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At least 19 recordsLinked to original sources
PubMed1985

N-Ethylmaleimide inhibition of the catalytic activities of the Dunaliella salina coupling factor 1 (CF1) and the restoration of the inhibition of the CF1 ATPase activity by N-ethylmaleimide.

The sensitivity of the catalytic activities of the D. salina chloroplast coupling factor 1 (CF1) to chemical modification by N-ethylmaleimide has been investigated. When D. salina thylakoid membranes are treated with N-ethylmaleimide, both photophosphorylation and the inducible CF1 ATPase activity are partially (approx. 60%) inhibited. The inhibition of both activities does not require the presence of a proton-motive force, and the inhibition of photophosphorylation is directly related to the N-ethylmaleimide-covalent modification of CF1 as shown by the time-course for the inhibition and the maximal extent of inhibition. Treatment of the purified, latent, D. salina CF1 with low concentrations of N-ethylmaleimide also results in the partial (approx. 60%) inhibition of the inducible ATPase activity (I50 approximately 50 microM). The inhibition does not require the presence of the chemical modifier during the activation of the enzyme. N-ethylmaleimide-induced inhibition of the ATPase activity of either membrane-bound or solubilized CF1 is partially reversed by either prolonged incubation at low concentrations of N-ethylmaleimide or short incubation times at high concentrations of N-ethylmaleimide. The results are interpreted as indicating multiple binding sites on the D. salina CF1 that have different rates of reactivity with N-ethylmaleimide. Those sites (or site) that react rapidly with N-ethylmaleimide cause(s) an inhibition of both ATP synthase and ATPase activities, whereas those sites (or site) that react more slowly partially restore(s) the original ATPase activity. The effects of N-ethylmaleimide on the catalytic activity of D. salina CF1 are probably mediated by N-ethylmaleimide-induced conformational changes of the enzyme.

Chlorophyta↗
PubMed1990

Chemical modification of pig liver initiation factor eIF-2 with N-ethylmaleimide. Amino acid sequences around the N-ethylmaleimide-reactive sulfhydryl groups and the effect of GDP on the modification.

The activity of eukaryotic initiation factor eIF-2 as to the formation of the ternary complex, eIF-2 GTP Met-tRNA(f), is inhibited by N-ethylmaleimide. Our preparation of pig liver eIF-2 contained alpha and gamma subunits and was inhibited by more than 90% by N-ethylmaleimide. Using our eIF-2, we determined the sequences around the N-ethylmaleimide-reactive sulfhydryl groups, studied the effect of GDP on the sulfhydryl modification and that of NEM on the [3H]GDP binding, and examined the protective effect of GTP against the inhibition of ternary complex formation by N-ethylmaleimide. Both subunits of native eIF-2 contained [14C]N-ethylmaleimide-reactive sulfhydryl groups. One N-ethylmaleimide-reactive sulfhydryl group was in the alpha subunit and 4 were in the gamma subunit. The sequence of the peptide of the alpha subunit was determined to be: Ala-Gly-Leu-Asn-Cys-Ser-Thr-Glu-Thr-Met-Pro-Ile. Two of the four [14C]N-ethylmaleimide-reactive sulfhydryl groups in the gamma subunit were highly reactive, their sequences being: Ile-Val-Leu-Thr-Asn-Pro-Val-Cys-Thr-Glu-Val-Gly-Glu-Lys (gamma 1); Ser-Cys-Gly-Ser-Ser-Thr-Pro-Asp-Glu-Phe-Pro-Thr-Asp-Ile-Pro-Gly-Thr-Lys (gamma 3a). Peptide gamma 3a contained the consensus sequence element (AspXaaXaaGly) of GTP-binding proteins. With preincubation of eIF-2 with GDP, the incorporation of [14C]N-ethylmaleimide into the gamma subunit was reduced to 40% of the control level, but the 14C-incorporation into the alpha subunit did not change.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1982

Incorporation of N-ethylmaleimide into the membrane-bound ADP/ATP translocator. Isolation of the protein labeled with N-[3H]ethylmaleimide.

