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PubMed1987

Comparative study on effect of N-ethylmaleimide on the superoxide-generating system of guinea-pig eosinophils stimulated by soluble stimuli.

1. The effect of N-ethylmaleimide (MalNEt) modification on O2- production by guinea-pig eosinophils mediated by different soluble stimuli was studied. 2. MalNEt pretreatment inhibited the O2- production stimulated by concanavalin A (Con A), cytochalasin E or digitonin, but not A23187 or sodium fluoride. 3. Particulate fractions from MalNEt-pretreated eosinophils before exposure to the stimulus showed the inhibition of the enhancement of NADPH-dependent O2- production induced by Con A, cytochalasin E or digitonin, but not A23187. 4. Treatment of eosinophils with MalNEt after stimulation had no effect on the NADPH oxidase activity. 5. These findings suggest that at least two pathways exist for the activation of the O2(-)-generating enzyme system, probably the NADPH oxidase system, in guinea-pig eosinophils.

Animals↗

PubMed1992

Capsaicin-like activity of N-ethylmaleimide in rat stomach.

1. N-Ethylmaleimide (NEM) and capsaicin induced a release of calcitonin gene-related peptide-like immunoreactivity (CGRP-li) in superfusates from rat gastric corpus. 2. The prior application of capsaicin completely blocked CGRP-li release by NEM while the exposure to NEM reduced the capsaicin-evoked CGRP-li outflow by about 51%. 3. These findings provide evidence that NEM might exert capsaicin-like activity in rat stomach and the release of CGRP-li from capsaicin-sensitive afferent fibres might influence the properties exerted by NEM in this tissue.

Animals↗

PubMed1984

Modification of radiation and dihydroxyanthraquinone-induced cell lethality by cysteamine and N-ethylmaleimide.

The effect of alteration of sulfhydryl levels on the cell lethality induced by ionizing radiation and dihydroxyanthraquinone (DHAQ) was investigated in cultured Chinese hamster V79 cells. DHAQ produces a potentiation of radiation-induced cell lethality, both by increasing the slope and decreasing the shoulder of the survival curve. It has been suggested that DHAQ functions through the production of free radicals which then produce DNA strand breaks and crosslinks, resulting in cytotoxicity. If this mode of action predominates, then one would expect to be able to change the degree of cell kill by modifying conditions such that free radical processes were altered. This was accomplished by the addition of N-ethylmaleimide (NEM) or Cysteamine (CYS) to the culture medium during treatment with DHAQ. It was observed that the combination of DHAQ and NEM did not produce more cytotoxicity than would be expected from an additive interaction. Likewise, CYS did not reduce the cytotoxicity induced by DHAQ. When cells were treated with DHAQ and radiation plus either NEM or CYS, the resultant survival was consistent with an additive interaction between the potentiation of DHAQ for radiation-induced cell kill and the extra effect of NEM or CYS. These results indicate that alterations of sulfhydryl levels do not produce changes in the cytotoxicity induced by DHAQ, nor in the enhancement by DHAQ of radiation-induced lethality. More investigation is required before definite conclusions can be reached as to the mechanisms of action by which DHAQ, alone or in combination with ionizing radiation, induces mammalian cell lethality.

Animals↗

PubMed1989

The effects of radical stress and N-ethylmaleimide on rat hepatic alpha 1-adrenergic receptors.

Liver plasma membranes of non-treated rats or rats treated with carbon tetrachloride were prepared. Liver plasma membranes obtained from non-treated rats were preincubated with cumene hydroperoxide, N-ethylmaleimide or hydrogen peroxide in the absence or presence of Fe2+. The effects of in-vivo or in-vitro treatment on hepatic alpha 1-adrenergic receptors were assessed in a [3H]prazosin binding assay. All treatments except preincubation with hydrogen peroxide alone caused a decrease in the number of alpha 1-adrenergic receptors without a change in the affinity of [3H]prazosin. The decrease in receptor number was accompanied by an increase in the level of lipid peroxidation in the plasma membranes. The results indicate that the hepatic alpha 1-adrenergic receptor is vulnerable to free radical stress.

