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PubMed1980

Effects of N-ethylmaleimide treatment on the action potential Na+-ionophore of cultured neuroblastoma cells.

Treatment of murine neuroblastoma N18 cells with N-ethylmaleimide under conditions that favor diminution of membrane potential leads to a 65% increase in the rate of veratridine-stimulated, tetrodotoxin-sensitive Na+ uptake and to a 50% decrease in the concentration of veratridine required for half-maximal stimulation. This modification does not appear to occur in N18 cells having a normal membrane potential of approx. -40 mV. The data are consistent with the involvement of a sulfhydryl group in the Na+ channel inactivation gate and in the conformational change in this gate which results in closing of the channel.

Action Potentials↗
PubMed1984

Differences and similarities in muscarinic receptors of rat heart and retina: effects of agonists, guanine nucleotides, and N-ethylmaleimide.

Muscarinic receptor stimulation inhibits cyclic AMP formation in rat atria but not in retina. We compared the properties of the muscarinic receptors in rat atrial and retinal membranes using the antagonist [3H]quinuclidinyl benzilate. In both atria and retina there is a single binding site for antagonists, while agonists appear to interact at two classes of binding sites. Muscarinic receptors in atria and retina have the same apparent affinities for several antagonists and for a series of muscarinic agonists. In both tissues N-ethylmaleimide decreases agonist affinity for the high-affinity binding sites. Muscarinic receptors in atria and retina differ, however, in several properties relating to the proportions of high- and low-affinity agonist sites. First, guanine nucleotides markedly increase the proportion of low-affinity binding sites in atria, but not in retina. Second, for all agonists there are fewer high-affinity binding sites in retina. Third, the "partial agonist" pilocarpine appears to interact with two classes of binding sites in atria, but with only a single class of sites in retina. Our data suggest that muscarinic receptors that inhibit cyclic AMP formation and those that do not share common properties that determine receptor affinity for agonists and classic antagonists. The differences between these receptors are manifest, however, in the effects of guanine nucleotides and the ability of agonists, especially those of low efficacy, to affect the proportion of high- and low-affinity sites and to effect a biological response.

Animals↗
PubMed1984

pH selectivity of N-ethylmaleimide reactions with opiate receptor complexes in rat brain membranes.

N-Ethylmaleimide (NEM) decreases opiate agonist binding presumably by blocking crucial sulfhydryl (SH) groups at receptor binding sites. At physiological pH, NEM decreased GTP and manganese regulation but increased sodium effects on [3H]D-Ala2-Met5-enkephalinamide (D-Ala enk) binding to rat brain membranes. To determine the apparent pK values of putative SH groups in opiate receptors that react with NEM, rat brain membranes were incubated with 100-250 microM NEM in buffers ranging from pH 4.5 to 8.0. Results showed that lowering pH below 6.5 reduced the NEM effect on opiate receptor functions and that the apparent pK values of NEM-reacting SH groups in binding and regulatory sites ranged between 5.4 to 6.0. Most of the total SH groups in brain membranes continued to react with NEM at low pH, so that when nonspecific SH groups were blocked by incubating membranes at pH 4.5 with NEM, opiate receptors became sensitive to very low concentrations (1 microM) of NEM.

Animals↗
PubMed1987

Uncoupling of rat cerebral cortical alpha 2-adrenoceptors from GTP-binding proteins by N-ethylmaleimide.