The incorporation of N-ethylmaleimide into the 30,000-Mr component of beef-heart mitochondria has been studied as a function of various ligands to the ADP/ATP carrier and the isolation of the N-ethylmaleimide-labeled protein is reported. 1. The incorporation of N-ethylmaleimide into the 30,000-Mr component is specifically stimulated by ADP and ATP. Thus by differential incorporation of N-ethylmaleimide, the 30,000-Mr component is preferentially labeled. 2. Addition of carboxyatractylate inhibits, whereas bongkrekate tolerates, the incorporation of N-ethylmaleimide. 3. After solubilization by Triton the purification of N-ethylmaleimide-labeled protein is facilitated in the presence of bongkrekate but not of carboxyatractylate, in agreement with the postulated existence of only a bongkrekate-N-ethylmaleimide-protein complex. The labeled protein was purified to homogeneity on hydroxyapatite in Triton and subsequently, after denaturation in dodecylsulfate, on Sepharose 6B. 4. The identify of the isolated labeled protein with the formerly isolated bongkrekate-protein or carboxyatractylate-protein complexes is confirmed by the isoelectric point and amino acid composition. 5. Two moles of N-ethylmaleimide must be incorporated into the 30,000-Mr component in order to inhibit fully the binding of one mole carboxyatractylate. This corresponds to one -SH group per unit.

Animals↗
PubMed1996

Studies on inhibition of mu and delta opioid receptor binding by dithiothreitol and N-ethylmaleimide. His223 is critical for mu opioid receptor binding and inactivation by N-ethylmaleimide.

The sensitivity of mu and delta receptor binding to dithiothreitol and N-ethylmaleimide was examined to probe receptor structure and function. Binding to both receptor types was inhibited by dithiothreitol (IC50 values = 250 mM), suggesting the presence of inaccessible but critical disulfide linkages. mu receptor binding was inhibited with more rapid kinetics and at lower N-ethylmaleimide concentrations than delta receptor binding. Ligand protection against N-ethylmaleimide inactivation suggested that alkylation was occurring within, or in the vicinity of, the receptor binding pocket. Sodium ions dramatically affected the IC50 of N-ethylmaleimide toward both receptor types in a ligand-dependent manner. Analysis of receptor chimeras suggested that the site of N-ethylmaleimide alkylation on the mu receptor was between transmembrane domains 3 and 5. Substitution of cysteines between transmembrane domains 3 and 5 and elsewhere had no effect on receptor binding or sensitivity toward N-ethylmaleimide. Serine substitution of His223 in the putative second extracellular loop linking transmembrane domains 4 and 5 protected against N-ethylmaleimide inactivation. The H223S substitution decreased the affinity of bremazocine 25-fold, highlighting the importance of this residue for the formation of the high affinity bremazocine binding site in the mu opioid receptor.

Amino Acid Sequence↗
PubMed1996

Soluble N-ethylmaleimide-sensitive-factor attachment protein and N-ethylmaleimide-insensitive factors are required for Ca2+-stimulated exocytosis of insulin.

Ca2+ stimulates exocytosis in permeabilized insulin-secreting cells. To investigate the putative cytosolic components involved in the Ca2+ response, HIT-T15 cells (a pancreatic B-cell line) were permeabilized with streptolysin-O, a procedure that allows rapid exchange of soluble components including macromolecules. We found that in this cell preparation the secretory response to Ca2+ but not to guanosine 5'-[gamma-thio]triphosphate was lost as a function of time and could be restored by rat brain cytosol in a concentration-dependent manner. Reconstitutive activity of rat brain cytosol was found in a high-molecular-mass heat-labile partially N-ethylmaleimide(NEM)-sensitive fraction. The NEM-sensitive factor (NSF) and the soluble NSF attachment protein (alpha-SNAP) were found to be expressed in HIT-T15 cells and largely lost (about 30% remaining) from porated cells. Recombinant alpha-SNAP partially reconstituted the Ca2+ response when added to the permeabilized cells. Moreover, alpha-SNAP restored the effect of NEM-treated cytosol to the level observed for untreated cytosol. In contrast, NSF was ineffective when preincubated alone or with NEM-treated cytosol. Our results indicate that both alpha-SNAP and NEM-insensitive cytosolic factors are involved in Ca2+-mediated exocytosis from endocrine HIT-T15 cells.

