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PubMed1991

Dual effects of N-ethylmaleimide on ethanol-induced gastric lesions in rats.

The effects of N-ethylmaleimide (NEM), a sulfhydryl (SH) blocker, on ethanol-induced gastric lesions were investigated in rats by varying the route of administration. Oral administration of acidified ethanol (60% ethanol in 150 mM HCl, 1 ml) produced hemorrhagic bandlike lesions in the gastric mucosa. Pretreatment of the animals with orally administered NEM (0.1-10 mg/kg) dose-dependently inhibited these lesions (the inhibition was over 80% at 1 mg/kg or greater), and the effects were partially reversed by indomethacin (5 mg/kg, subcutaneous). However, when NEM (10 mg/kg) was given subcutaneously, this agent significantly worsened the lesions. Intragastrically applied NEM produced a dose-dependent reduction of the transmucosal potential difference (PD) and the mucosal nonprotein SH levels, an increase of the volume of gastric contents, and an inhibition of gastric motility, while these parameters remained unaltered after subcutaneous administration of the agent. The microvascular permeability in the mucosa was significantly increased by both oral and subcutaneous administration of NEM (10 mg/kg) but remained unchanged in response to lower doses of orally administered (less than 3 mg/kg). These results suggest that NEM given orally is cytoprotective to the stomach against ethanol, probably by acting as a mild irritant and due to dilution of an irritant and inhibition of gastric motility (muscle relaxation), but when given subcutaneously it aggravates the lesions by unknown mechanisms.

Administration, Oral↗
PubMed1992

Effects of N-ethylmaleimide on the structure of skinned frog skeletal muscles.

The effects of N-ethylmaleimide (NEM) and other sulfhydryl modifiers on the structure of skinned frog skeletal muscles were studied using the X-ray diffraction technique. In sartorius muscle with full overlap between the thick and thin filaments, 0.1-1.0 mM NEM changed the intensity ratio of the (1,0) and (1,1) equatorial reflections from 4.35 to 0.72, and the (1,0) spacing of the hexagonal filament lattice from 40.4 to 41.4 nm. The axial X-ray diffraction pattern showed weak myosin layer-lines after the NEM treatment but enhancement of the actin layer-lines was not observed. In overstretched semitendinosus muscle, NEM did not affect the equatorial spacing but the myosin layer-lines were weakened. These results indicate that modification of myosin by NEM destroys the helical arrangement of myosin heads around the shaft of the thick filament and that when thin filaments are available, myosin heads move towards, and possibly bind to them. This binding is different from that in rigor since the 'ladder-like' appearance of the higher actin layer-lines, which is typical of patterns from rigor muscles, was not observed. On removal of ATP after the NEM treatment, the diffraction pattern showed features characteristic of that from normal rigor muscles but no tension was produced. The pattern showed well-defined samplings on layer-lines in the small-angle region, indicating the presence of an extensive lattice order and exact axial alignment of the filaments. The first actin layer-line did not show samplings from the superlattice of the thick filaments, which are observed on the myosin layer-lines in patterns from resting muscles.(ABSTRACT TRUNCATED AT 250 WORDS)

Actin Cytoskeleton↗
PubMed1987

Effect of the X-linked Hyp mutation on N-ethylmaleimide labelling of proteins in renal brush border membrane.

The X-linked dominant mutation, hypophosphataemia (gene symbol, Hyp) is expressed in the laboratory mouse as deficient phosphate transport at the renal brush border membrane (BBM) of proximal nephron. In an attempt to identify proteins which mediate phosphate transport, we treated renal BBM vesicles prepared from mutant male (Hyp/Y) and normal male (+/Y) littermates, with radiolabelled N-ethylmaleimide (NEM), in the presence or absence of arsenate of arsenate which is a competitive inhibitor of phosphate transport. Polyacrylamide gel electrophoresis revealed labelling of membrane proteins in the 40-45 kDa range; addition of arsenate during NEM treatment inhibited labelling. These findings indicate a 40-45 kDa protein as a component of the renal BBM phosphate transport system(s). We found no difference between protein labelling of the renal BBM from Hyp/Y and +/Y mice.

