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 217 records · Page 12Linked to original sources
PubMed1993

Identification of a hepatic plasma membrane glutathione S-transferase activated by N-ethylmaleimide.

Rat liver plasma membranes exhibit membrane-bound glutathione S-transferase activity. The specific activity in isolated canalicular membranes was 83 +/- 8 mU/mg protein and 50 +/- 3 mU/mg protein in the sinusoidal membranes. Whereas microsomal and outer mitochondrial glutathione S-transferases were stimulated seven and four-fold with N-ethylmaleimide, respectively, the plasma membrane activity was activated two-fold. Western blot analysis, using an antibody against the microsomal glutathione S-transferase, shows the presence of a 17 kDa protein in canalicular and sinusoidal membrane fractions. The antibody reaction was about three-fold higher in the canalicular compared to the sinusoidal membrane fraction. These data support the conclusion that the plasma membrane glutathione S-transferase is closely related to the microsomal and outer mitochondrial membrane enzyme.

Animals↗
PubMed1976

Ouabain-binding and phosphorylation of (Na+ + K+) ATPase treated with N-ethylmaleimide or oligomycin.

Ouabain-binding and phosphorylation of (Na+ mk+)-ATPase (EC 3.6.1.3) of the plasma membranes from kidney were investigated after treatment with N-ethylmaleimide or oligomycin. Either of these inhibitors brought about the following changes: the phosphoenzyme, formed in the presence of Na+, Mg2+ and ATP became essentially insensitive to splitting by K+ but was split by ADP. One mole of this ADP-sensitive phosphoenzyme bound one mole of ouabain but the enzyme-ouabain complex was less stable than in the native enzyme primarily because the rate of its dissociation increased. Ouabain was bound to the ADP-sensitive phosphoenzyme in the presence of Mg2+ alone and addition of inorganic phosphate enhanced both the rate of formation and the steady-state level of the enzyme-ouabain complex. The inhibitors did not affect the properties of this second type of complex. Both in the native enzyme and in the enzyme treated with the two inhibitors inorganic phosphate enhanced ouabain binding by phosphorylating the active center of the enzyme as shown (a) by mapping the labeled peptides from the enzyme after peptic digestion, (b) by inhibition of this phosphorylation with Na+ and (c) by the 1:1 stoichiometric relation between this phosphorylation and the amount of bound ouabain. Unlike the phosphoenzyme, the binding of ouabain remained sensitive to K+ in the enzyme treated with the inhibitors. K+ slowed ouabain-binding either in the presence of Na+, Mg2+ and ATP or of Mg2+ and inorganic phosphate. A higher concentration of K+ was needed to slow ouabain-binding either in the presence of Na+, Mg2+ and ATP or of Mg2+ and inorganic phosphate. A higher concentration of K+ was needed to slow ouabain-binding than to stimulate dephosphorylation. This finding is interpreted as being an indication of separate sites for K+ on the enzyme: a site(s) with high K+-affinity which stimulates dephosphorylation, another site(s) with moderate K+-affinity which inhibits ouabain-binding. Inhibitors may enhance formation of the ADP-sensitive phosphoenzyme by blocking interaction between K+ and the site(s) with high affinity.

Adenosine Triphosphatases↗
PubMed1995

Subcellular distribution of N-ethylmaleimide-sensitive and -insensitive phosphatidic acid phosphohydrolase in rat brain.

The dephosphorylation of phosphatidic acid by phosphatidic acid phosphohydrolase (PAP) is important in both cell-signalling and in glycerolipid metabolism. However, these roles are apparently performed by two different enzymes, which can be distinguished by their sensitivity in vitro to N-ethylmaleimide (NEM). Both of these enzymes are present in rat brain as well as a wide range of other rat tissues. However, the quantity and specific activity of each enzyme varies considerably between different tissues, as does the ratio of the two enzymes in each tissue. Tissues rich in glycerolipids are abundant in NEM-sensitive PAP, whereas there is no obvious pattern to the distribution of the NEM-insensitive enzyme in the different tissues tested. Studies on brain cortex, which is relatively rich in both forms of PAP, indicate that the NEM-insensitive PAP is located in the synaptosomes, and the NEM-sensitive enzyme present in the cytosol and microsomes. The NEM-sensitive PAP can also be translocated from the cytosol to the microsomes by oleate. When assayed against a range of phosphatidic acids, NEM-sensitive PAP showed a preference for phosphatidic acids with short acyl chains and for those containing arachidonate, whereas NEM-insensitive PAP had a preference for short and unsaturated acyl chains. The two isozymes also had different activity profiles against these substrates suggesting that they are in fact different enzymes. The implications for these results on the putative roles of the two forms of PAP are discussed.