Pretreatment of membranes from rat cerebral cortex with N-ethylmaleimide (NEM) decreased [3H]-clonidine binding in a concentration-dependent manner. The Bmax values of high-affinity sites for [3H]clonidine were reduced by 50 microM NEM treatment. Treatment with 500 microM NEM diminished the sum of Bmax of both high- and low-affinity components. GTP, Na+, and Mn2+ exerted little effect on [3H]clonidine binding in NEM-treated membranes. The addition of purified GTP-binding proteins caused an increase in the binding to the membranes pretreated with 50 microM NEM, but did not increase [3H]-clonidine binding in membranes treated with 500 microM NEM. In contrast, NEM pretreatment inhibited islet activating protein (IAP)-catalyzed ADP ribosylation of membrane-bound (41,000-dalton) and purified (39,000/41,000-dalton) GTP-binding proteins. From these results, it is suggested that two or three categories of essential sulfhydryl groups are involved in the coupling between agonist, alpha 2-adrenoceptor, and GTP-binding protein. One is a highly sensitive site to NEM (a concentration range of 1-50 microM), which is probably a cysteine residue, IAP-catalyzed ADP-ribosylating site on the alpha-subunit of GTP-binding protein. Other sites have low sensitivity to NEM (a concentration range of 0.1-1 mM), and are the binding domain of agonist and/or the coupling domain of GTP-binding protein on the alpha 2-adrenoceptor. In addition, Ki-ras p21 protein may lack the capacity to couple with the alpha 2-adrenoceptor.

Adenosine Diphosphate Ribose↗
PubMed1988

Selective protection of benzomorphan binding sites against inactivation by N-ethylmaleimide. Evidence for kappa-opioid receptors in frog brain.

Selective binding of [3H]bremazocine and [3H]-ethylketocyclazocine to kappa-opioid receptor sites in frog (Rana esculenta) brain membranes is irreversibly inactivated by the sulfhydryl group alkylating agent N-ethylmaleimide (NEM). Pretreatment of the membranes with kappa-selective compounds [ethylketocyclazocine (EKC), dynorphin (1-13), or U-50,488H] but not with [D-Ala2,N-Me-Phe4,Gly5-ol]enkephalin (DAGO; mu specific ligand) or [D-Ala2,N-Me-Phe4,Gly5-ol]enkephalin (DADLE; delta specific ligand) strongly protects the binding of the radioligands against NEM inactivation. These results provide more evidence for the existence of kappa-opioid receptors in frog brain. The relatively high concentrations of NEM that are needed to decrease the specific binding of [3H]bremazocine together with the observation of an almost complete protection of its binding sites by NaCl suggest that bremazocine may act as an opioid antagonist in frog brain.

Animals↗
PubMed1990

Functional reconstruction of purified Gi and Go with mu-opioid receptors in guinea pig striatal membranes pretreated with micromolar concentrations of N-ethylmaleimide.

Functional coupling between mu-opioid receptors and GTP-binding regulatory proteins (G proteins) was investigated in reconstituted membranes of the guinea pig striatum. Selective mu-opioid agonists stimulated low-Km GTPase in striatal membranes, in a Na(+)-dependent manner. The same mu-opioid agonist [( D-Ala2, N-Me-Phe4, Gly5-ol]-enkephalin (DAGO)] caused no stimulation when the membranes were exposed to islet-activating protein (IAP; pertussis toxin). There was also no DAGO stimulation in preparations pretreated with a lower concentration (5 microM) of N-ethylmaleimide (NEM), which abolished the ADP-ribosylation of purified Gi (the G protein that mediates inhibition of adenylate cyclase) and Go (a G protein of unknown function purified from bovine brain) by IAP. In addition, as the NEM treatment caused no change in the mu-agonist binding, NEM could probably substitute for IAP in inactivating native G proteins, without exhibiting effects on the receptor binding in membranes. The mu-agonist stimulation of low-Km GTPase activity in NEM-treated membranes was recovered by reconstitution with purified Gi or Go. The mu-agonist stimulation of low-Km GTPase was additive when Gi and Go were simultaneously reconstituted in NEM-treated membranes in amounts of 0.5 pmol/assay, which was required for maximal recovery, in either reconstitution experiment. The present findings provide the first evidence that the mu-opioid receptor may exist in at least two different forms, separately coupled to Gi or Go.

Adenylate Cyclase Toxin↗
PubMed1992

Activation of alpha 1- and alpha 2-adrenoceptors inhibits noradrenaline release in rabbit renal arteries: effects of pertussis toxin and N-ethylmaleimide.

