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PubMed1983

Inhibition of polyoma virus middle T antigen-associated tyrosyl kinase activity by N-ethylmaleimide.

The middle T antigen (MT Ag) encoded by polyoma virus has an associated protein kinase activity which transfers a phosphoryl group from ATP or GTP to a tyrosine residue on MT Ag in immunoprecipitates formed between polyoma virus-infected or transformed cell extracts and serum from animals bearing polyoma-induced tumors. Incubation of such immunoprecipitates or polyoma-transformed cell extracts prior to immunoprecipitation with the sulfhydryl reagent, N-ethylmaleimide (NEM), resulted in a significant inhibition of MT Ag-associated kinase activity. Inactivation of this enzyme activity by NEM was found to be dependent upon the incubation pH, time of incubation, and NEM concentration. ATP, GTP, and ADP in the presence of Mg2+ were found to decrease the rate of NEM-mediated inactivation of MT Ag-associated kinase activity, while CTP and UTP did not detectably alter the rate of enzyme inhibition by NEM. These results suggest that the MT Ag-associated kinase possesses at least one NEM-sensitive sulfhydryl group essential for phosphotransferase activity which may be present at or near the enzyme catalytic site.

Adenine Nucleotides↗
PubMed1983

[Effect of N-ethylmaleimide, introduced in to the serous fluid, on the active transport of sodium through the skin of Rana esculenta].

An SH reactive agent, N-ethylmaleimide (NEM), if introduced in the serosal bath (10(-4) M), stimulates the short circuit current (SCC) across the frog skin. This effect is due to an increase of Na active transport, because is inhibited by ouabain (10(-4) M). A stimulatory action on Cl- or HCO3- secretion can be ruled out because the rise in SCC occurs also in the absence of Cl- or HCO3-. It is known that NEM inhibits the ADH action by forming covalent bounds with SH groups of ADH membrane receptors (1). Thus it is possible that this binding should mimic the ADH action on SCC. In order to test this hypothesis, we studied the effect of serosal NEM on SCC both in the absence and in the presence of I midazol (20 mM), an activator of the cyclic AMP phosphodiesterase. Imidazol treatment decreases the effect of NEM on SCC. Thus we conclude that NEM serosal treatment is able to mimic some ADH effects, by using the same ways of the ormone action.

Animals↗
PubMed1983

Muscarinic receptors in porcine caudate nucleus. II. Different effects of N-ethylmaleimide on [3H]cis-methyldioxolane binding to heat-labile (guanyl nucleotide-sensitive) sites and heat-stable (guanyl nucleotide-insensitive) sites.

Heat treatment of membranes from porcine caudate nucleus (50 degrees for 7 min) caused a marked decrease in [3H]cis-methyldioxolane [( 3H]CD) binding without affecting seriously the binding of [3H]3-quinuclidinyl benzilate [( 3H]QNB). Approximately 20% of the [3H]CD binding at 5 nM [3H]CD remained after the heat treatment. The remaining binding was not affected by 0.1 mM guanylyl-5'-imidodiphosphate (GppNHp) or by nickel or other cations at concentrations below 10 mM. Treatment of the membranes with trypsin (30 micrograms/mg of protein) at 20 degrees for 20 min also caused a marked decrease in [3H]CD binding without affecting seriously the binding of [3H]QNB. About 20% of the original [3H]CD binding remained in the presence of trypsin at a high concentration of protein (90 micrograms/mg). N-Ethylmaleimide (NEM) affected [3H]CD binding in two different ways: (a) preincubation of the membranes with NEM caused a marked reduction in heat- and GppNHp-sensitive [3H]CD binding, and (b) treatment with NEM caused an enhancement of heat-, GppNHp-, and trypsin-insensitive [3H]CD binding. Neither of the NEM effects required the coexistence of agonists. The concentration of NEM required for the first effect was 10 times lower than that for the second effect, indicating the existence of two NEM-binding sites with different affinities for NEM. The equilibrium dissociation constant (Kd) for [3H]CD after NEM treatment was 33 nM and was not affected by GppNHp, Ni2+, or heat treatment; the Kd was only 4 times higher than that (8 nM) without NEM treatment. These findings indicated the existence of two kinds of [3H]CD binding sites with high affinities for agonists: one is sensitive to guanyl nucleotide and is abolished by NEM and the other is induced by NEM and insensitive to guanyl nucleotide.