Alkaline Phosphatase↗
PubMed1983

Thiol-dependent passive K/Cl transport in sheep red cells: III. Differential reactivity of membrane SH groups with N-ethylmaleimide and iodoacetamide.

Treatment with N-ethylmaleimide (NEM) is known to stimulate ouabain-insensitive, Cl- -dependent K+ transport in low K+ (LK) but not in high K+ (HK) sheep red cells (Lauf, P.K., and Theg, B.E., 1980, Biophys. Biochem. Res. Commun. 92:1422-1428). The dependence of this effect on the pH of pretreatment with NEM and/or iodoacetamide (IAA) was studied. Maximum stimulation of Cl- -dependent K+ transport in LK red cells was produced by prior treatment with 1-5 mM NEM at pH 6 at which only about 30-40% of the 10(7) SH groups present per membrane reacted. At pH 6 no NEM effect was seen on Nap K+ fluxes in HK red cells. Treatment with NEM below pH 6 enhanced Cl- -independent K+ transport in both LK and HK red cells. At higher pH values or higher concentrations the NEM-stimulation of K+ transport was reduced and absent at pH 8.7. Exposure of LK cells to 5 mM IAA prior to NEM abolished the stimulatory effect of NEM on K+ transport. Hence at least two different chemical groups were reacting with NEM, and more alkaline SH or NH2 groups whose reaction with NEM leads to an inhibition of the NEM effect brought about at pH 6.

Animals↗
PubMed1987

Furosemide-sensitive K+ (Rb+) transport in human erythrocytes: modes of operation, dependence on extracellular and intracellular Na+, kinetics, pH dependency and the effect of cell volume and N-ethylmaleimide.

The effect of extracellular and intracellular Na+ (Nao+, Nai+) on ouabain-resistant, furosemide-sensitive (FS) Rb+ transport was studied in human erythrocytes under varying experimental conditions. The results obtained are consistent with the view that a (1 Na+ + 1 K+ + 2 Cl-) cotransport system operates in two different modes: mode i) promoting bidirectional 1:1 (Na+-K+) cotransport, and mode ii) a Nao+-independent 1:1 ki+ exchange requiring Nai+ which, however, is not extruded. The activities of the two modes of operation vary strictly in parallel to each other among erythrocytes of different donors and in cell fractions of individual donors separated according to density. Rb+ uptake through Rbo+/Ki+ exchange contributes about 25% to total Rb+ uptake in 145 mM NaCl media containing 5 mM RbCl at normal Nai+ (pH 7.4). Na+-K+ cotransport into the cells occurs largely additive to K+/K+ exchange. Inward Na+-Rb+ cotransport exhibits a substrate inhibition at high Rbo+. With increasing pH, the maximum rate of cotransport is accelerated at the expense of K+/K+ exchange (apparent pK close to pH 7.4). The apparent KmRbo+ of Na+-K+ cotransport is low (2 mM) and almost independent of pH, and high for K+/K+ exchange (10 to 15 mM), the affinity increasing with pH. The two modes are discussed in terms of a partial reaction scheme of (1 Na+ + 1 K+ + 2 Cl-) cotransport with ordered binding and debinding, exhibiting a glide symmetry (first on outside = first off inside) as proposed by McManus for duck erythrocytes (McManus, T.J., 1987, Fed. Proc., in press). N-ethylmaleimide (NEM) chemically induces a Cl--dependent K+ transport pathway that is independent of both Nao+ and Nai+. This pathway differs in many properties from the basal, Nao+-independent K+/K+ exchange active in untreated human erythrocytes at normal cell volume. Cell swelling accelerates a Nao+-independent FS K+ transport pathway which most probably is not identical to basal K+/K+ exchange. Ko+ less than Nao+ less than Lio+ less than Mgo2+ reduce furosemide-resistant Rb+ inward leakage relative to cholineo+.