Amines↗
PubMed1986

N-ethylmaleimide desensitizes pH-dependence of K+/H+ antiporter in a marine bacterium, Vibrio alginolyticus.

The K+/H+ antiporter of a marine bacterium, Vibrio alginolyticus, is strongly dependent upon the cytoplasmic pH and functions only at an internal pH above 7.7. In alkaline buffer with an outwardly directed chemical gradient of K+ (delta pK), the internal pH was maintained at about 7.7. Addition of N-ethylmaleimide (NEM) released cellular K+ and acidified the cytosol below pH 7.7. The NEM effect was reversed by the addition of 2-mercaptoethanol: K+ efflux ceased, and the internal pH returned to about 7.7. In acidic buffer, the internal pH was also regulated at about 7.6 even in the absence of delta pK. Following addition of NEM, the internal pH decreased below 7.6, dissipating delta pH. These results suggest that NEM desensitizes the pH-dependence of the K+/H+ antiporter, allowing the antiporter to function at an internal pH below 7.7.

Carrier Proteins↗
PubMed1987

Stimulation of synaptosomal ATP-dependent Ca2+-uptake by N-ethylmaleimide.

Treatment of rat brain synaptosomal lysate with N-ethylmaleimide (NEM) was found to stimulate ATP-dependent Ca2+-uptake. This Ca2+-uptake stimulation was blocked by dithioerythritol (DTE), mitochondrial inhibitors oligomycin and sodium azide, but not by vanadate, an inhibitor of plasma membrane Ca2+ pump. Maximal stimulation of Ca2+-uptake was observed at a NEM/protein ratio of 0.1 mumole/0.5-1.0 mg. On fractionation, it was found that NEM did not affect synaptic plasma membrane Ca2+-uptake, but almost doubled it in synaptic mitochondria.

Adenosine Triphosphate↗
PubMed1989

Effects of bradykinin, GTP gamma S, R59022 and N-ethylmaleimide on inositol phosphate production in NG108-15 cells.

Accumulation of inositol phosphates in NG108-15 neuroblastoma x glioma hybrid cells, pre-labeled for 24h to equilibrium, was stimulated by bradykinin, guanosine 5'-O-(3-thiotriphosphate) and the diacylglycerol kinase inhibitor R59022. Only the stimulation by bradykinin was inhibited by the bradykinin receptor antagonist [D-Arg0, Hyp3, Phe7, Thi5,8] bradykinin. Neither bradykinin nor R059022 increased the labeling of the inositol phospholipids. The sulfhydryl-alkylating reagent N-ethylmaleimide at 100 microM essentially abolished the stimulation caused by all three agents, possibly by preventing the binding of GTP to a guanine nucleotide-binding regulatory protein of as yet unknown size.

Animals↗
PubMed1991

N-ethylmaleimide differentiates endothelin converting activity by two types of metalloproteinases derived from vascular endothelial cells.

We have recently found that cultured vascular endothelial cells (ECs) contain two types of metalloproteinases which convert big endothelin-1 (big ET-1) to endothelin-1 (ET-1) via a single cleavage between Trp21 and Val22. In the present study, two enzymes were clearly differentiated by using sulfhydryl blocking reagents and anion-exchange HPLC. As reported, the converting activity of the membrane fraction of ECs was specifically inhibited by phosphoramidon. N-ethylmaleimide (NEM) markedly enhanced the apparent converting activity of the membrane fraction. This enhancement was not due to the direct action on the converting enzyme, but rather to inhibition of the degradation of big ET-1 and/or ET-1. In contrast, the converting activity of the cytosolic fraction was abolished by NEM treatment. Effects of phosphoramidon and NEM on converting activities of both fractions were confirmed after anion-exchange HPLC of each fraction, using a COSMOGEL QA column. Our results provide new information on two types of metalloproteinases which convert big ET-1 to ET-1, in vascular ECs.

Animals↗
PubMed1989

Use of N-ethylmaleimide to directly determine the proportion of beta-adrenergic receptors exhibiting high affinity for agonists on human mononuclear leukocyte membranes.