Animals↗
PubMed1982

N-ethylmaleimide increases the facility of aqueous outflow of excised monkey and calf eyes.

Freshly enucleated eyes were quantitatively perfused via the anterior chamber at 15 mm Hg pressure with various concentrations of the sulfhydryl (-SH) agent. N-ethylmaleimide (NEM). In the monkey eye a dosage of 9.4 mM NEM produced a 108% increase in facility of outflow. In calf eyes a dosage of NEM of 4.7 mM or greater also produced a significant increase in outflow facility. At the threshold dosage of 4.7 mM NEM there was only slight (12%) inhibition of glycolysis in the calf meshwork, which was excised after perfusion. The results suggest that the effect of NEM on outflow facility is not caused by a metabolic inhibitory action. We propose that alteration of cell membrane -SH groups in the outflow pathways can influence aqueous outflow resistance.

Animals↗
PubMed1982

Acute effects of alkylating agents on canine renal function: N-ethylmaleimide and some of its potential biotransformation products.

Changes in urine flow rate, glomerular filtration rate and sodium, potassium and chloride excretion rates were monitored after the i.v. and renal arterial administration of N-ethylmaleimide (NEM) to dogs. Intravenous administration of NEM (72.9 and 117 mumol/kg) failed to induce an increase in any of the parameters mentioned. Renal arterial injection of 36.5 mumol/kg of NEM appeared to be nephrotoxic, whereas doses of 8.00 and 0.80 mumol/kg were associated with rapid and sustained increases in urine flow rate and the excretion rates of sodium, potassium and chloride. No changes in renal function were noted when the renal arterial dose of NEM was reduced to 0.08 mumol/kg. Three potential biotransformation products of NEM [i.e., N-ethylmaleamic acid; the cysteine adduct of NEM (NEM-cysteine); and the glutathione adduct of NEM (NEM-glutathione)] failed to induce an increase in any of the functional parameters studied regardless of the route of administration. We conclude that NEM is diuretic in the dog, but only after injection directly into the renal artery. The observed diuresis appears to be: 1) induced by NEM itself rather than by one of its potential biotransformation products, 2) due to its relatively irreversible alkylating property and 3) either a manifestation of the renal toxicity elicited by this agent or an independent effect that simply occurs at doses similar to those that induce renal injury.

Alkylating Agents↗
PubMed1980

[Stimulation and inhibition induced by N-ethylmaleimide in hydrolysis of ATP in rat liver mitochondria].

Data already reported have shown that in rat liver mitochondria N-ethylmaleimide (NEM) affects the energy dependent uptake of Ca2+ and K+ in a similar way as they do uncouplers and respiratory inhibitors. It was proposed that NEM could influence the energetic state of mitochondria, required to set up and maintain the gradient of cations across the inner membrane (1-3). In this paper the effect of NEM on the mitochondrial ATP-ase activity, has been tested. NEM added to intact mitochondria increases by more than 100% the hydrolysis of ATP supported by endogenous ATP-ase activity and inhibits the ATP-ase activity induced by couplers. Although the concentration of NEM is enought to inhibit the Pi-OH- exchange, in both cases all the Pi derived from ATP hydrolysis is found outside the mitochondria. The inhibition of uncoupler induced ATP-ase activity, the finding that the stimulatory effect of NEM is abolished by oligomycin and atractyloside and the observation that NEM does not influence the hydrolysis of ATP in sonic mitochondria, exclude the possibility of an unspecific damage of the inner membrane and of a direct effect on NEM on the ATP-ase system. The results obtained could be consistent with the possibility that in intact mitochondria nem may regulate the distribution across the inner membrane of H+ and/or K+. Concerning the problem of Pi transport our data are inconsistent with the existence of Pi-ADP cotransport (7) and with an involvement of dicarboxylate carrier.

Adenosine Triphosphate↗
PubMed1981

Muscarinic receptor: regulation by guanine nucleotides, ions, and N-ethylmaleimide.