Bumetanide↗
PubMed1989

Inactivation of the rabbit parotid Na/K/Cl cotransporter by N-ethylmaleimide.

The inactivation of the rabbit parotid Na/K/Cl cotransporter by the irreversible sulfhydryl reagent N-ethylmaleimide (NEM) is studied by monitoring its effect on high affinity bumetanide binding to the carrier. NEM reduces the number of bumetanide binding sites with no significant change in the affinity of those remaining. NEM also reduces KCl-dependent 22Na flux via the cotransporter by the same factor as the reduction in bumetanide binding sites. Both bumetanide and its analogue furosemide can protect against the effect of NEM. The concentration range over which this protection occurs is in good agreement with affinities of these two compounds for the high affinity bumetanide binding site (2.6 and 8.5 microM, respectively), indicating an association of this site with the site of action of NEM. Also consistent with this hypothesis are the observations that (i) sodium and potassium, both of which are required for high affinity bumetanide binding, increase the rate of inactivation of binding by NEM and (ii) chloride, at concentrations previously shown to competitively inhibit bumetanide binding, protects the cotransporter against NEM. The effects of NEM on bumetanide binding are mimicked by another highly specific sulfhydryl reagent, methyl methanethiolsulfonate. The apparent rate constant for inactivation of high affinity bumetanide binding by NEM is a hyperbolic function of NEM concentration consistent with a model in which the inactivation reaction is first order in [NEM] and proceeds through an intermediate adsorptive complex. The data indicate that the presence of a reduced sulfhydryl group at or closely related to the bumetanide binding site is essential for the operation of the parotid Na/K/Cl cotransporter.

Animals↗
PubMed1991

N-ethylmaleimide antagonizes stress-induced gastric ulcers in rats.

N-ethylmaleimide (NEM) 10 or 25 mg/kg b.wt, given s.c. 20 min beforehand, dose-dependently and significantly antagonizes the severity of gastric glandular ulcers produced by restraint at 4 degrees C (stress) for 2 h. These findings suggest that reduced activity of endogenous nonprotein sulfhydryl substances in gastric tissue does not worsen stress-induced ulceration in rat stomachs, unlike the deleterious effect its depletion is claimed to have on ethanol-evoked gastric mucosal damage. Thus, decreased SH activity appears not to play a role in the aetiology of mucosal ulcers due to stress.

Animals↗
PubMed1984

The effect of group selective reagents N-ethylmaleimide and dithiothreitol on histamine H1-receptor binding sites in the vascular smooth muscle membranes.

The chemical nature of the histamine H1-receptors of beef aortic membranes has been elucidated by introducing two group selective reagents in the [3H]-mepyramine binding studies: dithiothreitol (DTT), a protein-disulphide group reducing reagent, and N-ethylmaleimide (NEM), a protein-thiol group alkylating agent. In the binding experiments, NEM independently inhibits [3H]-mepyramine binding. The inhibition is time and concentration dependent. DTT on the other hand potentiates the binding of the radioligand to its receptor and changes the affinity of histamine in competing for [3H]-mepyramine binding site. In the DTT-pretreated membranes (100 microM), histamine shows a higher affinity for [3H]-mepyramine binding (Ki 0.35 microM) than in the untreated membranes (Ki 3.7 microM). Comparison of the pharmacological studies on the DTT-treated rabbit aortic strips and above binding studies, revealed a good correlation between the changes in the affinity of histamine for its receptor, when DTT was present. The results suggest an important role of the S-S and SH groups in the function of aortic histamine H1-receptor.

Animals↗
PubMed1997

Binding domains for blockers and substrates on the dopamine transporter in rat striatal membranes studied by protection against N-ethylmaleimide-induced reduction of [3H]WIN 35,428 binding.