Animals↗
PubMed1980

Inhibition of lactose transport in E. coli by N-ethylmaleimide-beta-galactoside, N-ethylsuccinimide-beta-galactoside and N-ethylmaleimide.

N-ethylmaleimide (NEM) inhibits lactose uptake in E. coli by reacting with the M protein component of the lac permease system. In an attempt to estimate the distance between the NEM reactive site and the substrate binding site, we have synthesized a beta-galactoside with NEM as the aglycon moiety (NEM-gal). NEM-gal was a more effective inhibitor of lactose transport than was NEM. Part of the inhibition by NEM-gal was caused by competition with lactose for the substrate binding site. To estimate this part of the inhibition, we synthesized the saturated and thus the unreactive N-ethylsuccinimide (NES) analog of NEM-gal. Nes-gal was a competitive inhibitor of lactose uptake. The remainder of the inhibition by NEM-gal followed first-order kinetics with the same rate constant as NEM. In addition, the protective effect of thiodigalactoside against the inhibition of transport by NEM was also observed against irreversible inhibition by NEM-gal. We suggest that the reactivity of NEM was unaltered by bringing it near the beta-galactoside binding site by way of covalent attachment to galactose. We conclude that the distance between the NEM reactive site and the position of the glycosidic oxygen of beta-galactosides bound to the lactose site is greater than 8 A.

Biological Transport↗
PubMed1986

Uncoupling of gamma-aminobutyric acid B receptors from GTP-binding proteins by N-ethylmaleimide: effect of N-ethylmaleimide on purified GTP-binding proteins.

Treatment of membranes from bovine cerebral cortex with N-ethylmaleimide (NEM) resulted in inhibition of gamma-aminobutyric acid (GABA) binding to GABAB receptors. The binding curve for increasing concentrations of agonist was shifted to the right by NEM treatment. Guanine nucleotide had little effect on the binding of GABA to NEM-treated membranes. The addition of purified GTP-binding proteins, which were the substrates of islet-activating protein (IAP), pertussis toxin, to the NEM-treated membranes caused a shift of the binding curve to the left, suggesting modification of GTP-binding proteins rather than receptors by NEM. Therefore, the effect of NEM on two purified GTP-binding proteins, Gi (composed of three subunits with molecular weight of alpha, 41,000; beta, 35,000; gamma, 10,000) and Go (alpha, 39,000; beta, 35,000; gamma, 10,000) was studied. NEM did not significantly change guanosine 5'-(3-O-thio)triphosphate (GTP gamma S) binding and GTPase activity of these two proteins. In contrast, NEM-treated Gi and Go were not ADP-ribosylated by IAP and did not increase GABA binding to NEM-treated membranes. When alpha and beta gamma subunits were treated with NEM and then mixed with nontreated alpha and beta gamma to form Gi or Go, respectively, both oligomers with NEM-treated alpha-subunits lost their abilities to be IAP substrates and to couple to receptors. These results indicate that NEM uncoupled GTP-binding proteins from receptors by modifying alpha-subunits of GTP-binding proteins, and the site seemed to be on or near the site of ADP-ribosylation by IAP. When alpha and beta gamma subunits were treated with NEM and then mixed to form Gi or Go, GTP gamma S binding in the absence of Mg2+ and GTPase activity were changed, although they were not affected when oligomers were treated with NEM. The results suggest the existence of another sulfhydryl group which is protected from NEM by the association of subunits. The modification of this sulfhydryl group by NEM appeared to interfere with the interaction between alpha and beta gamma.

Adenosine Diphosphate Ribose↗
PubMed2003

Role of vesicle-associated membrane protein-2, through Q-soluble N-ethylmaleimide-sensitive factor attachment protein receptor/R-soluble N-ethylmaleimide-sensitive factor attachment protein receptor interaction, in the exocytosis of specific and tertiary granules of human neutrophils.