Under the appropriate conditions, the binding of antagonists to the muscarinic receptor is consistent with the consequences of the law of mass action. In contrast, agonist binding is complex and can be rationalized in terms of multiple agonist binding sites having different affinities for agonists and equal affinity for antagonist. Certain correlations between the affinity constants and the pharmacological activities of agonists have led to the suggestion that differences among the subclasses of receptors are due to a single receptor molecule existing under varying degrees of conformational constraint. A variety of agents have been shown to modulate the binding properties of muscarinic receptors, including guanine nucleotides, sulfhydryl reagents, and ions. Guanine nucleotides reduce the binding of agonists by converting the heterogeneous agonist receptor population into a more homogeneous one of lower overall affinity. In contrast, antagonist binding is enhanced by guanine nucleotides. Alkylation sulfhydryl groups by N-ethylmaleimide causes a selective enhancement of agonist binding by converting low affinity sites into high affinity sites. In general, monovalent and divalent physiological ions perturb muscarinic receptor binding properties in a manner that is related to ionic strength. The modulatory effects of guanine nucleotides, sulfhydryl reagents, and ions make these agents useful tools for investigating the significance of muscarinic receptor heterogeneity.

Animals↗
PubMed1994

Modulation of Ca2+ channels by PTX-sensitive G-proteins is blocked by N-ethylmaleimide in rat sympathetic neurons.

The actions of N-ethylmaleimide (NEM), a sulfhydryl alkylating agent, on G-protein-mediated inhibition of N-type Ca2+ channels in adult rat superior cervical ganglion (SCG) neurons were studied using whole-cell voltage clamp. In SCG neurons, inhibition of ICa occurs by at least three separable pathways: one pertussis toxin (PTX) sensitive and voltage dependent, and two PTX insensitive and voltage independent. NEM blocked PTX-sensitive inhibition nearly completely, with only small effects on PTX-insensitive inhibition. Somatostatin inhibition is completely PTX sensitive and was wholly blocked by a 120 sec exposure to 50 microM NEM, with shorter exposure times producing a less complete block. Inhibition of ICa by norepinephrine (NE) is approximately half PTX sensitive and was also approximately half NEM sensitive. One component of muscarinic inhibition is PTX insensitive, voltage independent, and mediated by a diffusible cytoplasmic messenger; this pathway was largely spared by NEM treatment. Another pathway is also PTX insensitive and voltage independent, used by substance P, and was also largely NEM insensitive. Hence, in SCG neurons, NEM selectively inactivates PTX-sensitive G-proteins. We also find evidence that the PTX-insensitive action of NE is distinct from the other PTX-insensitive pathways, and therefore assign it to a fourth signaling pathway.

Animals↗
PubMed1995

The effect of N-ethylmaleimide on the Na(+)-dependent nucleoside transporter (N3) in rabbit choroid plexus.

The effect of the irreversible sulfhydryl-modifying agent, N-ethylmaleimide (NEM), on the transport of purine and pyrimidine nucleosides in choroid plexus was examined. [3H]thymidine and [3H]guanosine were used as model compounds to determine the effect of NEM on the Na(+)-dependent nucleoside transporter (N3) in rabbit choroid plexus tissue slices that were ATP-depleted with 2,4 dinitrophenol. Thymidine uptake in choroid plexus tissue slices preincubated with NEM was irreversibly inhibited in a concentration- (IC50 = 0.18 mM) and time-dependent fashion. NEM treatment also reduced the Na(+)-dependent uptake of other purine and pyrimidine nucleosides to a similar extent. In addition, an amine-selective modifying reagent, phenylisothiocyanate, had no effect on Na(+)-dependent thymidine uptake. Treatment of choroid plexus tissue slices with other sulfhydryl-modifying agents, including 4,4-dithiodipyridine, a reagent specific for cysteine residues, reduced the Na(+)-dependent uptake of thymidine. Preincubation of choroid plexus slices with NEM (2.5 mM) increased the Km of guanosine (control 64.5 +/- 5.7 microM; treated 120 +/- 23 microM) whereas the Vmax was unaffected (control 4.7 +/- 1 nmol/g/sec; treated 4.03 +/- 0.26 nmol/g/sec). These data suggest that covalent modification of sulfhydryl groups reduces the Na(+)-dependent uptake of nucleosides in choroid plexus slices by decreasing the affinity of nucleosides for the transporter.

Animals↗
PubMed1993

Characterization of cysteine residues of mitochondrial ADP/ATP carrier with the SH-reagents eosin 5-maleimide and N-ethylmaleimide.