Following incubation with the beta-adrenergic agonist isoproterenol (ISO) and the alkylating agent N-ethylmaleimide (NEM), the number of beta-adrenergic receptors measured on human mononuclear leukocyte (MNL) membranes decreased. Pretreatment induced an approximately 40% loss of measurable receptors. The proportion of receptors lost was that which formed the ternary complex of agonist-receptor-Gs protein (H-R-Gs) and exhibited high affinity for agonists. In comparison studies, the loss of receptors was found to be similar to the proportion of receptors that form the high affinity state of the receptor (%RH) for agonist in agonist competition binding studies. Acute exposure to agonist induced a similar reduction in ISO/NEM "sensitive" receptors and the %RH. These experiments indicate that pretreatment of MNL membranes with ISO/NEM is a powerful tool with which to directly determine the percentage of receptors able to form an agonist high affinity state.

Binding, Competitive↗
PubMed1990

Changes in inositol-1,4,5-trisphosphate binding to hepatic plasma membranes caused by temperature, N-ethylmaleimide and menadione.

We investigated the effects of the sulfhydryl-alkylating agent N-ethylmaleimide (NEM) and menadione--a sulfhydryl-arylating agent, which can undergo redox cycling--on the [3H]inositol-1,4,5-trisphosphate ([3H]IP3) binding properties of rat plasma membranes. Rat liver plasma membranes were incubated for 15 min at 37 degrees with 0.1 mM, 0.2 mM, 0.5 mM NEM or 0.3 mM menadione and subsequently diluted for use in [3H]IP3 binding studies. An incubation as such (15 min at 37 degrees) already caused the dissociation constant (Kd) of [3H]IP3 binding to increase from 1.9 +/- 0.2 nM to 3.4 +/- 0.2 nM, with only a small change in the maximal number of IP3 binding sites (Bmax-values of 401 +/- 32 and 349 +/- 13 fmol/mg protein, respectively). Incubation with NEM (0.1, 0.2 and 0.5 mM) resulted in a dose dependent decrease in the Bmax with 41, 87 and 99%, respectively, without a significant change in the Kd compared to the time matched controls. Menadione (0.3 mM) decreased the Bmax with 54% without affecting the Kd. In contrast to our findings at 37 degrees, incubation of the plasma membranes with NEM (0.5 mM) at 0 degrees for 30 min did not affect [3H]IP3 binding. In order to account for this discrepancy, the reaction rate of NEM with glutathione was examined at both 0 degrees and 37 degrees by recording the changes in the UV-spectrum of NEM (lambda max = 302 nm) after addition of 1 mM NEM to 1 mM glutathione. A similar reaction rate was observed at both temperatures. These data suggest that alkylation of a sulfhydryl-moiety in the IP3-receptor molecule causes inactivation of the receptor function. Since at 0 degrees NEM is still able to react with sulfhydryl groups, but not able to inactivate the IP3-receptor, it can be suggested that the sulfhydryl-moiety of the IP3-receptor is masked and cannot be reached by a sulfhydryl-alkylating agent at 0 degrees.

Animals↗
PubMed1981

Dissociation of frog olfactory epithelium with N-ethylmaleimide.

Treatment of frog olfactory epithelium with 8 mM N-ethylmaleimide for 2 min results in extensive dissociation of the epithelium. The resulting cell suspension contains single olfactory receptor neurons, sustentacular cells, respiratory epithelial cells, and cells of Bowman's glands. The cells in suspension exhibit the same morphologies seen in histological sections of intact epithelium.

Animals↗
PubMed1985

The aminoglycoside antibiotic, gentamicin, fails to block increases in miniature endplate potential frequency induced by the sulfhydryl reagent, N-ethylmaleimide, in low calcium solutions.

N-ethylmaleimide (NEM) increases the frequency of miniature endplate potentials (MEPPs) at the adult rat hemidiaphragm. This sulfhydryl-alkylating agent produces comparable effects in the absence of added calcium (2 mM EGTA), suggesting that the drug releases calcium from internal stores, or promotes calcium-independent release by depolarizing the nerve terminal or interacting more directly with the release mechanism. These increases in frequency are not blocked by the aminoglycoside antibiotic, gentamicin; although the latter agent reduces quantal content and the elevations in MEPP frequency induced by high potassium solutions. The results suggest that gentamicin and NEM act at different sites at the presynaptic terminal, and that the aminoglycosides block voltage-dependent presynaptic calcium influx.

Action Potentials↗
PubMed1986

Heterogeneity of binding sites for N-ethylcarboxamido[3H]adenosine in rat brain: effects of N-ethylmaleimide.