Binding sites for 2 beta-carbomethoxy-3 beta-(4-fluorophenyl)[3H]tropane ([3H]WIN 35,428) on rat striatal membranes were alkylated with N-ethylmaleimide (NEM), and the protective potency was measured of the blockers cocaine, N[1-(2-benzo[b]thiophenyl) cyclohexyl]piperidine (BTCP), benztropine, WIN 35,428; and nomifensine, and of the substrates dopamine, norepinephrine, S(+)-amphetamine, tyramine, and metaraminol. In general, the protective potency was lower (at least 3 times) than the potency in inhibiting [3H]WIN 35,428 binding with the compounds present under the same experimental conditions used for the NEM-induced alkylation. However, the disparity was substantially greater for all substrates tested (10- to 93-fold) than for the blockers (2- to 6-fold), especially cocaine and BTCP (3-fold). [3H]WIN 35,428 binding was best described by a l-site model under the present conditions. The results are discussed in terms of models involving blocker-induced conformational changes and overlapping nonidentical binding domains for blockers and substrates.

Amphetamine↗
PubMed2002

Inhibition of transport function and desipramine binding at the human noradrenaline transporter by N-ethylmaleimide and protection by substrate analogs.

N-ethylmaleimide (NEM) inhibits [(3)H]desipramine binding and [(3)H]noradrenaline uptake at the rat noradrenaline transporter (rNET) by covalently modifying cysteine residues. We report here that NEM also inhibits [(3)H]desipramine binding and [(3)H]noradrenaline uptake at the cloned human noradrenaline transporter (hNET) stably expressed in C6 glial cells. The IC(50) for NEM inhibition of [(3)H]noradrenaline uptake was 43.6+/-5.5 microM. We tested several compounds for their abilities to inhibit [(3)H]noradrenaline uptake via the hNET and for their abilities to protect against NEM inactivation of [(3)H]desipramine binding. We found that the substrate analogs bupropion, 3-bromomethcathinone, and 4-bromomethcathinone all inhibit uptake at the hNET with IC(50) values of 1370+/-140, 158+/-20, and 453+/-30 nM, respectively. These compounds as well as methamphetamine, methcathinone, and desipramine also protected the hNET from NEM inactivation of [(3)H]desipramine binding. The ability of substrate analogs and desipramine to protect the [(3)H]desipramine binding site is consistent with the hypothesis that the desipramine binding site and the substrate binding site are mutually exclusive. It also supports the use of structure-activity relationships derived from substrate analogs in the rational design of hNET uptake inhibitors. The hNET contains 10 cysteine residues whereas the rNET contains 12 cysteine residues. Since the hNET and the rNET are both inhibited by NEM, and because the NEM inhibition is protectable by desipramine and substrate analogs, we conclude that the two additional cysteine residues (C28 and C447) present in the rNET are not likely to be involved in desipramine binding or uptake function.

Animals↗
PubMed2000

Stimulation of human erythrocyte K-Cl cotransport and protein phosphatase type 2A by n-ethylmaleimide: role of intracellular Mg++.

An increase in the activity of membrane-associated protein phosphatase type 1 (mb-PP1) is associated with stimulation of erythrocyte K-Cl cotransport (KCC). We have recently proposed that membrane-associated protein phosphatase type 2A (mb-PP2A) is also involved in KCC regulation by cell swelling (Bize et al., 1999. Am. J. Physiol. 277:C899-C912). We used two protein phosphatase inhibitors, okadaic acid (OA) and calyculin A (CalA), and two KCC activating treatments, n-ethylmaleimide (NEM) and Mg(i)++-depletion, and determined KCC transport activity and mb-PP1 and mb-PP2A activities. OA, an inhibitor of erythrocyte mb-PP2A, partially prevents stimulation of KCC activity by NEM but not by Mg(i)++-depletion. CalA, an inhibitor of both mb-PP1 and mb-PP2A prevents stimulation of KCC activity by both treatments. NEM and Mg(i)++-depletion inhibit mb-PP1 activity, suggesting that activation of KCC can take place in the absence of mb-PP1 activation. Mb-PP2A activity is stimulated in NEM-treated cells but not in Mg(i)++-depleted cells. In NEM-treated cells, Mg(i)++-depletion inhibits both KCC and mb-PP2A. In Mg(i)++-depleted cells, NEM does not stimulate KCC or mb-PP2A. The strong correlation between KCC stimulation and mb-PP2A stimulation provides further support to the idea that mb-PP2A plays an important role in KCC regulation. Our results are consistent with the hypothesis that KCC regulation involves at least two distinguishable phosphorylation sites.