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 very rapidly, Cys56 slowly, and Cys256 very slowly and did not label Cys128. Its labeling of Cys159 was associated with inhibition of the ADP transport, suggesting that the peptide segment containing Cys159 is involved in the transport of adenine nucleotides. In contrast to the very rapid binding of EMA to Cys159, NEM bound to Cys56 more slowly and labeled Cys159 and Cys256 much more slowly. Like EMA, it did not react with Cys128. The labeling of Cys56 with NEM also inhibited ADP transport. From these results, the locations of these cysteine residues in the ADP/ATP carrier are discussed in relation to the transport of adenine nucleotides mediated by the ADP/ATP carrier.

Animals↗
PubMed1994

Studies of the mechanism for enhanced cell surface factor VIIa/tissue factor activation of factor X on fibroblast monolayers after their exposure to N-ethylmaleimide.

Fibroblast monolayers constitutively expressing surface membrane tissue factor (TF) were treated with 0.1 mM N-ethylmaleimide (NEM) for 1 min to inhibit aminophospholipid translocase activity without inducing general cell damage. This resulted in increased anionic phospholipid in the outer leaflet of the cell surface membrane as measured by the binding of 125I-annexin V and by the ability of the monolayers to support the generation of prothrombinase. Specific binding of 125I-rVIIa to TF on NEM-treated monolayers was increased 3- to 4-fold over control monolayers after only brief exposure to 125I-rVIIa, but this difference progressively diminished with longer exposure times. A brief exposure of NEM-treated monolayers to rVIIa led to a maximum 3- to 4-fold enhancement of VIIa/TF catalytic activity towards factor X over control monolayers, but, in contrast to the binding studies, this 3- to 4-fold difference persisted despite increasing time of exposure to rVIIa. Adding prothrombin fragment 1 failed to diminish the enhanced VIIa/TF activation of factor X of NEM-treated monolayers. Moreover, adding annexin V, which was shown to abolish the ability of NEM to enhance factor X binding to the fibroblast monolayers, also failed to diminish the enhanced VIIa/TF activation of factor X. These data provide new evidence for a possible mechanism by which availability of anionic phospholipid in the outer layer of the cell membrane limits formation of functional VIIa/TF complexes on cell surfaces.

Annexin A5↗
PubMed1997

N-ethylmaleimide blocks depolarization-induced suppression of inhibition and enhances GABA release in the rat hippocampal slice in vitro.

Regulation of synaptic, GABAA receptor-mediated inhibition is a process of critical importance to normal brain function. Recently, we have described a phenomenon in hippocampus of a transient, yet marked, decrease in spontaneous, GABAA receptor-mediated IPSCs after depolarization activated Ca2+ influx into a pyramidal cell. This process, depolarization-induced suppression of inhibition (DSI), is absent in hippocampal cells that previously had been exposed to pertussis toxin in vivo, implicating a G-protein in the DSI process. To circumvent the problem that a single cell cannot be studied before and after G-protein block using the pertussis toxin pretreatment method, we have used the sulfhydryl alkylating agent N-ethylmaleimide (NEM), which blocks pertussis toxin-sensitive G-proteins, to determine whether acute inhibition of G-proteins can eliminate DSI of spontaneous IPSCs (sIPSCs). In whole-cell recordings from CA1 pyramidal cells that were first determined to express DSI, we have found that NEM does block DSI of sIPSCs. We also report that DSI of monosynaptic, evoked IPSCs is blocked by NEM, suggesting that a similar mechanism underlies both forms of DSI. It was of interest that DSI was abolished at a time when NEM had increased, not decreased, GABA transmission. Indeed, NEM greatly increased quantal GABA release by a Ca(2+)-independent mechanism, an observation with potentially important implications for understanding synaptic GABA release.

Analysis of Variance↗
PubMed2005

N-Ethylmaleimide sensitive factor in the cortex of subjects with schizophrenia and bipolar I disorder.

N-Ethylmaleimide sensitive factor (NSF) is a presynaptic protein that has been suggested to be differentially expressed in the cortex of schizophrenic subjects through both high-throughput proteomic and genomic screening studies. Thus, to expand upon these studies we measured NSF using Western blotting in four regions of the cortex (BA9, 10, 40 and 46), in a cohort comprising 20 schizophrenic subjects, 8 bipolar I disorder subjects, and 20 control subjects. There was no significant difference in NSF levels between diagnostic cohorts in any of the four cortical regions. These findings highlight the importance of validating findings from high-throughput screening studies and do not support changes in cortical NSF as being of significance in schizophrenia or bipolar 1 disorder.

