition site.

N-Ethylmaleimide, a nonspecific protein modifier which reacts selectively with the sulfhydryl group of cysteinyl residues under controlled conditions, irreversibly inhibited the binding of [3H]threo-(+/-)-methylphenidate to a subset of stimulant binding sites in striatal tissue membranes from the rat in vitro. The inhibition was marked by a decrease in the Bmax of binding of the radiolabelled stimulant drug, while the KD remained unchanged. Pretreatment with excess unlabelled methylphenidate afforded complete protection from inactivation of the binding site by N-ethylmaleimide. Uptake of [3H]dopamine into striatal synaptosomes was likewise reduced after treatment with N-ethylmaleimide; pretreatment with large concentrations of methylphenidate provided partial protection from inactivation of transport. These findings suggest that the stimulant recognition site on the dopamine transport complex contains one or more cysteinyl residues.

Animals↗
PubMed1990

Opposing effects of N-ethylmaleimide on the affinity of carbachol for muscarinic cholinoceptors of guinea-pig atrium.

1. Inhibition of the binding of [3H]quinuclidinyl benzilate to homogenates of guinea pig right atrium (M2 receptors) by varying concentrations of carbachol was studied. 2. Pretreatment of membranes with 5 x 10(-5) M N-ethylmaleimide at 2 degrees C shifted the carbachol inhibition curve to the right, indicating decreased affinity of the receptor for carbachol. However pretreatment at 37 degrees C moved the curve to the left. 3. The ability of guanyl-5'-yl imidodiphosphate to reduce agonist affinity was largely eliminated by treatment with N-ethylmaleimide at both temperatures. 4. Conflicting reports in the literature and the present results can be explained by invoking a model in which N-ethylmaleimide has a high affinity for a heat-labile site and a lower affinity for a heat-insensitive site. Reaction with the first site decreases agonist affinity, but at 37 degrees C this site is largely inactivated and reaction with the second site, which leads to increased agonist affinity, predominates.

Animals↗
PubMed1992

Cocaine and dopamine differentially protect [3H]mazindol binding sites from alkylation by N-ethylmaleimide.

The binding of cocaine, d-amphetamine and dopamine to the site on the dopamine transporter labeled by [3H]mazindol was investigated in rat striatal membranes. N-Ethylmaleimide inhibited about 95% of the specific binding of 5 nM [3H]mazindol in a concentration-dependent manner. The effect of 10 mM N-ethylmaleimide was completely prevented by cocaine (EC50 of 3 microM), but neither 300 microM dopamine nor d-amphetamine afforded any significant protection. On the other hand, high concentrations of cocaine, d-amphetamine and dopamine provided similar protection against inhibition by 0.1 mM N-ethylmaleimide. Taken together these data support the hypothesis that a significant portion of the cocaine binding domain on the transporter is distinct from that of either dopamine or amphetamine. This distinction may be sufficient to allow properly designed drugs to prevent cocaine binding without inhibiting DA transport.

Alkylation↗
PubMed1983

Potentiation of monovalent cation effects on ligand binding to cardiac muscarinic receptors in N-ethylmaleimide treated membranes.

Guanine nucleotides and monovalent cations decrease the affinity of cardiac muscarinic receptors for agonists and are required for muscarinic receptor mediated inhibition of adenylate cyclase. N-ethylmaleimide abolished the effects of Gpp(NH)p on the ability of the agonist oxotremorine to inhibit the binding of the antagonist [3H]quinuclidinyl benzilate to purified chick heart membranes. However, the effects of NH4+ to decrease the IC50 for oxotremorine were retained in N-ethylmaleimide treated membranes. The N-ethylmaleimide treatment mimicked the effects of Gpp(NH)p and the oxotremorine inhibition curves obtained with treated membranes in the presence of NH4+ were identical to those obtained in control membranes in the presence of NH4+ and Gpp(NH)p. The results suggest that monovalent cation effects on muscarinic receptors are mediated at a site distinct from effects produced by guanine nucleotides and are greater on free receptors than on receptors coupled to guanine nucleotide binding proteins.

Ammonia↗