Binding sites for N-ethylcarboxamido[3H]adenosine (NECA) in rat brain membranes and cryostat sections were examined in relation to their sensitivities to displacement by unlabeled NECA and R(-)-phenylisopropyladenosine (R-PIA). In membrane fractions from cerebral cortex, cerebellum, hippocampus and striatum, nanomolar concentrations of these adenosine receptor agonists displaced [3H]NECA such that R-PIA was more effective than NECA, consistent with the presence of an A1-adenosine receptor. At concentrations of displacing agent higher than 1 microM, R-PIA was unable to displace [3H]NECA further in cerebral cortex, cerebellum and hippocampus. In striatum, a second R-PIA-sensitive component of [3H]NECA binding was evident which was more sensitive to NECA than to R-PIA, i.e. it showed the characteristics of an A2-adenosine receptor. In striatum, however, R-PIA was also unable to displace [3H]NECA binding completely. Similar results were obtained in quantitative autoradiographic studies. Preincubation of cryostat sections with N-ethylmaleimide (NEM) abolished both the A1- and R-PIA-insensitive binding components such that both NECA and R-PIA were able to displace [3H]NECA binding completely. The remaining sites showed IC50 values of 0.13 and 3.68 microM for NECA and R-PIA, respectively. These A2-like [3H]NECA binding sites had a highly specific distribution in the brain, being concentrated in the striatum, nucleus accumbens and olfactory tubercle. The results indicate the presence in brain tissue of at least 3 classes of [3H]NECA binding sites, an A1-site, an A2-site and a third, unidentified R-PIA-insensitive site.

2-Chloroadenosine↗
PubMed1986

Molecular distinction between calf heart and brain muscarine receptors: different N-ethylmaleimide modulation of agonist binding.

Muscarinic acetylcholine receptors in calf heart and forebrain membranes were identified by binding of 1-[benzilic-4,4'-3H]quinuclidinyl benzilate ([3H]QNB). We were able to solubilise these receptors with a yield of 50% of the proteins by treatment of the membranes with digitonin. The existence of two or more receptor subclasses with different agonist affinity in the heart membranes was evidenced by the shallow carbachol/[3H]QNB competition binding curves. The receptors displayed only low agonist affinity, in the presence of GTP as well as after solubilisation. The alkylating reagent N-ethylmaleimide (NEM) caused a 70-fold increase in agonist affinity for the solubilised receptors whereas GTP was ineffective. A similar difference in affinity was observed for the membranes when agonist competition curves in the presence of NEM were compared to those in the presence of GTP. NEM caused only a 2- to 3-fold increase of the agonist affinity for solubilised brain cortex membranes. These data suggest that heart and brain muscarine receptors are structurally different.

Animals↗
PubMed1987

N-ethylmaleimide blocks the modulatory effects of divalent cations and guanine nucleotides on the brain substance P receptor.

The binding of [3H]physalaemin ([3H]PHY) to rat brain substance P receptors is modulated by cations and guanine nucleotides. [3H]PHY binding in the presence of either monovalent or divalent cations (125 mM Na2SO4 or 2.5 mM MnCl2) shows a KD of 5.9 and 5.5 nM and a Bmax of 44.4 and 63.9 fmol/mg protein respectively. In the presence of both, there is a 2-fold increase in the affinity (KD 2.8 nM) and a 25-80% increase in the Bmax (81.6 fmol/mg protein). Addition of 100 microM GTP or Gpp(NH)p in either 125 mM Na2SO4 or 2.5 mM MnCl2 or both decreases the Bmax by 25-55%. However, the receptor affinity for [3H]PHY is not significantly altered by guanine nucleotides. N-Ethylmaleimide (NEM) irreversibly inhibits the receptor binding with an IC50 of 1.0 mM, demonstrating that SH groups play a critical role in the interaction of the ligand with the receptor. If the SP receptors are protected with 1 microM PHY, NEM irreversibly inhibits the effect of divalent cations and guanine nucleotides. Analysis of [3H]PHY binding in 125 mM Na2SO4, 2.5 mM MnCl2 on membranes that were protected with 1 microM PHY and then preincubated with NEM demonstrates a variable decline in receptor number and a 2-fold decrease in the affinity (KD, from 2.8 to 6.9 nM). These observations indicate the existence of a second class of SH groups that are essential for the interaction of divalent cations and guanine nucleotides with the receptor. The blockade of the modulatory effects of divalent cations and guanine nucleotides by NEM treatment further suggests that brain SP receptors are coupled to a guanine nucleotide binding regulatory protein.

Animals↗
PubMed1988

Uncoupling of the platelet alpha 2-adrenoceptor adenylate cyclase system by N-ethylmaleimide and the resulting effect on receptor density and sensitivity.

