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PubMed1982

Inhibition of N-ethylmaleimide of the MgATP-driven proton pump of the chromaffin granules.

The thiol reagent N-ethylmaleimide (NEM) completely inhibits the proton pump activity of the H+-ATPase in chromaffin granule 'ghosts' at concentrations which only partly (approximately 20%) inhibit the Mg2+-dependent ATP hydrolysis. Half-maximal inhibition was obtained at approximately 13 microM NEM as compared to 18 microM for the classical proton channel inhibitor N,N'-dicyclohexylcarbodiimide (DCCD), and the apparent stoichiometry of the inhibitors at complete inhibition was NEM : DCCD congruent to 1 : 2. HIgh concentrations of NEM (greater than 100 microM) induce a dissipation of the transmembrane potential generated by MgATP. These findings establish NEM as a valuable proton channel inhibitor in chromaffin granules and explain the rather complex effect of NEM previously reported for catecholamine accumulation in this organelle.

Adenosine Triphosphatases↗
PubMed1990

Identification of sites for alkylation by N-ethylmaleimide and pertussis toxin-catalyzed ADP-ribosylation on GTP-binding proteins.

An alpha beta gamma-trimeric GTP-binding protein (Go) serving as the substrate of pertussis toxin-(IAP) catalyzed ADP-ribosylation was purified from rat brain membranes. The constituent alpha-subunit (alpha o) was alkylated with N-ethylmaleimide (NEM), and the functionally important sulfhydryl groups were investigated. There were at least two cysteine residues highly reactive to NEM on the GDP-bound form of alpha o. These alkylations resulted in loss of its ability to be ADP-ribosylated by IAP and to associate with beta gamma, but leaving the GTP-binding site of alpha o intact. The reacted cysteine residues were identified by the sequencing of tryptic fragments of alpha o. One of the alkylation sites was Cys-351, which was four amino acid residues away from the carboxyl-terminus of the molecule. The Cys-351 was proven to be also a site for IAP-catalyzed ADP-ribosylation. Possible roles of cysteine residues on the alpha-subunit of Go are discussed in the functions of the signal transducing protein.

Adenosine Diphosphate Ribose↗
PubMed1993

Chemical modification of rat liver cytosolic NADP(+)-linked isocitrate dehydrogenase by N-ethylmaleimide. Evidence for essential sulphydryl groups.

Incubation of rat liver cytosolic isocitrate dehydrogenase with N-ethylmaleimide (NEM) resulted in the inactivation of the enzyme following pseudo-first order kinetics. Isocitrate affords considerable protection against inactivation whereas NADP+ enhances modification of the enzyme, suggesting localization of the modified group at the active site. Correlation of loss of activity with incorporation of [14C]NEM indicated that two sulphydryl residues/sub-unit are modified of which only one is shown to be involved in catalysis. pH dependence of the inactivation process implicates a reactive group of pKa 8.1 in catalysis. We conclude that a unique cysteine residue is essential for maximal catalytic activity of isocitrate dehydrogenase.

Animals↗
PubMed1994

Association of N-ethylmaleimide-sensitive factor with synaptic vesicles.

N-Ethylmaleimide-sensitive factor (NSF) mediates docking and/or fusion of transport vesicles in the multi-pathways of vesicular transport. NSF was highly expressed in brain and adrenal gland. Immunostaining of cerebellum with an anti-NSF monoclonal antibody showed that NSF is predominantly localized in the molecular layers and the glomeruli of the granule cell layers. This distribution coincided well with that of synaptophysin, a marker protein of synaptic vesicles. Purification and immunoprecipitation revealed that NSF is associated with brain synaptic vesicles. The present results suggest that NSF is associated with synaptic vesicles without Ca2+ influx.

Animals↗
PubMed1994

N-ethylmaleimide-sensitive mutant (beta Val-153-->Cys) Escherichia coli F1-ATPase: cross-linking of the mutant beta subunit with the alpha subunit.