We have examined the role of the R-soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) synaptobrevin-2/vesicle-associated membrane protein (VAMP)-2 in neutrophil exocytosis. VAMP-2, localized in the membranes of specific and gelatinase-containing tertiary granules in resting human neutrophils, resulted translocated to the cell surface following neutrophil activation under experimental conditions that induced exocytosis of specific and tertiary granules. VAMP-2 was also found on the external membrane region of granules docking to the plasma membrane in activated neutrophils. Specific Abs against VAMP-2 inhibited Ca(2+) and GTP-gamma-S-induced exocytosis of CD66b-enriched specific and tertiary granules, but did not affect exocytosis of CD63-enriched azurophilic granules, in electropermeabilized neutrophils. Tetanus toxin disrupted VAMP-2 and inhibited exocytosis of tertiary and specific granules. Activation of neutrophils led to the interaction of VAMP-2 with the plasma membrane Q-SNARE syntaxin 4, and anti-syntaxin 4 Abs inhibited exocytosis of specific and tertiary granules in electropermeabilized neutrophils. Immunoelectron microscopy showed syntaxin 4 on the plasma membrane contacting with docked granules in activated neutrophils. These data indicate that VAMP-2 mediates exocytosis of specific and tertiary granules, and that Q-SNARE/R-SNARE complexes containing VAMP-2 and syntaxin 4 are involved in neutrophil exocytosis.

Antibodies, Monoclonal↗
PubMed1990

Prejunctional beta-adrenoceptors, angiotensin II and neuropeptide Y receptors on sympathetic nerves in mouse atria are linked to N-ethylmaleimide-susceptible G-proteins.

We used the alkylating agent N-ethylmaleimide in order to investigate G-proteins linked to release-modulating prejunctional receptors of sympathetic nerves in mouse atria incubated with [3H]-noradrenaline. The receptors tested were facilitatory beta-adrenoceptors and angiotensin II receptors and inhibitory neuropeptide Y receptors. In order to evaluate the specificity of the N-ethylmaleimide treatment, we tested N-ethylmaleimide against the second messenger pathways that are linked to beta-adrenoceptors (adenylate cyclase) and angiotensin II (protein kinase C). The results show that a 60-min preincubation with N-ethylmaleimide (3 microM) abolished the facilitatory effect of isoprenaline (0.1 microM) and angiotensin II (0.1 microM) on the stimulation-induced release of noradrenaline and reduced the inhibitory action of neuropeptide Y (0.3 microM). N-ethylmaleimide had no effect on the stimulatory action of either phorbol dibutyrate (0.01, 0.1 microM), forskolin (10 microM), or a combination of 8-bromo adenosine-3'5'-monophosphate (90 microM) and 3-isobutyl-1-methylxanthine (100 microM). However, at a higher concentration (10 microM), N-ethylmaleimide reduced the facilitatory effect of phorbol dibutyrate (0.1 microM) and the combination of 8-bromo adenosine-3',5'-monophosphate (90 microM) and 3-isobutyl-1-methylxanthine (100 microM). This suggests that N-ethylmaleimide at 3 microM but not 10 microM was selective for receptor-mediated modulation of noradrenaline release without directly affecting the adenylate cyclase (forskolin, 8-bromo adenosine-3',5'-monophosphate + 3-isobutyl-1-methylxanthine) or protein kinase C (phorbol dibutyrate) transduction pathways. In atria from mice pretreated with pertussis toxin (1.5 micrograms/mouse), N-ethylmaleimide preincubation (1 and 3 microM) resulted in a more pronounced reduction of the inhibitory action of neuropeptide Y (0.3 microM). The nature of this interaction is unclear. Since N-ethylmaleimide has been shown in other studies to inactivate G-proteins, the inhibitory effect of N-ethylmaleimide on prejunctional beta-adrenoceptors, angiotensin II receptors and neuropeptide Y receptors of sympathetic nerves may suggest that G-proteins are involved with these receptors, although other effects of N-ethylmaleimide on the receptor coupling processes cannot be ruled out. Moreover, it appears that the concentration of N-ethylmaleimide used is critical since a higher concentration (10 microM) resulted in non-specific effects on signal transduction mechanisms in the present experimental conditions.