Preincubation of platelet-rich plasma with N-ethylmaleimide (NEM) attenuated the inhibitory effect of the alpha 2-adrenoceptor agonist UK 14304 on basal and forskolin-stimulated adenylate cyclase activities. NEM also led to concomitant marked reductions of the specific binding of [3H]UK 14304 to platelet membranes and of the primary aggregation response induced by UK 14304. These results indicate that uncoupling of the receptor adenylate cyclase system by NEM induces down-regulation of platelet alpha 2-adrenoceptor density (3H-agonist binding sites) and of the associated functional response (platelet aggregation).

Adenylyl Cyclases↗
PubMed1989

Intracerebroventricular N-ethylmaleimide differentially reduces supraspinal opioid analgesia in mice.

I.c.v. injection of 1 nmol N-ethylmaleimide (NEM) into mice interfered with opioid-induced supraspinal analgesia, as assessed in the warm water tail-flick test. This effect of NEM was long-lasting (more than 3 days), non-competitive and differentially inhibited by the opioids studied. The analgesia induced by [D-Ala2,D-Leu5]enkephalin (DADLE), [D-Ala2,Met5]enkephalinamide (DAME) and [D-Pen2,D-Pen5]enkephalin (DPDPE) was greatly reduced in NEM-treated mice. The antinociception elicited by [D-Ala2,N-MePhe4,Gly-ol5]enkephalin (DAGO) was also impaired although to a lesser extent. In contrast, the activity of morphine and etorphine remained unchanged. NEM-sensitive opioids coadministered with morphine antagonized the analgesia elicited by the alkaloid in NEM-treated mice. The administration of naltrexone or DADLE, DAGO, [D-Ala2,N-MePhe4,Met-(O)5-ol]enkephalin (FK-33824) and morphine in doses equivalent to the ED90 doses for inducing analgesia, a few minutes before NEM prevented it from interfering with DADLE-elicited supraspinal analgesia when evaluated 24 h later. In contrast, the selective delta antagonist, ICI 174864, did not protect the DADLE-induced analgesia against the effect of NEM. We suggest that NEM produced its effect by acting upon a site that appears to be distal to the receptor binding site, presumably located on the guanine nucleotide binding regulatory proteins, Gi/Go. Therefore, these transducer proteins might play a key role in the effects displayed by opioids when acting via the mu receptor-Gi/Go complexes.

Analgesics↗
PubMed1990

Pre- and postjunctional effects of N-ethylmaleimide in the isolated mouse vas deferens.

The influence of N-ethylmaleimide (NEM) on contractions due to exogenously applied noradrenaline and bethanechol and on the inhibitory effects of clonidine, of the enkephalin derivative, FK 33-824, and 2-chloroadenosine (2-CLA) on field stimulation-response curves and [3H]noradrenaline [( 3H]NA) release was studied in the isolated mouse vas deferens. Exposure to NEM (60 microM: 10 min) caused a 30% reduction of the maximal contraction due to NA but nearly abolished the response to bethanechol. NEM partially reversed the depression of the pulse width-response curves by clonidine and FK 33-824 but was without effect with 2-CLA. The contractions evoked by stimulation frequencies above 20 Hz were depressed by NEM both in presence and absence of the agonists. NEM diminished the inhibition of the stimulation-evoked release of [3H]NA by the three agonists. The prejunctional effect of NEM was markedly influenced by the stimulation parameters. These findings support the suggestion that the inhibition mediated by alpha 2-adrenoceptors, mu- and P1-receptors in the mouse vas deferens is NEM-sensitive and possibly transmitted by a pertussis toxin-sensitive G-protein.

2-Chloroadenosine↗
PubMed1985

Changes in the organization of actin and myosin in non-muscle cells induced by N-ethylmaleimide.

There is evidence from in vitro studies that the SH reagent N-ethylmaleimide (NEM) causes the formation of ATP-resistant rigor-complexes between actin and myosin, and NEM-modified heavy meromyosin has been used by Cande et al. to study the contractile process during cytokinesis. It is reported here that treatment of tissue-cultured cells with NEM causes an immediate cessation of all motile activities and a simultaneous stabilization of the ultrastructure of the cell visualized on lysis with detergent-containing buffers. After NEM treatment a 5- to 10-fold increase in the amount of myosin was found associated with the detergent-resistant cell residues. As suggested by immunoelectron microscopy, using antibodies to non-muscle myosin together with gold-labelled protein A, increasing amounts of myosin filaments became associated with the microfilament assemblies of the cell with time of NEM treatment. In addition to this there was a slow, progressive reorganization of the cortical wave of microfilaments. The structures interpreted as myosin filaments were visualized at relatively high resolution. The immunoelectron microscopy finally also indicated the presence of a non-filamentous form of myosin in agreement with the results of others.

Actins↗