Animals↗

PubMed1990

Selective coupling of purified alpha-subunits of pertussis toxin-substrate GTP-binding proteins to endogenous receptors in rat brain membranes treated with N-ethylmaleimide.

The membrane fraction prepared from rat brain was incubated with 0.5 mM N-ethylmaleimide (NEM) for 10 min. 3H-labelled agonist binding to muscarinic, A1-adenosine, opiate and alpha 2-adrenergic receptors was markedly inhibited by this NEM treatment of membranes, which interfered with the subsequent ADP-ribosylation of endogenous G-proteins by pertussis toxin. This indicated that the toxin target cysteine residues of the G-protein were modified by NEM. The NEM-induced inhibition of agonist bindings was mostly reversed by reconstitution of the alpha-subunits of purified Gi or Go into the membranes. The NEM-induced inhibition, together with the reversal by the G alpha reconstitution, was due to changes in the relative number of high- to low-affinity receptors solely without change in the total (high- plus low-affinity) receptor number. Thus, in NEM-treated membranes endogenous G-proteins become uncoupled from receptors, which were coupled to either Gi alpha or Go alpha. Reconstitution of NEM pre-treated membranes showed that Go acted in preference to Gi in interaction with muscarinic receptors and vice versa in interaction with three other types of receptor. The possible involvement of Go in mediating phospholipase C activation and Gi in mediating adenylate cyclase inhibition is discussed.

Adenosine Diphosphate Ribose↗

PubMed1994

Induction of the alkylation-inducible aidB gene of Escherichia coli by cytoplasmic acidification and N-ethylmaleimide.

We have studied the effects of changes in intracellular pH and the influence of thiol reagents on the induction of the DNA damage-inducible genes of Escherichia coli, aidB, alkA, and alkB. Under aerobic conditions in the absence of alkylating agents aidB, but not alkA or alkB, was induced by an acidification of cytoplasm or by treatment with the sulfhydryl reagent N-ethylmaleimide. Alkaline shift and thiosalicyclic acid did not affect the induction of aidB and alkB. The induction of alkA increased under the alkaline shift but not in the case of treatment with reducing agents. Compared with the aidB gene, a component of the SOS system, the sulA (sfiA) gene, responded to changes in cytoplasmic pH and in the level of intracellular thiols in an opposite way. SulA induction was observed under alkaline shift and after treatment with thiosalicylic acid.

Acetates↗

PubMed2006

N-ethylmaleimide-resistant acyl-coenzyme A oxidase from Arthrobacter ureafaciens NBRC 12140: molecular cloning, gene expression and characterization of the recombinant enzyme.

N-ethylmaleimide (NEM)-resistant acyl-coenzyme A oxidase (ACO) has been desired for the determination of free fatty acids (FFAs). In order to meet this demand, we prepared recombinant ACO from Arthrobacter ureafaciens NBRC 12140. The coding region of the gene was 2109, encoding a protein of 703 amino acids with a predicted molecular mass of 76.5 kDa. The heterologous expression level in Escherichia coli was 520-fold higher than that in the native strain. The purified enzyme retained more than 60% activity after incubation in the presence of 10 mM NEM at 37 degrees C for 4 h, while other commercially available ACOs showed only less than 10% activities after the same NEM treatment. We presume that this is due to the presence of only three cysteines in ACO from A. ureafaciens. Site-directed mutagenesis studies and close scrutiny of the three-dimensional structures of other related ACOs suggested that these cysteines were buried in the protein and unreactive to NEM. The recombinant enzyme was used for the colorimetric determination of free fatty acid, which gave a linear calibration.

Acyl-CoA Oxidase↗

PubMed2005

Distinct effects of N-ethylmaleimide on formyl peptide- and cyclopiazonic acid-induced Ca2+ signals through thiol modification in neutrophils.