A beta subunit mutation, beta Val-153-->Cys, in the glycine-rich sequence (phosphate-binding loop) of Escherichia coli F1 was constructed. Like vacuolar-type ATPase, the mutant enzyme was inhibited by N-ethylmaleimide (NEM) and labeled with [14C]NEM. The inhibition and labeling were prevented by ATP. m-Maleimidobenzoyl-N-hydroxysuccinimide (MBS) (3 microM) almost completely inhibited the mutant enzyme, and cross-linked one pair of alpha and beta subunits. These results suggest that the interaction of the domain near beta Val-153 with the alpha subunit is essential for catalytic cooperativity of the enzyme and that beta Val-153 is within 10 A of the alpha subunit.

Adenosine Triphosphate↗
PubMed1995

Substrate-induced acceleration of N-ethylmaleimide reaction with the Cys-65 mutant of the transposon Tn10-encoded metal-tetracycline/H+ antiporter depends on the interaction of Asp-66 with the substrate.

We previously reported that the reaction of [14C]N-ethylmaleimide (NEM) with the S65C mutant of the transposon Tn10-encoded metal-tetracycline/H+ antiporter (TetA(B)) is competitively inhibited by tetracycline [Yamaguchi, A. et al., FEBS Lett. 322 (1993) 201-204]. However, this observation has been revealed to be a mistake. The reaction of [14C]NEM with S65C TetA(B) was significantly and reproducibly accelerated by tetracycline, i.e. not inhibited. When Asp-66 was replaced by Ala, the reaction of NEM with the Cys-65 residue was no longer affected by tetracycline. In contrast, when Arg-70 was replaced by Ala, the acceleration of the reaction was unaltered. The tetracycline acceleration of the reaction to the Cys-65 residue was further stimulated with energization of the membrane on the addition of NADH. On the other hand, the tetracycline-induced acceleration was not observed in the absence of a divalent cation. These observations indicated that the Cys-65 locus is exposed to the medium according to the interaction of a divalent cation-tetracycline chelation complex with Asp-66.

Amino Acid Sequence↗
PubMed1986

Effects of organic co-solvents on the oxygen equilibrium of N-ethylmaleimide-treated human haemoglobin.

We have studied the effects of monohydric alcohols and formamide on the oxygen equilibrium of native and N-ethylmaleimide-treated human haemoglobin. Comparison of the results obtained for the two haemoglobins gives further and compelling evidence in favour of the model, proposed recently by our group, on the role played by the solvent in the conformational equilibria of haemoglobin; moreover the results provide direct functional evidence of the relevance of the electrostatic free energy of salt bridges to the T in equilibrium with R equilibrium of haemoglobin.

1-Propanol↗
PubMed1985

Racial differences in bumetanide-sensitive cotransport and N-ethylmaleimide-stimulated potassium efflux.

Racial differences in erythrocyte potassium effluxes mediated by two loop-diuretic sensitive modes of cotransport were compared. In red cells loaded to contain approximately equimolar amounts of sodium and potassium, black subjects had lower bumetanide-sensitive sodium-dependent net potassium effluxes as compared to whites. In fresh, washed erythrocytes pretreated with N-ethylmaleimide (NEM), maximal net potassium efflux was greater in blacks than in whites. NEM-stimulated potassium efflux was partially inhibited by bumetanide but only at very high concentrations. The quantitative differences in these two modes of potassium efflux suggest that NEM-stimulated potassium efflux is not an altered mode of sodium-dependent potassium efflux.

Biological Transport↗
PubMed1986

Effects of N-ethylmaleimide on muscarinic acetylcholine receptor subtype autoradiography and inositide response in rat brain.

Chemical modification of brain muscarinic acetylcholine receptors (mAChr) with N-ethylmaleimide (NEM) has been employed to investigate mAChr-subtype distribution and mediation of the inositide response. 3H-Pirenzepine and 3H-oxotremorine-M were used to autoradiographically localize the M1- and M2-AChr subtypes, respectively, in brain slices. M1- and M2-AChr distribution were observed to be distinct from each other. The presence of 1 mM NEM selectively reduced the labeling of M2-, but not of M1-AChr. These data support the notion that NEM converts the high-affinity M2-AChr to a lower affinity state, without affecting the affinity of the M1-AChr. Also, regional analysis indicated that the M1- and M2-AChr subtypes were not interconvertible by NEM. NEM at 30 microM enhanced the carbamylcholine stimulated labeling of phosphatidic acid from 32Pi in nerve endings from rat forebrain, suggesting that the low affinity M2-AChr may mediate at least a part of the inositide response to cholinergic stimulation.