1. The effects of selective alpha 1- and alpha 2-adrenoceptor agonists and antagonists on the stimulation-induced (S-I) outflow of radioactivity at 2 Hz were investigated in superfused rabbit renal arteries incubated with [3H]-noradrenaline. 2. The alpha 1-adrenoceptor agonist methoxamine (10 microM) inhibited S-I outflow of radioactivity and this effect was abolished by the alpha 1-adrenoceptor antagonist prazosin (0.1 microM) but not by the alpha 2-adrenoceptor antagonist rauwolscine (1 microM). Neither the prostaglandin synthesis inhibitor indomethacin (10 microM) nor the adenosine receptor antagonist 8-phenyl-theophylline (1 microM) prevented the inhibitory effect of methoxamine. 3. The alpha 2-adrenoceptor agonists clonidine (0.1 microM) and UK 14304 (0.1 microM) both inhibited S-I outflow of radioactivity. The inhibitory effect of clonidine was blocked by rauwolscine but not by prazosin. The inhibitory effect of UK 14304 was markedly reduced by rauwolscine. 4. Prazosin (0.1 microM) alone did not enhance the S-I outflow of radioactivity at 2 Hz and slightly enhanced S-I outflow at 4 Hz. Rauwolscine (1 microM) alone markedly enhanced S-I outflow of radioactivity at 2 and 4 Hz. 5. Pretreatment of the arteries with pertussis toxin (1 microgram ml-1) did not significantly alter the inhibitory effects of methoxamine or UK 14304 or the potentiation by rauwolscine. However, pretreatment of the arteries with a higher concentration of pertussis toxin (5 micrograms ml-1) prevented the inhibitory effect of methoxamine but still did not affect the responses to UK 14304 and rauwolscine. 6. Pretreatment of the arteries with N-ethylmaleimide (NEM, 10 microM) for 30 min did not alter the inhibitory effect of methoxamine but markedly attenuated the inhibitory effect of UK 14304 and the facilitatory effect of rauwolscine. 7. The results suggest that both alpha 1- and alpha 2-adrenoceptors take part in the modulation of noradrenaline release from sympathetic nerves in rabbit renal arteries. Alpha 1-adrenoceptor mediated inhibition may be coupled to G-proteins which are pertussis toxin sensitive and alpha 2-adrenoceptor mediated inhibition to G-proteins which are NEM-sensitive.

Adrenergic alpha-Agonists↗
PubMed1970

Effects of sulphydryl inhibitors on frog sartorius muscle: N-ethylmaleimide.

1. The characteristic action of the -SH inhibitor, N-ethylmaleimide (NEM), is muscle rigour. The dose-response curve indicates a biphasic effect with maximum rigour tension produced by 1.0 mM NEM; beyond 1.0 mM there was an inverse relationship between dose and response.2. NEM produces a membrane depolarization unrelated to rigour development.3. NEM causes a sustained increase in (45)Ca efflux from whole muscle. Pretreatment of a muscle with ethylenediamine tetra-acetic acid (EDTA, 5 mM) to remove membrane calcium does not alter the NEM induced (45)Ca efflux.4. It is suggested that the primary site of NEM action is inhibition of calcium uptake by the sarcoplasmic reticulum thereby producing rigour. At concentrations above 1.0 mM, NEM may affect the myofilaments.

Action Potentials↗
PubMed1985

Treatment with N-ethylmaleimide selectively reduces adenosine receptor-mediated decreases in cyclic AMP accumulation in rat hippocampal slices.

N-ethylmaleimide (NEM) has been reported to interact with the GTP-binding Ni-protein; we have examined its effect on adenosine receptor binding in feline cortical membranes and on adenosine-receptor mediated effects on cyclic AMP accumulation in rat hippocampal slices. Treatment of cortical membranes with NEM (100 microM for 5 min) altered the binding of [3H]-phenylisopropyladenosine (PIA) from being almost exclusively to a single class of high affinity sites (KD = 1.65 nM) to binding at two classes of sites (KDH = 2.1 nM, KDL = 102 nM). The total number of binding sites was similar (825-845 fmol mg-1 in control membranes, 944-1428 fmol mg-1 in NEM-treated membranes). In rat hippocampal slices treated with forskolin (0.3 microM) L-PIA produced a biphasic effect on cyclic AMP accumulation: an inhibition at 0.03 to 1 microM and at higher concentrations, a stimulation. Treatment with 50 microM NEM selectively inhibited the inhibitory phase, causing stimulation at lower concentrations of L-PIA. At 50 microM, NEM did not alter basal or forskolin-stimulated cyclic AMP accumulation but at higher concentrations inhibition was observed. It is concluded that NEM can, in certain doses, selectively block adenosine A1-receptor-mediated effects without affecting A2-receptor-mediated actions in the same tissue. It is suggested that this is due to NEM affecting the Ni guanine nucleotide binding protein.