In this study, we demonstrate that N-ethylmaleimide (NEM), a cell permeable thiol-alkylating agent, enhanced the [Ca2+]i rise caused by stimulation with cyclopiazonic acid (CPA), a sarcoplasmic-endoplasmic reticulum Ca2+-ATPase inhibitor, in rat neutrophils. In addition, NEM attenuated the formyl-Met-Leu-Phe (fMLP)-induced [Ca2+]i rise whether NEM was added to cells prior to or after fMLP stimulation. Moreover, application of NEM after fMLP activation in the absence of external Ca2+ inhibited the Ca2+ signal upon addition of Ca2+ to the medium. Similar patterns were also obtained by using 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB), a cell impermeable dithiol-oxidizing agent, which replaced NEM in the CPA- and fMLP-induced [Ca2+]i rise experiments. Treatment with dithiothreitol (DTT), a cell permeable dithiol-reducing agent, N-acetyl-l-cysteine (NAC), a cell permeable monothiol-reducing agent, and tris-(2-carboxyethyl)phosphine (TCEP), a cell impermeable reductant without a thiol group, all rescued the fMLP-induced Ca2+ signal from NEM. Rat neutrophils express the mRNA encoding for transient receptor potential (TRP) C6, inositol trisphosphate receptor (IP3R) 2 and IP3R3. NEM had no effect on the mitochondrial membrane potential. NEM could restore the polarization and F-actin accumulation of fMLP-treated cells to those of the control. In the absence of external Ca2+, NEM rendered the CPA-induced [Ca2+]i elevation persistently but inhibited the fMLP-induced Ca2+ spike, which was reversed by tris-(2-cyanoethyl)phosphine (TCP), a cell permeable reductant without a thiol group. DTNB did not affect the Ca2+ spike caused by fMLP. These results indicate that through protein thiol oxidation, NEM affects the receptor-activated and the store depletion-derived Ca2+ signals in an opposing manner.

Actins↗

PubMed2005

Entamoeba histolytica: identification and characterization of an N-ethylmaleimide sensitive fusion protein homologue.

Entamoeba histolytica is a phagocytic cell with numerous vesicles of different sizes and shapes but without a well-defined Golgi apparatus. Despite this, genes implied in membrane trafficking have been identified in the genome of this parasite. One of these genes is homologous to the N-ethylmaleimide sensitive fusion factor (NSF), whose protein has been shown to play an important role in vesicle fusion in other eukaryotic cells. In this report, we investigated the NSF homologue gene from a pathogenic E. histolytica, characterized its protein product and two of its activities, ATPase and in vitro intra-Golgi transport. The finding of an active NSF protein in E. histolytica indicates that a simple or primordial Golgi apparatus probably exists in this microorganism, as has been proposed by others.

Amino Acid Sequence↗

PubMed2006

Spatial and temporal expression of N-ethylmaleimide-sensitive factor gene in the nervous system of the cotton bollworm, Helicoverpa armigera.

N-ethylmaleimide-sensitive factor (NSF) is a component required for vesicular transport and release of neurotransmitters or neurohormones in the constitutive secretory pathways. Here, the spatial and temporal expression of NSF gene was investigated in Helicoverpa armigera (Helar). Reverse transcription-polymerase chain reaction analysis reveals that Helar-NSF is transcribed preferentially in the nervous system of H. armigera. Using in situ hybridization, the Helar-NSF mRNA is further localized in the superficial layer or deep layer cells of the brain, subesophageal ganglion (SG) and other ganglia. The developmental profiles of Helar-NSF show that both mRNA and protein in pupal brain-SG complexes are significantly higher in nondiapause-destined individuals than in diapause-destined individuals. The expression patterns are consistent with those of two neurohormones, prothoracicotropic hormone (PTTH) and diapause hormone (DH). These data suggest a central function of Helar-NSF in developmental process through regulating neurohormone release.

Animals↗

PubMedDate not supplied

Multiple binding proteins suggest diverse functions for the N-ethylmaleimide sensitive factor.