Adenylate Cyclase Toxin↗
PubMed1985

Effects of N-ethylmaleimide on ouabain-insensitive cation fluxes in human red cell ghosts.

In red cells of several species, the sulfhydryl reagent N-ethylmaleimide activates a Cl- -dependent, ouabain-resistant K+ transport pathway. Here we report our attempts to demonstrate ouabain-resistant Cl- -dependent K+ fluxes stimulated by N-ethylmaleimide in resealed human red cell ghosts using Rb+ as a K+ analogue. In contrast to intact cells, the rate constants of the base level Rb+ efflux in ghosts were similar in NaNO3 and NaCl (okRb = 0.535 +/- 0.079 h-1 and 0.534 +/- 0.085 h-1, respectively), while 1 mM N-ethylmaleimide stimulated Rb+ efflux strongly in NaNO3 (okRb = 14.26 +/- 1.32 h-1) and moderately in NaCl (okRb = 2.73 +/- 0.54 h-1). This effect was dependent on the presence of internal ATP. Stimulation of Rb+ efflux was observed in the presence of greater than or equal to 0.2 mM N-ethylmaleimide and increased at pH values approaching 8.0, consistent with titration of SH groups. N-Ethylmaleimide-stimulated Rb+ efflux was approx. 50% inhibited by 100 microM quinine sulfate whereas 1 microM bumetanide had no effect. In NaCl the N-ethylmaleimide-stimulated efflux saturated with initial internal ghost Rb+ concentration, but rates increased linearly in NaNO3. Replacement of external Na+ with glucamine or choline decreased the N-ethylmaleimide-stimulated Rb+ efflux, suggesting a role for external Na+. N-Ethylmaleimide-stimulated Rb+ efflux was greater in buffers with lipophilic anions such as SCN- or NO3- than in solutions with Cl- or acetate. However, the cation selectivity of the pathway studied was low, as Li+ efflux was also stimulated by N-ethylmaleimide. We conclude that the effect of N-ethylmaleimide on ouabain-resistant cation effluxes of human red cell ghosts is very different from the selective action of N-ethylmaleimide on Rb+ influxes in intact red cells.

Acetates↗
PubMed1984

Inhibitory effects of N-ethylmaleimide on insulin- and oxidant-stimulated sugar transport and on 125I-labelled insulin binding by rat soleus muscle.

These experiments examined the effects of N-ethylmaleimide on insulin- and oxidant-stimulated sugar transport in soleus muscle in terms of the Thiol-Redox model for insulin-stimulated adipocyte sugar transport (Czech, M.P. (1976) J. Cell. Physiol. 89, 661-668). Brief exposure (1 min) to N-ethylmaleimide (0.3-10 mM) inhibited the stimulatory effect of insulin (0.1 U/ml) on D-[U-14C]xylose uptake by rat soleus muscle. N-Ethylmaleimide also inhibited the stimulatory effects of H2O2 (5 mM), diamide (0.2 mM) and vitamin K-5 (0.05 mM). This effect of N-ethylmaleimide on insulin action was paralleled by the inhibition of 125I-labelled insulin binding by the muscle. N-ethylmaleimide lowered muscle ATP; however, its effects on sugar transport and 125I-labelled insulin binding could be dissociated from its effect on ATP. Exposing muscles to insulin prior to N-ethylmaleimide did not abolish the inhibitory effect of sulphydryl blockade on insulin-stimulated sugar transport, but did reduce the effect of the inhibitor by 20-30%. Conversely, when muscles were first allowed to bind 125I-labelled insulin and then exposed to the inhibitor, there was no effect of N-ethylmaleimide on pre-bound insulin. Exposure to diamide or vitamin K-5 before N-ethylmaleimide (1 mM) attenuated the inhibitory effect of sulphydryl blockade but no protective effect was observed with H2O2. None of the oxidants protected against the inhibitory effect of 3 mM N-ethylmaleimide. It is concluded that there are two N-ethylmaleimide-sensitive sites involved in the activation of muscle sugar transport at the post-receptor level. One of these would appear to be similar to the Thiol-Redox site described in the adipocyte; the other site appears to be an essential sulphydryl group whose function does not involve oxidation to a disulphide.