Animals↗
PubMed1990

Effect of ICV pertussis toxin and N-ethylmaleimide on the levels of substance P and serotonin in brain and spinal cord of the rat.

The effects of single intracerebroventricular (icv) injections of either 0.5 microgram pertussis toxin or 5 micrograms N-ethylmaleimide (NEM) on the levels of immunoreactive substance P (ir-SP) and serotonin (5-HT) in the brain and spinal cord of rats have been assessed. At two and six days after pertussis toxin injection, the levels of ir-SP appeared significantly diminished in the spinal cord (about 34%). This reduction was even greater at two days after NEM injection (43%). These two agents did not alter the ir-SP of the midbrain and thalamus, whereas NEM increased the neuropeptide content in the pons-medulla. On the other hand, the thalamic content of serotonin was reduced two days after pertussis toxin (32%) or NEM (20%) injection. The indoleamine levels of the spinal cord were reduced by these treatments (20%), while in the midbrain a slight decrease could be observed. These findings suggest that pertussis toxin and NEM produce these effects by acting upon a common neural substrate.

5-Hydroxytryptophan↗
PubMed1993

Inhibition by N-ethylmaleimide of H(+)-ATPase reduces the cellular action of arginine vasopressin in cultured rat renal papillary collecting tubule cells.

We examined whether H(+)-ATPase is involved in the control of cellular action of arginine vasopressin (AVP) to produce adenosine 3', 5'-monophosphate (cAMP) and mobilize cellular free calcium ([Ca2+]i) in rat renal papillary collecting tubule cells in culture. N-Ethylmaleimide (NEM), an inhibitor of H(+)-ATPase, reduced the cellular pH (pHi) dose-dependently. AVP increased cellular cAMP production in a dose-dependent manner. 5 x 10(-6) and 1 x 10(-5) M NEM significantly diminished the AVP-induced increase in cAMP production. 1 x 10(-7) M AVP also increased [Ca2+]i from 111.2 to 189.3 nM, which was significantly reduced by NEM in a dose-dependent manner. These results indicate that H(+)-ATPase participates the cellular action of AVP mediated via the pHi control in renal papillary collecting tubule cells.

Animals↗
PubMed1988

Role of an N-ethylmaleimide-sensitive transport component in promoting fusion of transport vesicles with cisternae of the Golgi stack.

An N-ethylmaleimide-sensitive transport component (NSF) has been purified on the basis of its ability to support transport between Golgi cisternae. We now report that NSF is needed for membrane fusion. Thus, when NSF is withheld from incubations of Golgi stacks with cytosol and ATP, uncoated transport vesicles accumulate. Biochemical experiments confirm this conclusion and reveal that NSF is needed to form the first of two previously described prefusion complexes. NSF, therefore, acts within a cascade in which a vesicle-cisterna complex is matured until it is competent for fusion. We suggest that this reflects the stepwise assembly of a multisubunit "fusion machine" following vesicle attachment.

Animals↗
PubMed1992

Guanine nucleotide stimulation of norepinephrine secretion from permeabilized PC12 cells: effects of Mg2+, other nucleotide triphosphates and N-ethylmaleimide.