Adult↗
PubMed2001

Reversible conformational change in beta-lactoglobulin A modified with N-ethylmaleimide and resistance to molecular aggregation on heating.

beta-Lactoglobulin A (beta LG A) modified with N-ethylmaleimide (NEM-beta LG A) was purified by ion exchange chromatography, and modification of beta LG A by NEM was confirmed by time of flight mass spectrometry and 5,5'-dithiobis(2-nitrobenzoic acid) methods. The fluorescent spectrum of NEM-beta LG A was slightly different from that of native beta LG A. NEM-beta LG A gave no polymerization after heating at 80 degrees C and pH 7.5, as shown by polyacrylamide gel electrophoresis. Conformational change of NEM-beta LG A was observed at 80 degrees C by ultraviolet differential spectra, whereas after cooling it recovered to its original state as before heating, indicating apparent reversible thermal denaturation. Native beta LG A is resistant to pepsin hydrolysis, whereas heated beta LG A was easily hydrolyzed by pepsin. NEM-beta LG A before heating was also resistant to pepsin hydrolysis, and after heating NEM-beta LG A was still resistant to pepsin hydrolysis. These results indicate that NEM-beta LG A maintained a conformation similar to its native form even after heating. Addition of 0.2 M NaCl to the beta LG A heated under salt-free condition induced polymerization of heated beta LG A molecules, but not that of heated NEM-beta LG A. This seemed to indicate that the formation of inter- or intramolecular disulfide linkage made the heat-induced conformational change of beta LG A irreversible.

Chromatography, Ion Exchange↗
PubMed2005

Solvent effects on the 9-hydroxymethylanthracene + N-ethylmaleimide Diels-Alder reaction. A theoretical study.

[structure: see text] The origin of the high reaction rates of the 9-hydroxymethylanthracene + N-ethylmaleimide Diels-Alder reaction in fluorous solvents and supercritical carbon dioxide is analyzed through a combination of regression analyses and theoretical calculations. Both approaches allow the solvent effects on the activation barrier (a decrease by solvophobic interactions, an increase by dipolarity-polarizability) to be attributed to the existence of a hydrogen bond between the two reactants in the transition state, refuting a previous hypothesis based on strong solvophobic interactions.

Journal Article↗
PubMed2006

Water in oil microemulsions as reaction media for a Diels-Alder reaction between N-ethylmaleimide and cyclopentadiene.

The Diels-Alder reaction between N-ethylmaleimide and cyclopentadiene in water/AOT/isooctane microemulsions, where AOT denotes sodium bis(2-ethylhexyl)sulfosuccinate, was studied. The rate of the reaction was found to be higher than that obtained in pure isooctane, irrespective of the particular microemulsion composition used. The efficiency of this catalytic action ranged from a factor of 3 at low water contents (viz., W = [H2O]/[AOT] = 2) to 15 at W = 35. On the basis of these results, the reaction takes place simultaneously in the continuous medium and at the microemulsion interface. The favorable arrangement of the reactants at the interface results in more than 95% of the reaction occurring in this microenvironment. The kinetic analysis revealed the rate constant at the microemulsion interface to change with the water content. For small W values a bimolecular rate constant at the interface close to that observed in hexane was obtained. This value increases with W and for W > 20, a value close to that obtained in ethanol was found. This can be ascribed to the absence of hydrogen bonding at the microemulsion interface as well as the accelerating effects due to enforced hydrophobic interactions.

Journal Article↗
PubMed2006

AOT-based microemulsions accelerate the 1,3-cycloaddition of benzonitrile oxide to N-ethylmaleimide.

We studied the 1,3-dipolar cycloaddition of benzonitrile oxide to N-ethylmaleimide in AOT/isooctane/water microemulsions at 25.0 degrees C and found the reaction rate to be roughly 150 and 35 times greater than that in isooctane and pure water, respectively. The accelerating effect of the microemulsion is the combined result of an increase in the local concentrations of the reactants through incorporation into the interface and of the intrinsic rate of the process through electrostatic interactions with the headgroups in the surfactant.

Journal Article↗