Animals↗
PubMed1975

Interaction of actin with N-ethylmaleimide modified heavy meromyosin in the presence and absence of adenosine triphosphate.

N-Ethylmaleimide modified heavy meromyosin in only 3-fold activated by actin rather than 200-fold as is normal heavy meromyosin (Silverman, R., Eisenberg, E., and Kielley, W. W. (1972), Nature (London) 240, 207). Ultracentrifuge studies demonstrated that in the absence of ATP the N-ethylmaleimide modified heavy meromyosin binds to actin at a ratio of 2 actins to 1 N-ethylmaleimide modified heavy meromyosin. However, it was found that most of the N-ethylmaleimide modified heavy meromyosin was not bound to actin during ATP hydrolysis. Ultracentrifuge studies demonstrated that in the presence of 25 or 50 mM KCl under conditions where the ATPase is maximally activated by actin, less than 5% of the N-ethylmaleimide modified heavy meromyosin was bound to actin. In the absence of KCl there was limited binding but even this binding did not appear to correlate with the N-ethylmaleimide modified heavy meromyosin ATPase rate. Turbidity and viscosity studies also indicated that in the presence of ATP under conditions of maximal actin activation the N-ethylmaleimide modified heavy meromyosin and actin are almost completely dissociated, whereas there is a marked increase in turbidity and viscosity after all of the ATP is hydrolyzed. These results suggest that in the presence of ATP and actin N-ethylmaleimide modified heavy meromyosin exists most of the time in a refractory state unable to bind to actin and only a small part of the time in a nonrefractory state which can interact with actin. It follows that the major rate-limiting step during actin activation is the transition from the refractory to the nonrefractory state. Since the actin activation of N-ethylmaleimide modified heavy meromyosin is lower than that of normal heavy meromyosin this transition may be slower for N-ethylmaleimide modified heavy meromyosin than for normal heavy meromyosin.

Actins↗
PubMed1979

Intramitochondrial phospholipase activity and the effects of Ca2+ plus N-ethylmaleimide on mitochondrial function.

Liver mitochondria treated with N-ethylmaleimide can accumulate Ca2+ but cannot retain it. Ca2+ loss following uptake occurs in parallel with a proton uptake and collapse of the membrane potential. Respiration is not activated during Ca2+ release and cannot be stimulated by uncoupler. After Ca2+ release and accompanying phenomena are nearly complete, the mitochondria undergo a large amplitude swelling. Nupercaine inhibits the premature release of Ca2+, proton uptake, decline in membrane potential, inhibition of uncoupler-stimulated respiration, and large amplitude swelling. Ruthenium red also prevents these effects. Neither Sr2+ or Mn2+ will substitute for Ca2+ to induce these effects in N-ethylmaleimide-treated mitochondria. The effects of N-ethylmaleimide plus Ca2+ on mitochondria are not accompanied by a significant alteration in the content or composition of phospholipids but are accompanied by small increases in the mitochondrial content of free fatty acids. Free fatty acids accumulate more rapidly in response to limited Ca2+ loading in the absence of N-ethylmaleimide than they do in its presence. In the absence of N-ethylmaleimide, polyunsaturated fatty acids and saturated plus monounsaturated fatty acids accumulate at nearly equal rates. In the presence of N-ethylmaleimide, polyunsaturated fatty acids accumulate more rapidly than saturated plus monounsaturated fatty acids. Any condition or agent tested which inhibited swelling and the other effects produced by Ca2+ plus N-ethylmaleimide also prevented the more rapid accumulation of polyunsaturated, compared to saturated plus monounsaturated, fatty acids. In the light of a positional analysis of phospholipid acyl moieties, these data suggest that 1-acyllysophospholipids accumulate in swelling mitochondria but not in response to noraml Ca2+ loading or when swelling is blocked by other agents. The free fatty acid accumulation, per se, is not responsible for swelling, but levels of exogenous palmitic acid as low as 1 nmol/mg of protein dramatically alter the dependence of swelling velocity on Ca2+ concentration, producing a shift from a sigmoidal- to a hyperbolic-like relationship. This same alteration is brought about by aging the mitochondrial preparation at 0 degrees C. Either pyruvate or DL-carnitine prevents the effect of exogenous palmitate and restores the Aa2+ swelling dependence of aged N-ethylmaleimide-treated mitochondria to that of fresh N-ethylmaleimide-treated mitochondria. Intramitochondrial acylcoenzyme A or acylcarnitine, or both, therefore, to be the modulator of Ca2+ sensitivity rather than free fatty acid. The findings are discussed in terms of the role of intramitochondrial phospholipase and other phospholipid metabolizing enzymes in the mechanisms of N-ethylmaleimide plus Ca2+ effects on mitochondria.