The addition of either Ca2+ or guanosine 5'-O-3-(thiotriphosphate), GTP gamma S, to digitonin-permeabilized rat pheochromocytoma PC12 cells stimulates norepinephrine release. Unlike Ca(2+)-stimulated release, there is a delay between the time of addition of GTP gamma S to digitonin-permeabilized PC12 cells and stimulation of norepinephrine release. Preincubation of the permeabilized cells in the absence of Mg2+ eliminates this lag and increases the initial rate of GTP-gamma S-stimulated norepinephrine secretion. This suggests that the rate of GDP dissociation from the GTP-binding protein responsible for this stimulation is faster in the absence of Mg2+ than in its presence. While an equimolar concentration of GTP gives 50% inhibition of GTP gamma S-stimulated release, 100-fold excesses of ITP, ATP, UTP and CTP gave no inhibition of GTP gamma S-stimulated release. Both the inability of ITP to inhibit GTP gamma S-stimulated secretion and the increase in GTP gamma S-stimulated secretion caused by preincubation in the absence of Mg2+ indicate that some of the properties of the GTP-binding protein responsible for this stimulation are more like those of the low molecular weight GTP-binding proteins rap1 and ras than those of a heterotrimeric G-protein. Low concentrations of N-ethylmaleimide gave more inhibition of GTP gamma S-stimulated release than Ca(2+)-stimulated release which suggests that the mechanisms by which Ca2+ and GTP gamma S stimulate norepinephrine release are at least in part distinct.

Animals↗
PubMed1995

Sensitivity to inhibition by N-ethylmaleimide: a property of nitrobenzylthioinosine-sensitive equilibrative nucleoside transporter of murine myeloma cells.

Murine myeloma SP2/0-Ag14 cells possess both nitrobenzylthioinosine (NBMPR)-sensitive and NBMPR-insensitive equilibrative uridine transport systems. No Na(+)-dependent uridine transport system was detected. The NBMPR-insensitive transport system is similarly insensitive to inhibition by dilazep and dipyridamole. Dose-response curve for the inhibition of equilibrative uridine transport by N-ethylmaleimide (NEM), a sulfhydryl reagent, in these cells was biphasic. About 30-40% of the uridine transport was inhibited by NEM at IC50 value of 0.15 mM. The other 60-70% of the transport activity remained insensitive to NEM at concentration as high as 3 mM. The decrease in NBMPR-sensitive uridine transport in the presence of 0.3 mM NEM was due to a 3-fold decrease in transport affinity. Apparent Km values of 500 and 1600 microM and Vmax values of 13 and 12 microM/s were obtained for untreated and NEM-treated cells, respectively. NEM (0.3 mM) has little effect on the Km of NBMPR-insensitive transporter, with apparent Km values of 100 and 110 microM and Vmax values of 3.0 and 2.5 microM/s for untreated and NEM-treated cells, respectively. High sensitivity of NBMPR-sensitive transporter to NEM inhibition was also observed in HL-60 and MCF-7 cells. Decrease in specific 3H-NBMPR equilibrium binding affinity in myeloma cells was observed after treatment with 0.3 mM NEM. Apparent Kd values of 0.32 and 2.3 nM with Bmax values of 48,000 and 44,000 sites/cell were obtained for untreated and NEM-treated cells, respectively. NBMPR, dilazep and dipyridamole at 30 microM, and uridine at 10 mM failed to protect the NBMPR-sensitive transporter against NEM inhibition. It is possible that a critical sulfhydryl residue is closed to substrate binding/transporting site of the NBMPR-sensitive transporter. NEM, a sulfhydryl reagent containing an activated double bond, hinders the affinity of this transporter by forming a stable thiol ether bond with the reactive residue.

Animals↗
PubMed1989

The inhibitory effects of N-ethylmaleimide, colchicine and cytochalasins on human T-cell functions.

The thiol-alkylating agent, N-ethylmaleimide (NEM), was found to inhibit the response of human peripheral blood mononuclear cells to the T-cell mitogen, concanavalin A (Con A). NEM (10 microM) blocked Con A-induced agglutination, production of interleukin-2 (IL-2) and expression of the IL-2 receptors (Tac) without toxicity. In order to determine whether the effects of NEM on lymphokine production were related to inhibition of agglutination, we compared the immunosuppressive effects of NEM with those of cytochalasin A, cytochalasin B and colchicine. NEM did not inhibit E-rosette (ER) formation, suggesting that it does not interfere with actin filaments. Low concentrations of NEM (4 microM) inhibited IL-2 production and Tac expression without inhibiting agglutination, while 6-10 microM NEM blocked agglutination and DNA synthesis as well. In contrast, 5-10 microM cytochalasin B (CB) inhibited ER formation, agglutination, Tac expression and DNA synthesis, but augmented IL-2 production by three- to ten-fold. Colchicine (0.1-10 microM) had no effect on ER formation or agglutination and augmented IL-2 production by as much as 18-fold. However, colchicine blocked Tac expression by greater than 40% and DNA synthesis by greater than 80%. Cytochalasin A (CA), which has the thiol-reactive properties of NEM, the actin filament-disrupting properties of CB, and the microtubule-disrupting properties of colchicine, exhibited the immunosuppressive effects of all three compounds. These studies suggest that the inhibitory effects of NEM on IL-2 production do not appear to be due to reactivity with the cytoskeleton, but are probably due to effects on signal transduction pathways leading to IL-2 production and expression of IL-2 receptors.