The hexameric ATPase, N-ethylmaleimide sensitive factor (NSF), is essential to vesicular transport and membrane fusion because it affects the conformations and associations of the soluble NSF attachment protein receptor (SNARE) proteins. NSF binds SNAREs through adaptors called soluble NSF attachment proteins (alpha- or beta-SNAP) and disassembles SNARE complexes to recycle the monomers. NSF contains three domains, two nucleotide-binding domains (NSF-D1 and -D2) and an amino terminal domain (NSF-N) that is required for SNAP-SNARE complex binding. Mutagenesis studies indicate that a cleft between the two sub-domains of NSF-N is critical for binding. The structural conservation of N domains in NSF, p97/VCP, and VAT suggests that a similar type of binding site could mediate substrate recognition by other AAA proteins. In addition to SNAP-SNARE complexes, NSF also binds other proteins and protein complexes such as AMPA receptor subunits (GluR2), beta2-adrenergic receptor, beta-Arrestin1, GATE-16, LMA1, rabs, and rab-containing complexes. The potential for these interactions indicates a broader role for NSF in the assembly/disassembly cycles of several cellular complexes and suggests that NSF may have specific regulatory effects on the functions of the proteins involved in these complexes. The structural requirements for these interactions and their physiological significance will be discussed.

Binding Sites↗

PubMed2005

Direct interaction of N-ethylmaleimide-sensitive factor with GABA(A) receptor beta subunits.

GABA(A) receptors mediate most of the fast inhibitory neurotransmission in the brain, and are believed to be composed mainly of alpha, beta, and gamma subunits. It has been shown that GABA(A) receptors interact with a number of binding partners that act to regulate both receptor function and cell surface stability. Here, we reveal that GABA(A) receptors interact directly with N-ethylmaleimide-sensitive factor (NSF), a critical regulator of vesicular dependent protein trafficking, as measured by in vitro protein binding and co-immunoprecipitation assays. In addition, we established that NSF interacts with residues 395-415 of the receptor beta subunits and co-localizes with GABA(A) receptors in hippocampal neurons. We also established that NSF can regulate GABA(A) receptor cell surface expression depending upon residues 395-415 in the beta3 subunit. Together, our results suggest an important role for NSF activity in regulating the cell surface stability of GABA(A) receptors.

Amino Acid Sequence↗

PubMed2004

Alterations of N-ethylmaleimide-sensitive atpase following transient cerebral ischemia.

Neuronal repair following injury requires recruitment of large amounts of membranous proteins into synaptic and other cell membranes, which is carried out by the fusion of transport vesicles to their target membranes. A critical molecule responsible for assemblage of membranous proteins is N-ethylmaleimide-sensitive factor (NSF) which is an ATPase. To study whether NSF is involved in ischemic neurological deficits and delayed neuronal death, we investigated alterations of NSF after transient cerebral ischemia by means of biochemical methods, as well as confocal and electron microscopy. We found that transient cerebral ischemia induced depletion of free NSF and concomitantly relocalization of NSF into the Triton X-100-insoluble fraction including postsynaptic densities in CA1 neurons during the postischemic period. The NSF alterations are accompanied by accumulation of large quantities of intracellular vesicles in CA1 neurons that are undergoing delayed neuronal death after transient cerebral ischemia. Therefore, permanent depletion of free NSF and relocalization of NSF into the Triton X-100-insoluble fraction may disable the vesicle fusion machinery necessary for repair of synaptic injury, and ultimately leads to synaptic dysfunction and delayed neuronal death in CA1 neurons after transient cerebral ischemia.

Adenosine Triphosphatases↗

PubMed2005

Pronociceptive role of dynorphins in uninjured animals: N-ethylmaleimide-induced nociceptive behavior mediated through inhibition of dynorphin degradation.