Animals↗
PubMedDate not supplied

N-ethylmaleimide increases P-glycoprotein photoaffinity labeling with iodoaryl-azidoprazosin in multidrug resistant cells.

P-glycoprotein (P-gp) mediates a multidrug resistance (MDR) phenotype in tumor cell lines selected with lipophilic cytotoxic drugs. Transport studies using purified P-glycoprotein reconstituted into defined liposomes have shown energy-dependent drug efflux of structurally dissimilar drugs. In this report, we have examined the effects of N-ethylmaleimide, a potent inhibitor of the P-gp ATPase, on P-gp drug binding in intact MDR cells and in plasma membranes. Our results show that short term treatment of MDR cells with 1-50 microM N-ethylmaleimide led to a concentration dependent increase in P-gp photoaffinity labeling with iodoaryl-azidoparazosin (IAAP). In addition, N-ethylmaleimide increases [3H] vinblastine accumu-lation in drug-resistant but not in sensitive cells. Comparison of IAAP photolabeled P-gp from intact cells with or without N-ethylmaleimide treatment did not show differences in the pattern of IAAP photolabeled peptides. Thus, the observed increase in P-gp photolabeling with IAAP in N-ethylmaleimide treated cells is not due to photolabeling at different sites. Incubation of MDR cells with [14C] N-ethylmaleimide showed that P-gp is directly modified at several Cysteine residues, as found from a complete proteolytic digestion of [14C] Nethylmaleimide labeled P-gp. The comparison of V8 staphylococcus aureas peptides from [14C] Nethylmaleimide or IAAP modified P-gp showed some peptides to co-migrate on SDS PAGE. However, modification of plasma membranes from drug resistant cells treated with N-ethylmaleimide did not show a dose-dependent increase in P-gp photolabeling with IAAP as seen with intact MDR cells. Interestingly, N-ethylmaleimide increases P-gp phosphorylation by inhibiting the turnover of Pgp phosphates. However, inhibition of P-gp phosphorylation with calyculin A did not show an increase in P-gp photolabeling in MDR cells. Taken together, the results of this study suggest that N-ethylmaleimide potentiates P-gp photolabeling with IAAP by inhibiting P-gp ATPase thereby increasing the local concentration of IAAP in intact MDR cells. Furthermore, inhibition of P-gp ATPase by N-ethylmaleimide does not lead to conformational changes that affects P-gp drug binding.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1994

Effects of N-ethylmaleimide on dopamine release in the rat striatum after repeated treatment with methamphetamine.