Animals↗
PubMed1989

Effects of N-ethylmaleimide on 5-hydroxytryptamine transport and sodium content in rabbit platelets.

1. The present study analysed the mechanism underlying the inhibitory action of N-ethylmaleimide (NEM) on the 5-hydroxytryptamine (5-HT) uptake by blood platelets. 2. Rabbit platelets suspended in protein-free buffer were first preincubated for 45 min in the absence and presence of NEM (20 to 160 microM) or ouabain (0.5-2.0 microM) and then either analysed for their Na+ and K+ content or incubated (15s) with various concentrations of [3H]-5-HT (0.13-4.03 microM) to determine Km and Vmax for 5-HT uptake. 3. Both NEM and ouabain produced concentration-dependent decreases in Vmax with IC50 values of 52 and 0.58 microM, respectively. Neither drug changed Km significantly. 4. Both NEM and ouabain increased the Na+ and decreased the K+ content of platelets in a concentration-dependent manner. 5. There was a linear correlation between Vmax (expressed in % of control) and the reciprocal cellular Na+ content, with the results for both drugs falling onto one and the same regression line (r = 0.992; n = 8). This regression showed that an increase in Na+ content by 69% sufficed to reduce Vmax by 50%. 6. At concentrations that reduced 5-HT uptake by about 60%, neither NEM nor ouabain altered the potency of imipramine for inhibition of 5-HT uptake. 7. Hence, NEM inhibits 5-HT transport by inhibiting the Na+/K+-ATPase and not by a direct interaction with the 5-HT carrier. The consequential increase in the intracellular Na+ concentration reduces the transmembrane Na+ gradient and, therefore, hinders 5-HT inward transport. This action of the drug does not affect the ability of the carrier to bind 5-HT or imipramine.

Animals↗
PubMed1986

Effects of N-ethylmaleimide and forskolin on glutamate release from rat hippocampal slices. Evidence that prejunctional adenosine receptors are linked to N-proteins, but not to adenylate cyclase.

In the present experiments we have examined the effect of N-ethylmaleimide (NEM) on the release of [3H]glutamate from rat hippocampal slices. Pretreatment of slices with NEM in a concentration between 50 microM and 200 microM, can inhibit the GTP-binding protein (Ni) that transmits receptor signals into inhibitions of adenylate cyclase, without affecting the Ns-protein, that transmits signals into stimulation of the cyclase, or the cyclase. The adenosine receptor agonist R-phenylisopropyladenosine (R-PIA, 1 microM) caused an approximately 50% inhibition of the evoked [3H]glutamate release. This effect was completely prevented by NEM treatment, which did not affect basal or stimulated release of the amino acid. By contrast, the effect of R-PIA was unaffected by adding an adenylate cyclase stimulator (forskolin 1 microM) and a phosphodiesterase inhibitor (rolipram, ZK 62.711, 30 microM) which raised the cyclic AMP content of the slices approximately 10-fold. In conclusion, these results suggest that the adenosine receptor that mediates prejunctional inhibition of glutamate release is coupled to a protein similar to the Ni-protein, but that another effector than adenylate cyclase is involved.

Adenylyl Cyclases↗
PubMed1987

Effects of N-ethylmaleimide and forskolin on noradrenaline release from rat hippocampal slices. Evidence that prejunctional adenosine and alpha-receptors are linked to N-proteins but not to adenylate cyclase.