Intrathecal (i.t.) administration into mice of N-ethylmaleimide (NEM), a cysteine protease inhibitor, produced a characteristic behavioral response, the biting and/or licking of the hindpaw and the tail along with slight hindlimb scratching directed toward the flank. The behavior induced by NEM was inhibited by the intraperitoneal injection of morphine. We have recently reported that dynorphin A and, more potently big dynorphin, consisting of dynorphins A and B, produce the same type of nociceptive response whereas dynorphin B does not [Tan-No K, Esashi A, Nakagawasai O, Niijima F, Tadano T, Sakurada C, Sakurada T, Bakalkin G, Terenius L, Kisara K. Intrathecally administered big dynorphin, a prodynorphin-derived peptide, produces nociceptive behavior through an N-methyl-d-aspartate receptor mechanism. Brain Res 2002;952:7-14]. The NEM-induced nociceptive behavior was inhibited by pretreatment with dynorphin A- or dynorphin B-antiserum and each antiserum also reduced the nociceptive effects of i.t.-injected synthetic big dynorphin. The characteristic NEM-evoked response was not observed in prodynorphin knockout mice. Naloxone, an opioid receptor antagonist, had no effects on the NEM-induced behavior. Ifenprodil, arcaine and agmatine, antagonists at the polyamine recognition site on the N-methyl-D-aspartate (NMDA) receptor ion-channel complex, and MK-801, an NMDA ion-channel blocker inhibited the NEM-induced effects. Ro25-6981, an antagonist of the NMDA receptor subtype containing NR2B subunit was not active. NEM completely inhibited degradation of dynorphin A by soluble and particulate fractions of mouse spinal cord. Collectively, the results demonstrate that endogenous prodynorphin-derived peptides are pronociceptive in uninjured animals, and required for the NEM-induced behavior. The NEM effects may be mediated through inhibition of the degradation of endogenous dynorphins, presumably big dynorphin that in turn activates the NMDA receptor ion-channel complex by acting on the polyamine recognition site.

Agmatine↗

PubMed2005

Molecular characterization and functional distribution of N-ethylmaleimide-sensitive factor in Helicoverpa armigera.

N-ethylmaleimide-sensitive fusion protein (NSF) is an essential component for the neurotransmitter or neurohormone release apparatus present in all eukaryotic cells. Here, a new NSF orthologue was characterized from the cotton bollworm, Helicoverpa armigera (Har). Northern blot exhibited a high expression in larval brain. Southern analysis indicated that a single copy of the gene is present in a haploid genome. Using antibodies labeled with fluoresceins, we directly proved that NSF is co-localized with two crucial neurohormones, prothoracicotropic hormone and diapause hormone, both of which regulate insect development. These findings suggest that Har-NSF may be involved in regulating insect neurohormone release.

Amino Acid Sequence↗

PubMed2002

Complex effects of sulfhydryl reagents on ligand interactions with nucleoside transporters: evidence for multiple populations of ENT1 transporters with differential sensitivities to N-ethylmaleimide.

Functional studies have implicated cysteines in the interaction of ligands with the ENT1 nucleoside transporter. To better define these interactions, N-ethylmaleimide (NEM) and p-chloromercuribenzylsulfonate (pCMBS) were tested for their effects on ligand interactions with the [(3)H] nitrobenzylthioinosine (NBMPR) binding site of the ENT1 transporters of mouse Ehrlich ascites cells and human erythrocytes. NEM had biphasic, concentration-dependent effects on NBMPR binding to intact Ehrlich cells, plasma membranes, and detergent-solubilized membranes, with about 35% of the binding activity being relatively insensitive to NEM inhibition. NBMPR binding to human erythrocyte membranes also displayed heterogeneity in that about 33% of the NBMPR binding sites remained, albeit with lower affinity for NBMPR, even after treatment with NEM at concentrations in excess of 1 mM. However, unlike that seen for Ehrlich cells, no "reversal" in NBMPR binding to human erythrocyte membranes was observed at the higher concentrations of NEM. pCMBS inhibited 100% of the NBMPR binding to both Ehrlich cell and human erythrocyte membranes, but had no effect on the binding of NBMPR to intact cells. The effects of NEM on NBMPR binding could be prevented by coincubation of membranes with nonradiolabeled NBMPR, adenosine, or uridine. Treatment with NEM and pCMBS also decreased the affinity of other nucleoside transport inhibitors for the NBMPR binding site, but enhanced the affinities of nucleoside substrates. These data support the existence of at least two populations of ENT1 in both erythrocyte and Ehrlich cell membranes with differential sensitivities to NEM. The interaction of NEM with the mouse ENT1 protein may also involve additional sulphydryl groups not present in the human ENT1.