Effects of N-ethylmaleimide or forskolin on electrically evoked dopamine release were investigated in striatal slices of rats pretreated with methamphetamine. N-Ethylmaleimide and forskolin both enhanced the evoked dopamine release in a concentration-dependent manner. The enhancement by N-ethylmaleimide of spontaneous dopamine release was not abolished by tetrodotoxin; the electrically evoked release was abolished, irrespective of its magnitude. Moreover, N-ethylmaleimide prevented the inhibitory effect of the dopamine receptor agonist, 2-amino-6-allyl- 5,6,7,8-tetrahydro-4H-thiazolo(5,4-d)-azepine dihydrochloride (B-HT 920) and the stimulatory effect of the dopamine receptor antagonist, (-)-sulpiride, on the evoked dopamine release. In contrast, forskolin had no effect on the B-HT 920-induced inhibition and (-)-sulpiride-induced enhancement of the evoked dopamine release. These data indicate that release-modulating dopamine autoreceptors are N-ethylmaleimide-sensitive and forskolin-insensitive. As N-ethylmaleimide has been reported to inactivate Gi protein and to block the regulation of noradrenaline release by alpha 2-adrenoceptors, the present results suggest that N-ethylmaleimide inactivates inhibitory GTP binding proteins to block the regulation by dopamine autoreceptors of evoked dopamine release. Methamphetamine pretreatment, which caused behavioral sensitization to a challenge dose of methamphetamine, attenuated the stimulatory effect of N-ethylmaleimide but not forskolin on the evoked dopamine release. The data indicate that the repeated administration of methamphetamine reduces a function of N-ethylmaleimide sensitive signal transduction system, probably including inhibitory GTP binding protein.

Animals↗
PubMed1989

Differential labeling of the erythrocyte hexose carrier by N-ethylmaleimide: correlation of transport inhibition with reactive carrier sulfhydryl groups.

Inhibition of hexose transport by N-ethylmaleimide was studied with regard to alkylation of different types of sulfhydryl group on the hexose carrier of the human erythrocyte. Uptake of 3-O-methylglucose was progressively and irreversibly inhibited by N-ethylmaleimide, with a half-maximal effect at 10-13 mM. A sulfhydryl group known to exist on the exofacial carrier was not involved in transport inhibition by N-ethylmaleimide, since reversible protection of this group by the impermeant sulfhydryl reagent 5,5'-dithiobis(2-nitrobenzoic acid) had no effect on the ability of N-ethylmaleimide to inhibit transport, or on its ability to decrease the affinity of the exofacial carrier for maltose. Nevertheless, the exofacial sulfhydryl was quite reactive with N-ethylmaleimide, since it was possible using a differential labeling technique to specifically label this group in protein-depleted ghosts with a half-maximal effect at 0.3 mM N-[3H]ethylmaleimide, and to localize it to the Mr 19,000 tryptic carrier fragment. Transport inhibition by N-ethylmaleimide correlated best with labeling of a single cytochalasin B-sensitive internal sulfhydryl group on the glycosylated Mr 23,000-40,000 tryptic fragment of the carrier, which was half-maximally labeled at about 4 mM reagent. Whereas N-ethylmaleimide readily alkylates the exofacial carrier sulfhydryl, it inhibits transport by reacting with at least one internal carrier sulfhydryl located on the glycosylated tryptic carrier fragment.

3-O-Methylglucose↗
PubMed1992

Assessment of a model for the opposing effects of N-ethylmaleimide on the affinity of muscarinic agonists for M2 receptors.

1. Homogenates of guinea pig right atrium (M2 receptors) were treated with N-ethylmaleimide, after which the ability of carbachol to inhibit binding of [3H]quinuclidinyl benzilate was studied. 2. At 37 degrees C, 10(-4) M and 10(-3) M, but not 10(-5) M, N-ethylmaleimide increased the affinity of carbachol for the receptor. At 2 degrees C, 10(-5) M and 10(-4) M, but not 10(-3) M, N-ethylmaleimide decreased carbachol affinity. At 2 degrees C, after the homogenate had been incubated at 37 degrees C without N-ethylmaleimide, 10(-5) M N-ethylmaleimide decreased carbachol affinity, as at 2 degrees C without preincubation, but 10(-3) M N-ethylmaleimide increased carbachol affinity, as at 37 degrees C. 10(-4) M N-ethylmaleimide was without effect. 3. The results are discussed with respect to a previously proposed model in which N-ethylmaleimide interacts with two sites, causing an increase or decrease in agonist affinity respectively.

Animals↗