N-ethylmaleimide (NEM) treatment has been shown to inactivate regulatory GTP-binding N (G)-proteins in many preparations, including slices of rat hippocampus. NEM-treatment (100 microM for 15 min) has been used to examine the possible involvement of a N-protein in the prejunctional inhibitory effect of an adenosine analogue, R-PIA acting on A1-receptors, and of clonidine acting on alpha 2-adrenoceptors in this tissue. NEM treatment significantly enhanced basal overflow of [3H]NA and the overflow stimulated by low (0.3 Hz) frequency stimulation, but not the overflow stimulated by higher (1-10 Hz) frequency stimulation. The prejunctional inhibitory effect of R-PIA (1 microM) on NA release, stimulated by a 3 Hz stimulation, was abolished by NEM pretreatment, which also eliminated the dose-dependent prejunctional effect of clonidine and reduced the facilitatory effect of yohimbine. Forskolin had a small, but significant stimulatory effect on NA overflow, but did not reduce the prejunctional inhibitory effect of R-PIA. The adenylate cyclase inhibitor SQ 22, 536 did not reduce NA overflow. These results show that NEM blocks a critical step in the prejunctional action of both adenosine- and alpha 2-receptor agonists, which may be a N-protein. The possibility is discussed that the prejunctional A1- and alpha 2-receptors couple to a N-protein that controls a different effector than adenylate cyclase.

Adenylyl Cyclases↗
PubMed1968

Uptake of N-ethylmaleimide and I-Fluoro-2, 4-dinitrobenzene in relation to the irreversible inhibition of glucose transfer in the human erythrocyte.

1. The uptake of the inhibitors N-ethylmaleimide (NEM) and 1-fluoro-2,4-dinitrobenzene (DNFB) by human red cells has been correlated with the inhibition of glucose exit.2. With both inhibitors there was an initial rapid uptake by the cells with little inhibition; this was followed by a phase when inhibition was developing rapidly but uptake continued at a steady rate even after the development of inhibition had flattened off.3. The rate of uptake of DNFB during the rapid development of inhibition corresponded to about 4 x 10(8) molecules/cell for 100% inhibition, irrespective of the temperature of incubation. This cannot be used as an estimate of the number of glucose transfer sites in the cell membrane because of the lack of specificity.4. In an examination of lipids from red cells incubated with [(14)C]DNFB, labelling associated with lipids was eluted with peaks in chloroform-methanol 4:1 and 1:4 respectively. Thus, although DNFB is normally regarded as a protein reagent, involvement of lipids in the transfer of glucose could not be excluded.

Biological Transport, Active↗
PubMed1993

N-ethylmaleimide discriminates between two lysine transport systems in human erythrocytes.

1. The sulfhydryl reagent N-ethylmaleimide (NEM) was shown to inactivate the low affinity lysine transporter in human erythrocytes (system y+) without affecting the high affinity transporter (system y+L). 2. Pre-treatment of the cells with NEM reduced the rate of entry of L-[14C]lysine (1 microM) by approximately 50% (maximum effect). 3. NEM (0.2 mM) inhibited the NEM-sensitive component of the flux with mono-exponential kinetics. The inactivation rate constant (k, +/- S.E.M.) was 0.53 +/- 0.027 min-1 (25 degrees C). The substrate did not protect against inactivation. 4. Lysine self-inhibition experiments revealed two transport systems in untreated cells (half-saturation constants Km; +/- S.E.M.), 12.0 +/- 1.7 microM and 109 +/- 15.6 microM) and only one high affinity system in NEM-treated cells (Km 9.5 +/- 0.67 microM), indicating that NEM inactivates system y+. 5. The NEM-insensitive L-[14C]lysine influx (system y+L) was inhibited with high affinity by unlabelled neutral amino acids. The inhibition constant for L-leucine in sodium medium (Ki +/- S.E.M.) was 10.7 +/- 0.72 microM (37 degrees C). The system was also strongly inhibited by L-methionine, L-glutamine and with less affinity by L-phenylalanine and L-serine. N-methyl-L-leucine, L-proline and 2-amino-2-norbornane-carboxylic acid, a bicyclic analogue of leucine, did not exert a significant effect. 6. Lysine transport through system y+L occurred at the same rate in Na+, K+ or Li+ medium and the binding of lysine to the transporter was unaffected by Na+ replacement. 7. The interaction of system y+L with neutral amino acids was dependent on the cation present in the medium. The inhibition constant for leucine and glutamine increased approximately 90- and 60-fold respectively when Na+ was replaced by K+. Li+ was shown to be a very good substitute for Na+.