Carrier Proteins↗
PubMed2000

Functional coupling of GABA(B) receptors with G proteins that are sensitive to N-ethylmaleimide treatment, suramin, and benzalkonium chloride in rat cerebral cortical membranes.

Although it is known that GABA(B) receptors are negatively coupled to adenylyl cyclase, the detailed selectivity of functional interaction between GABA(B) receptors and Gi subfamily members is still ambiguous. (+/-)-Baclofen-stimulated high-affinity GTPase activity, which was competitively antagonized by 2-hydroxy-saclofen, was attenuated by pretreatment of the membranes with N-ethylmaleimide (NEM) in a concentration- and incubation period-dependent manner. The NEM-pretreated (50 microM, 15 min at 4 degrees C) membranes restored the (+/-)-baclofen-sensitive high-affinity GTPase activity when reconstituted with pertussis toxin-sensitive bovine brain G proteins. Among recombinant rat Galpha subunits, G(i alpha(-2)) appeared most effective as compared with other subunits (G(i alpha(-2)) > G(i alpha(-3) > G(i alpha(-1) = G(o alpha). The GABA(B) receptor-mediated high-affinity GTPase activity was also completely eliminated by 100 microM suramin and by 100 microM benzalkonium chloride. These results indicate that GABA(B) receptors in rat cerebral cortex couple to NEM-sensitive G proteins, in particular Gi2, which are sensitive to suramin and benzalkonium chloride.

Adenylate Cyclase Toxin↗
PubMed1986

Alkaline hydrolysis of N-ethylmaleimide allows a rapid assay of glutathione disulfide in biological samples.

The estimation of glutathione disulfide (GSSG) is based on the NADPH-dependent glutathione reductase reaction. A new method has been developed to eliminate the inactivating effect of N-ethylmaleimide (NEM), added to prevent glutathione oxidation, on glutathione reductase. This method takes advantage of instability of NEM in alkaline solutions. The product of NEM hydrolysis, N-ethylmaleamic acid, obtained under accurate pH-controlled conditions, is compatible with a good activity of glutathione reductase which allows total recovery and measurement of GSSG. The method, applied to estimation of GSSG content in human erythrocytes and rat liver, gives results in optimum agreement with values reported in literature. Because of its simple performance and rapidity, the procedure can be considered an improved method in removing NEM and is particularly advantageous when a large number of biological samples must be treated and estimated.

Animals↗
PubMed1992

Assay of thiols and disulfides based on the reversibility of N-ethylmaleimide alkylation of thiols combined with electrolysis.

A simple and specific method for analyzing thiols and disulfides on the basis of the reversibility of N-ethylmaleimide (NEM) alkylation of thiols is described. When the adduct of NEM and glutathione (GSH) was electrolyzed at neutral pH, all of the GSH was recovered. When the adduct was exposed to pH 11.0 for 15 min at 30 degrees C before electrolysis, GSH was not detected. The same behavior was observed after protein thiols reacted with NEM. This pH-dependent production of thiol from the adduct was used to assay GSH and oxidized glutathione in yeast cells, to assay sulfhydryl groups and disulfide bonds in authentic proteins, and to protect thiols from oxidation during enzymatic digestion of protein. This method is useful for assay of thiols and disulfides of both small and large molecules and can be used to identify labile thiols in biological samples that are oxidized during extraction procedures.

Alkylation↗
PubMed1977

The effects of N-ethylmaleimide on active amino acid transport in Escherichia coli.