Adenosine↗

PubMed1997

Mitochondrial membrane protein thiol reactivity with N-ethylmaleimide or mersalyl is modified by Ca2+: correlation with mitochondrial permeability transition.

The content of mitochondrial membrane protein thiol groups accessible to react with the monofunctional thiol reagents mersalyl or N-ethylmaleimide (NEM) was determined using Ellman's reagent. Deenergized mitochondria incubated in the presence of Ca2+ (0-500 microM) undergo a very significant decrease in the content of membrane protein thiols accessible to NEM, and an increase in the content of thiols accessible to mersalyl. This process is time-dependent and inhibited by Mg2+, ruthenium red and ADP, but not by cyclosporin A. This suggests that Ca2+ binding to the inner mitochondrial membrane promotes extensive alterations in the conformation of membrane proteins that result in location changes of thiol groups. The relationship between these alterations and mitochondrial membrane permeability transition was studied through the effect of NEM and mersalyl on mitochondrial swelling induced by Ca2+ plus t-butyl hydroperoxide (t-bOOH) or Ca2+ plus the thiol cross-linkers 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) or phenylarsine oxide (PhAsO). We observed that the hydrophobic thiol reagent NEM inhibits the effects of t-bOOH, DIDS and PhAsO, while the hydrophilic thiol reagent mersalyl inhibits only the effect of DIDS. Permeability transition in all the situations studied is accompanied by a significant decrease in the total membrane protein thiol content. In addition, mitochondrial membrane permeabilization induced by PhAsO is inhibited by EGTA, but not by ruthenium red. This result suggests that PhAsO leads to permeability transition through a mechanism independent of intramitochondrial Ca2(+)-induced alterations of thiol group reactivity, but dependent on Ca2+ binding to an extramitochondrial site. This site is sensitive to extramitochondrial Ca2+ concentrations in range of 1-50 microM.

Animals↗

PubMed1998

Induction of the mitochondrial permeability transition by N-ethylmaleimide depends on secondary oxidation of critical thiol groups. Potentiation by copper-ortho-phenanthroline without dimerization of the adenine nucleotide translocase.

Addition to energized rat liver mitochondria of low micromolar concentrations of the thiol oxidant, copper-o-phenanthroline [Cu(OP)2], causes opening of the permeability transition pore, a cyclosporin A-sensitive channel. The effects of Cu(OP)2 can be reversed by reduction with dithiothreitol (DTT), suggesting that a dithiol-disulfide interconversion is involved. However, at variance with all pore inducers known to act through dithiol oxidation, the effects of Cu(OP)2 are not prevented by treatment of mitochondria with low (10-20 microM) concentrations of N-ethylmaleimide (NEM). Rather, these concentrations of NEM potentiate the inducing effects of Cu(OP)2. We show that this enhancing effect of NEM is blocked by the subsequent addition of DTT, indicating that potentiation by NEM is mediated by an oxidative event rather than by substitution as such. We find that also pore induction by high (0.5-1.0 mM) concentrations of NEM in the absence of oxidants is completely blocked by reduction with DTT or beta-mercaptoethanol. These results underscore the unexpected importance of oxidative events in pore opening by substituting agents. Since we find that pore opening by Cu(OP)2 or by high concentrations of NEM is not accompanied by dimerization of the adenine nucleotide translocase, we conclude that the translocase itself is not the target of the pore-inducing oxidative events triggered by Cu(OP)2 and NEM.

Animals↗