Amino Acids↗
PubMed2005

A novel inhibitor of N-ethylmaleimide-sensitive factor decreases leukocyte trafficking and peritonitis.

Endothelial exocytosis is an early stage in the process of leukocyte trafficking. N-ethylmaleimide-sensitive factor (NSF) plays a critical role in regulating exocytosis. We hypothesized that inhibitors of NSF decrease endothelial exocytosis and vascular inflammation. We designed a novel fusion polypeptide consisting of a human immunodeficiency virus transactivator of transcription (TAT) protein transduction domain joined to a NSF homohexamerization domain. We show that this TAT-NSF polypeptide inhibits the ATPase activity and the disassembly activity of NSF. Furthermore, the TAT-NSF polypeptide decreases endothelial cell exocytosis and reduces leukocyte adherence to endothelial cells in culture. Finally, the TAT-NSF polypeptide inhibits leukocyte rolling on murine venules in vivo and inhibits leukocyte trafficking into the peritoneal cavity in a murine model of experimental peritonitis. These data suggest that NSF is a critical regulator of leukocyte trafficking in vivo. Novel compounds that inhibit the exocytic machinery in endothelial cells may be useful anti-inflammatory drugs.

Animals↗
PubMed1964

N-ETHYLMALEIMIDE INHIBITION OF HORSESHOE CRAB HEMOCYTE AGGLUTINATION.

The sulfhydryl inhibitor N-ethylmaleimide (NEM) inhibits the agglutination of horseshoe crab hemocytes. The inhibitory action is neutralized by adding cysteine to the NEM before it is mixed with the hemolymph. This new method for arresting horseshoe crab hemocyte agglutination suggests the participation of sulfhydryl groups in the process.

Agglutination↗
PubMed2000

Glutathione-dependent conversion of N-ethylmaleimide to the maleamic acid by Escherichia coli: an intracellular detoxification process.

The electrophile N-ethylmaleimide (NEM) elicits rapid K(+) efflux from Escherichia coli cells consequent upon reaction with cytoplasmic glutathione to form an adduct, N-ethylsuccinimido-S-glutathione (ESG) that is a strong activator of the KefB and KefC glutathione-gated K(+) efflux systems. The fate of the ESG has not previously been investigated. In this report we demonstrate that NEM and N-phenylmaleimide (NPM) are rapidly detoxified by E. coli. The detoxification occurs through the formation of the glutathione adduct of NEM or NPM, followed by the hydrolysis of the imide bond after which N-substituted maleamic acids are released. N-ethylmaleamic acid is not toxic to E. coli cells even at high concentrations. The glutathione adducts are not released from cells, and this allows glutathione to be recycled in the cytoplasm. The detoxification is independent of new protein synthesis and NAD(+)-dependent dehydrogenase activity and entirely dependent upon glutathione. The time course of the detoxification of low concentrations of NEM parallels the transient activation of the KefB and KefC glutathione-gated K(+) efflux systems.

Escherichia coli↗
PubMed1973

Kinetics of reaction of Escherichia coli ribosomal proteins toward N-ethylmaleimide.

The kinetics of reaction of the exposed ribosomal proteins from Escherichia coli toward N-ethylmaleimide (NEM) have been studied. While most of the proteins from the 30S subunit react rapidly with NEM and plateau levels can be reached, most of the proteins from the 50S subunit show biphasic uptake kinetics. Furthermore, differences have been observed in the reactivity of proteins toward NEM depending on whether the protein is in the free subunit or in the 70S ribosome.

Bacterial Proteins↗