N-Ethylmaleimide (MalNEt) binds covalently and without specificity to accessible sulfhydryl residues in proteins. In some cases specificity has been imposed on this reaction by manipulating reaction conditions, yielding information concerning both enzyme mechanism and the identity of specific proteins (for example C.F. Fox and E.P. Kennedy (1965) Proc. Natl. Acad. Sci. u.s. 54, 891-899) and R.E. McCarty and J. Fagan (1973) Biochemistry 12, 1503-1507). We have examined the effects of MalNEt on the active accumulation of nine amino acids by Escherichia coli strains ML 308-225 and DL 54. Whole cells have been used in order that transport systems both dependent on and independent of periplasmic binding proteins could be studied under various conditions of energy supply for transport. Our results suggest that the systems transporting ornithine, phenylalanine and proline are those most likely to undergo inactivation by direct reaction of MalNEt with the transport apparatus, rather than merely via side effects such as interruption of their energy supply. The inhibition of proline transport is specifically enhanced by the presence of proline, competitive inhibitors of proline transport, or carbonylcyanide p-trifluoromethyoxyphenylhydrazone during MalNEt treatment. The other eight systems tested showed no analogous effects.

Amino Acids↗
PubMed1989

Accessibility of the N-ethylmaleimide-unreactive sulfhydryl of human erythrocyte Band 3.

The human erythrocyte anion exchange protein, Band 3, was reacted with N-ethylmaleimide (NEM) in cells to a stoichiometry of 5.3 mol NEM per mol Band 3, indicating that all NEM-reactive cysteines in Band 3 were labeled. Quantitatively NEM-blocked Band 3 was still able to bind to and be eluted by reducing agents from a mercurial affinity resin, [p-(chloromercuribenzamido)ethylene]amino-Sepharose. Reaction of NEM-blocked Band 3 with p-chloromercuribenzoate (pCMB) did not prevent binding to the resin due to exchange of pCMB for the immobilized mercurial. pCMB has been reported to inhibit water and urea permeation across the red cell membrane, and this has been attributed to reaction with a NEM-reactive sulfhydryl in Band 3. The interaction of Band 3 with the immobilized ligand directly demonstrates the reaction of NEM-blocked Band 3 with a mercurial and indicates that the NEM-unreactive, pCMB-reactive sulfhydryl residue is accessible to within approximately equal to 12 A (the distance from the solid support to the Hg) of the surface of the solubilized Band 3 protein.

Anion Exchange Protein 1, Erythrocyte↗
PubMed1989

Cation transport in oxidant-stressed human erythrocytes: heightened N-ethylmaleimide activation of passive K+ influx after mild peroxidation.

Normal and chronically dehydrated (hereditary xerocytosis) human red cells were subjected to mild peroxidative treatment (315 microM hydrogen peroxide (H2O2), 15 min) in the presence of azide. The subsequent expression of passive (ouabain-resistant) K+ transport activities was analyzed by measurement of 86Rb+ influx. Peroxidation of normal red cells did not affect basal K+ transport activity, but the increment in K+ influx elicited by 0.5 mM N-ethylmaleimide (NEM) was increased 3-fold. The enhanced K+ influx was chloride-dependent, but only partially inhibited by 0.1 mM furosemide. Stimulated activity declined progressively after NEM activation, but could be restored by a second NEM treatment. Prior conversion of hemoglobin to the carbonmonoxy form abolished the response to peroxide, while 200 microM butylated hydroxytoluene (BHT) exerted only partial inhibition, suggesting that the effect of H2O2 requires interaction of activated, unstable hemoglobin species with the membrane, but that lipid peroxidation is not sufficient. Peroxidation following NEM treatment also enhanced NEM activation, indicating that enhancement does not require altered NEM reactions with stimulatory or inhibitory sites. Passive K+ transport in hereditary xerocytosis red cells was not activated by NEM, with or without H2O2 pretreatment. The results demonstrate that modest peroxidative damage to red cells can heighten the activation of a transport system that is thought to be capable of mediating net K+ efflux and volume reduction in cells that express it. Models are proposed in which the effects of NEM, H2O2, cell swelling and other factors are mediated by conformational changes in a postulated subpopulation of anion channel (Band 3) molecules that bind the K+ transporter.

Butylated Hydroxytoluene↗