Agglutination↗
PubMed1992

An N-ethylmaleimide-sensitive G-protein modulates Aplysia Ca2+ channels.

The sulfhydryl alkylating agent N-ethylmaleimide (NEM) was used to probe the possible modulation of calcium current (ICa) by G-proteins in identified neurons of Aplysia californica. ICa recorded with conventional two-electrode voltage clamp was irreversibly suppressed by bath applied NEM in a concentration-dependent manner. This effect was fully blocked by addition of dithiothreitol or intracellular pressure injection of GTP gamma S but was unaffected by pre-treatment with pertussis toxin. These findings suggest that NEM inhibits ICa by causing persistent activation of an inhibitory G-protein in the absence of applied agonist. It appears that alkylation of key cysteine residues involved in G-protein deactivation underlie this effect.

Action Potentials↗
PubMed1992

Reversal by pertussis toxin and N-ethylmaleimide of the facilitation of baroreceptor reflex response by somatostatin in the rat.

We evaluated the transmembrane signaling mechanism that may underlie the facilitatory action of somatostatin (SOM) on baroreceptor reflex (BRR), using adult, male, Sprague-Dawley rats anesthetized with pentobarbital sodium (40 mg/kg, i.p.). Intracerebroventricular (i.c.v.) application of SOM (2 nmol) promoted a significant elevation in BRR response, induced by phenylephrine (5 micrograms/kg, i.v.). This potentiatory action of the tetradecapeptide was significantly reversed after pretreating animals with bilateral microinjection of pertussis toxin (25 ng) or N-ethylmaleimide (2 nmol) into the nucleus tractus solitarius (NTS), the terminal site for baroreceptor afferents. These results suggest that a pertussis toxin-sensitive GTP-binding regulatory protein, possibly Gi, may be involved in the modulation of the BRR by SOM at the NTS.

Afferent Pathways↗
PubMed1994

Potassium efflux from infant and adult rat choroid plexuses: effects of CSF anion substitution, N-ethylmaleimide and Cl transport inhibitors.

To analyze interdependent transport of K and Cl, we investigated Rb (K) efflux from in vitro choroid plexus (CP) in isotonic artificial CSF (aCSF) medium containing anions or agents that alter KCl transport. Lateral ventricle CP was loaded with 86Rb for release to enable calculation of the efflux rate coefficient, k. With Cl as the main anion in control aCSF, the k value for 86Rb (K) in CP of 1 week infant rats (0.177 min-1) was 19% lower than in adults (0.218 min-1) (P < 0.005). Replacing CSF Cl with NO3 or SCN, respectively, reduced k for K in infant CP by 73% and 43%; similar anion selectivity was observed in adult tissues (P < 0.05). N-Ethylmaleimide (NEM), which stimulates KCI cotransport, significantly enhanced K efflux in infants and adults. In adult CP, the KCl cotransport inhibitor, furosemide (1 mM), decreased K efflux by 23% or 65%, respectively, when aCSF had Cl or NO3 as the main anion. In infant rat CP, 0.1 mM bumetanide (another KCl cotransport inhibitor), reduced k for K by 65%, whereas the Cl channel blocker diphenylamine carboxylate (1 mM) did not significantly alter K efflux. The collective findings for rat CP indicate a substantial component of K efflux that is associated with Cl concentration and the Cl transport protein sensitive to loop diuretics and NEM. The Cl-dependent K efflux is present in infants.

Aging↗